Bostanci N, Belibasakis G (Eds.) - Pathogenesis of Periodontal Diseases. Biological Concepts For Clinicians (Springer, 2018)

Nagihan Bostanci
Georgios Belibasakis
Editors
Pathogenesis of
Periodontal Diseases
Biological Concepts
for Clinicians
123
Pathogenesis of Periodontal Diseases
Nagihan Bostanci
Georgios N. Belibasakis
Editors
Pathogenesis of
Periodontal Diseases
Biological Concepts for Clinicians
Editors
Nagihan Bostanci Georgios N. Belibasakis
Department of Dental Medicine Department of Dental Medicine
Karolinska Institute Karolinska Institute
Stockholm Stockholm
Sweden Sweden
ISBN 978-3-319-53735-1 ISBN 978-3-319-53737-5 (eBook)

DOI 10.1007/978-3-319-53737-5
Library of Congress Control Number: 2017953835
© Springer International Publishing AG 2018

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Contents
1 Periodontal Pathogenesis: Definitions and Historical

Perspectives�� 1
Nagihan Bostanci and Georgios N. Belibasakis
2 Diversity of Oral Biofilms in Periodontal Health
and Disease �� 9
Purnima S. Kumar
3 Subgingival Biofilms as Etiological Factors
of Periodontal Disease�� 21
Thomas Thurnheer, Kai Bao, and Georgios N. Belibasakis
4 Bacterial Virulence Factors that Contribute
to Periodontal Pathogenesis�� 31
Anders Johansson and Gunnar Dahlén
5 Active Matrix Metalloproteinase-8: Contributor
to Periodontitis and a Missing Link Between
Genetics, Dentistry, and Medicine�� 51
Timo Sorsa, Anna Maria Heikkinen, Jussi Leppilahti, Taina
Tervahartiala, Solomon Nwhator, Nilminie Rathnayake, Päivi
Mäntylä, Dirk-Rolf Gieselmann, and Lutz Netuschil
6 Inflammatory Pathways of Bone Resorption
in Periodontitis �� 59
Franco Cavalla, Claudia C. Biguetti, Thiago P. Garlet, Ana
Paula F. Trombone, and Gustavo P. Garlet
7 Genetic Influences on the Periodontal
Microbial-Host Crosstalk�� 87
Luigi Nibali
8 Antimicrobial Peptides: Roles in Periodontal
Health and Disease�� 97
Daniel Jönsson
9 Periodontal Pathogenesis: Conclusions
and Future Directions�� 111
Georgios N. Belibasakis and Nagihan Bostanci
Periodontal Pathogenesis:
Definitions and Historical
1
Perspectives
Nagihan Bostanci and Georgios N. Belibasakis
1.1 Introduction: Realizations increases with age. Taking also under consideration
from an Epidemiological the increasing life expectancy, periodontitis is a
Perspective growing health problem.
Despite the persistence of severe periodontitis
Periodontal disease, or periodontitis, is a globally even in the twenty-first century, the last 100 years
widespread pathology of the human oral cavity. have witnessed a significant progress in our
Indeed, approximately 10% of the global adult understanding of its pathogenesis, that is, the con-
population is highly vulnerable to severe peri- glomerate of biological processes that lead to the
odontitis. Another 10–15% appears to be com- disease. Nevertheless, the actual “coordinator(s)”
pletely resistant to it, while the remainder vary of the disease is still an issue of intense debate.
between these two extremes [1, 2]. Moreover, the Microbiology researchers place emphasis in seek-
prevalence of periodontitis is peaking at the fourth ing species, or combinations of them, associated
decade of life and increasing to 70–85% in the age with different clinical forms of the disease. On the
group of 60–65 [3]. Strikingly, despite major other side, immunologists are in pursuit of cells
improvements in oral hygiene practices today, and molecules that orchestrate the tissue-destruc-
these proportions are not far from what was tive inflammation, as a result of the bacterial chal-
reported in possibly the first epidemiological lenge. These are the two sides of the same coin,
report of periodontitis in humans back in 1918 and only when studied together can we appreciate
(then defined as periodontoclasia or pyorrhea the complexity of the periodontal pathogenesis.
alveolaris). According to that, the prevalence of Understanding the variants, uniqueness, and
the disease in the Chicago area was 13% in the redundancies of these biological mechanisms is a
age range of 20–24, 68% in 30–39, and 88% over preamble for thinking in new ways for preventing
50. Much recent data from USA showed that there and managing these diseases.
is not a drop but rather an increase in these num-
bers among the older individuals [4]. That means
that periodontitis is an inevitable oral pathology
1.2 efinition of Periodontal
D
of the human population, and its prevalence
Disease from a Historical
Perspective
N. Bostanci (*) • G.N. Belibasakis
Department of Dental Medicine,
The human interest in defining and understanding
Karolinska Institute, Stockholm, Sweden
e-mail: [email protected]; periodontal disease spans over the centuries, and
[email protected] many paradigm shifts occurred concerning its
© Springer International Publishing AG 2018 1

N. Bostanci, G.N. Belibasakis (eds.), Pathogenesis of Periodontal Diseases,
DOI 10.1007/978-3-319-53737-5_1
2 N. Bostanci and G.N. Belibasakis
epidemiology and pathogenesis. Pioneers in the 1746. He supported that the disease does not have
field have left us with a heritage of historical in systemic causes, since he observed that “inter-
knowledge that has been transforming over the nal remedies” were not successful as a cure.
years. Their views and contributions were direct Hence, it had to be a local or accidental origin that
reflection of the knowledge that was available at caused the disease [6]. More than half a century
the time and an important testament for the gen- later, physiologist and surgeon John Hunter pro-
erations to come. Although it won’t be possible to posed that alveolar bone around the teeth is dis-
single out all great contributors, we will try to solved due to inflammation occurring in the
summarize some hallmarks in chronological order. gingiva, coining the term “periodontosis” to the
disease [7]. In 1882, American dentist John Riggs
historically named the disease “pyorrhea alveo-
1.3 he Eras of “Pitius”
T laris,” later known as “Riggs’ disease,” which he
and “Calculus” described as a suppurative inflammation of the
gums and alveolar process [8]. Although inflam-
While ancient Greeks knew nothing about the mation of gingival tissue was acknowledged,
nature of periodontal disease, let alone their Riggs still advocated for calculus being the local
pathogenesis, they used their senses to “diagnose” etiological factor of the disease, based on the
it by its malodor and proposed the etiological fac- observation that it was cured following the
tors. Hippocrates wrote in his scripts that the “evil removal of calculus by a scaler.
malodor” is as result of “pitius.” He even a pro-
posed a therapy, which involved rinsing the mouth
with a solution containing oil from anise seeds 1.5 he Eras of Histopathology
T
and white wine, possibly one of the pioneer and Microbial Causation
mouthwashes [5]. Moving into the Roman Era,
the disease was still not named. “Wobbly” The period between 1880 and 1920 was marked
(mobile) teeth were observed as a result of “calcu- by an expansion of the understanding of the sci-
lus,” a Latin word meaning “a pebble or stone.” It entific discipline of microbiology and the recog-
was possibly then when calculus was placed on nition of resided oral bacteria as a causative
the map as an etiological factor of periodontal dis- factor for pyorrhea alveolaris. With that, the
ease, something that emerged again in the early notion of dental plaque being a causative factor
eighteenth century. for the disease was on the rise. In 1890, W.D.
Miller hypothesized that several remote causal
factors weakened periodontal tissues, rendering
1.4 he Era of “Periodontosis”
T them susceptible to challenge by bacteria inhabit-
and “Pyorrhea Alveolaris” ing the mouth [9], a concept of great resemblance
to the ecological plaque hypothesis that devel-
While there were no known scientific attempts to oped more than a century later [10]. During the
prove the causative relationship between calculus same period, G.V. Black, a renowned restorative
and the disease for over a millennium, this was the dentist, detailed the structure of the periodontal
dominating dogma due to the casual observation tissues and named their pathological destruction
that routine calculus removal improves this condi- “periodontoclasia-calcic inflammation of the
tion. French pathologist Pierre Fauchard, the periodontal membrane.” His descriptions were
“father of modern dentistry,” was possibly the first based on clinical characteristics, and he was the
to discuss this periodontal pathology in detail and first to use a primordial-type periodontal probe
to describe it as “a distinct type of scurvy” in and X-rays to assess the disease [11].
1 Periodontal Pathogenesis: Definitions and Historical Perspectives 3
From the mid-1920s to early 1950s, the under- inflammation occurs as short as 3 days follow-
standing of periodontal pathology shifted toward ing abstinence of oral hygiene, and an inflam-
the degeneration of the periodontium with oral matory exudate of the periodontal tissues
bacteria considered merely as secondary invaders (gingival crevicular fluid) accompanies the
of degenerated periodontium [12]. Hence, this clinical signs of inflammation. Within days of
period was dominated by histopathology obser- reinstating oral hygiene practices, the inflam-
vations, with the two most profound break- mation subsides, and gingival tissue health is
throughs being (a) the description of the epithelial restored. Since then, experimental gingivitis
attachment on the tooth as a “sealer tissue” to studies in humans have been the hallmark of
protect the underlying connective tissue and (b) clinical, microbiological, and histopathological
the definition of gingival pocket [13]. These find- investigations, more recently also in conjunc-
ings paved the way for modern periodontal tion to cutting-edge proteomic techniques [16].
pathogenesis research, with implications in rou- Although many believed that gingivitis was
tine clinical practice. harmful and would indifferently lead to the
From the mid-1940s onward, dental research- destruction of the periodontal tissues, today we
ers once again rediscovered bacteria in the etiol- know that not all gingivitis cases will pro-
ogy of periodontal disease, with the predominant gresses to periodontitis. Additionally, the clini-
notion of a nonspecific infection. This provided cal signs of gingivitis alone are not adequate to
the first seeds for the conception of the nonspe- identify the risk of transition to periodontitis
cific plaque hypothesis [14]. It was suggested [17, 18], and it is still debatable whether gingi-
that microorganisms within the mass of dental val inflammation in response to plaque accumu-
plaque, rather than the calculus, were responsi- lation is an interim stage between health and
ble for causing the disease. They were not periodontitis.
thought to be exogenous pathogens but rather
overgrown indigenous oral species. Because it
was unclear which organisms were pathogenic, 1.7 he Era of “Specific Plaque”
T
treatment approaches during that era were Hypothesis and Periodontal
directed at suppressing all of them, a concept Pathogenesis
largely applicable in today’s preventive
techniques. In the mid-1960s and 1970s, the next major
breakthrough came by attempts to demonstrate
microbial specificity of subgingival plaque at
1.6 he Era of “Nonspecific
T sites with “periodontosis” [19]. A number of pio-
Plaque” Hypothesis neers, including Max Listgarten [20, 21],
and Experimental Gingivitis Sigmund Socransky [22], and Jorgen Slots [23],
have identified and implicated specific microor-
In the era of “nonspecific plaque hypothesis,” ganisms as etiologic agents for periodontal dis-
several epidemiological studies showed a close ease. These findings led to the revision of older
relationship between poor oral hygiene and favorites, such as Capnocytophaga spp., and the
periodontal disease [14]. In the mid-1960s, the emergence of new species as more significant
landmark studies of Harald Löe convincingly contributors to the disease, such as Bacteroides
demonstrated that plaque accumulation directly gingivalis, Bacteroides forsythus, and
preceded gingivitis, in a volunteer human Actinobacillus actinomycetemcomitans (today
experimental model known as “experimental known as Porphyromonas gingivalis, Tannerella
gingivitis” [15]. According to this, gingival forsythia, and Aggregatibacter actinomycetem-
comitans, respectively). The specific plaque the beginning of the accumulation of micro-
hypothesis was further corroborated by studies bial plaque, as an acute exudative vasculitis in
on the quality and quantity of serum antibodies, the plexus of venules lateral to the junctional
showing, for instance, that patients with severe epithelium. This response coined the term
forms of periodontitis have high serum antibody “initial lesion” and includes features, such as
titers to selected species [24]. migration of PMNs via the junctional epithe-
The unravelling of the microbial constitu- lium into the gingival sulcus, co-exudation of
ents of supra- and subgingival plaque has natu- fluid from the sulcus, and loss of perivascular
rally led to the investigation of their role in the collagen. This subsequent stage is the “early
initiation and establishment of the disease. It lesion,” which develops within 4–10 days. It is
was now clear that microbial plaque initiated a characterized by a dense infiltrate of mainly T
series of as-yet-undefined events that led to the lymphocytes (T cells) and other mononuclear
destruction of the periodontium. The efforts to cells, as well as pathologic alteration of fibro-
understand the pathogenic mechanisms of peri- blasts. The early lesion is followed by the
odontitis were intensified in the 1970s, as “established lesion,” which develops within
favored by the boost of immunology and immu- 2–3 weeks. It is dominated by activated B cells
nopathology fields and the establishment of the (plasma cells) and accompanied by further
concept of “host response.” It was now becom- loss of the marginal gingival connective tissue
ing evident from several in vitro and in vivo matrix, but no bone loss is yet detectable. A
studies that bacteria or their products may number of PMNs continue to migrate through
affect the gingival epithelium, the underlying the junctional epithelium, and the gingival
connective tissue, as well as the enclosed pocket is gradually established. The estab-
immune cells. The instigated pathological alter- lished lesion, clinically manifesting as moder-
ations are reflected by physical breach of the ate to severe gingivitis, may remain stable for
epithelium, impairment of phagocytosis by years or even decades, or it may progress to a
polymorphonuclear neutrophils (PMNs), break- destructive “advanced lesion.” In the
down of the connective tissue extracellular “advanced lesion,” plasma cells continue to
matrix, activation of resident macrophages and predominate as the architecture of the gingival
osteoclasts, and destruction of the alveolar tissue disturbed, together with destruction of
bone [25, 26]. the alveolar bone and periodontal ligament. In
summary, the conversion from the established
to the advanced lesion is characterized by (a)
1.8 Periodontal Pathogenesis conversion of junctional (eventually ulcer-
Based on Description of ated) epithelium to pocket epithelium, (b) for-
Histopathological Features mation of denser inflammatory infiltrate
composed of plasma cells and macrophages,
In vivo experimental studies in dogs have (c) loss of collagen attachment to the root sur-
enabled the study of the conversion of estab- face, and (d) resorption of the alveolar bone
lished gingivitis to destructive periodontitis, [26]. The initial, early, and established lesions
leading to pioneering concepts in the patho- represent sequential stages in gingivitis,
genesis of periodontal disease, which hold up whereas the advanced lesion clinically mani-
to date [26]. These studies have shown that fests as periodontitis. These events are sum-
gingival tissues respond within 2–4 days from marized in Fig. 1.1.
Initital lesion Early lesion Established lesion Advanced lesion

(2 to 4 days) (2 to 3 days) (?)
(4 to 10 days)
Classic acute exudative Dense infiltrate of Predominance of plasma Plasma cells continue to
vasculitis with loss of lymphocytes and other cells but no bone loss yet. predominate. Loss of
perivascular collagen in mononuclear cells, may remain stable for alveolar bone and
gingival tissue (like acute fibroblast morphology years or decades, or may periodontal ligament
injury) alteration, initiation of become converted into an occures. Disruption of the
connective tissue loss advanced lesion gingival tissue architecture
Fig. 1.1 Histopathological stages of periodontal disease according to Page and Schroeder (1976)
1.9 Current Paradigm classic risk factors. Although the proposed model
of Periodontal Pathogenesis is reasonable, our understanding of how inflam-
matory host response is regulated is far from com-
According to the most well-established patho- plete. New discoveries are needed to augment the
genesis paradigm, periodontal disease is the information obtained from traditional indicators
result of a complex interplay between microbial and to better illuminate the disease mechanisms.
challenge, host response, and other modifying As a result, there is still an intense interest in
factors [26]. The microbial challenge triggers an applying emerging technologies to achieve a bet-
inflammatory response by the host, which is ter understanding of the disease processes.
meant to be protective in first place. Yet, if it
becomes excessive, it will lead to gingival con-
nective tissue and alveolar bone damage, culmi- 1.10 P
ending Clinical Questions
nating to periodontitis. Environmental/habitual in the Era Systems Biology
and genetic host factors (risk factors) render
some patients more susceptible than others to the Based on the progress in our understanding of
inflammatory response and consequently to the etiology and pathogenesis of the disease, several
disease. This may explain the observed large classification systems have been proposed and
variations in the destruction patterns and suscep- came to clinical use [27–34]. Yet, based on clini-
tibilities to the disease. However, many individu- cal parameters alone, we still cannot predict
als with disease have only one, or none, of the when a periodontal pocket is about to be formed,
whether it will progress over time, or whether 5. Mitsis FJ. Hippocrates in the golden age: his life, his
indeed it will resume clinically healthy levels work and his contributions to dentistry. J Am Coll
Dent. 1991;58(1):26–30.
once treated. Additionally, the current classifica- 6. Barasch A, Cunha-Cruz J, Curro F, DeRouen T,
tion system in practice refers to “different” forms Gilbert GH, Hujoel P, et al. Dental risk factors for
of periodontitis that, paradoxically, can have sim- osteonecrosis of the jaws: a CONDOR case-control
ilar clinical presentation. We therefore need more study. Clin Oral Investig. 2013;17(8):1839–45.
7. Loe H. Periodontal diseases: a brief historical per-
robust diagnostic and prognostic tools, which can spective. Periodontol. 1993;2:7–12.
only come with the incorporation of molecular 8. Baer PN, Iacono V. John Riggs said it first. Periodontal
parameters into the daily practice. It is very much Clin Investig. 1999;21(1):4.
anticipated that the enormous advances in 9. Ring ME, Miller WD. The pioneer who laid the foun-
dation for modern dental research. N Y State Dent J.
genomic and proteomic technologies will have an 2002;68(2):34–7.
impact in bridging these gaps between our scien- 10. Marsh PD. Are dental diseases examples of ecological
tific knowledge and clinical practice. catastrophes? Microbiology. 2003;149(Pt 2):279–94.
One of the most crucial open questions in peri- 11. Gold SI. Diagnostic techniques in periodontology: a
historical review. Periodontol. 1995;7:9–21.
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the conversion from gingivitis to periodontitis. A Periodontol. 1980;7(4):276–88.
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its nature, meaning that the disease does not cause ogy, deepening and elimination. Odontol Tidskr.
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Tidskr. 1965;73(4):458–63.
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immune regulation mechanisms exist, all leading Jonsson D, Barnes VM, et al. Label-free quantita-
to similar or comparable clinical manifestations tive proteomics reveals differentially regulated pro-
of periodontitis. Understanding the uniqueness teins in experimental gingivitis. J Proteome Res.
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and redundancies among these mechanisms via 17. Lang NP, Joss A, Orsanic T, Gusberti FA, Siegrist
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Diversity of Oral Biofilms in
Periodontal Health and Disease
2
Purnima S. Kumar
2.1 The Oral Real Estate ments such as the subgingival crevice, tongue,
buccal and alveolar mucosa, and tonsils. The sub-
Our knowledge of the human microbiome is gingival crevice provides 12 cm2 of surface area
changing on an almost daily basis. Although the for bacterial colonization [5], while the oral
number of bacteria that colonize the human mucosa hosts a real estate of more than 200 cm2
biome is still being debated [1], we now have evi- [6]. Together with the tooth surfaces, there is half
dence that areas in the human body that were nor- a square foot of space available for bacterial
mally considered sterile under conditions of colonization.
health, for example, the placenta, joint cavities,
and brain, do in fact contain bacteria [2, 3].
The oral cavity is one of the first portals of 2.2 Bacterial Diversity in Health
entry for bacteria into the human body; therefore,
at any given time, there are over two million Colonization determinants: Colonization of the
organisms in this environment, representing tooth surface is a complex interplay between both
nearly 700 distinct species [4]. Each individual inter-bacterial and host-bacterial interactions.
carries about 70–120 different species in differ- Inter-bacterial interactions promote colonization
ent oral niches. Several of these species are tran- by providing structural and metabolic support.
sient (allochthonous) members, while most are For example, Streptococcus, Actinomyces,
stable colonizers (autochthonous species). Within Haemophilus, Neisseria, and Veillonella are con-
the mouth, bacteria form organized, cooperating sidered pioneer organisms, because they adhere
communities linked through energy flow, nutri- to the acquired salivary pellicle on enamel by
tion, and metabolic networks. These communi-
specific and non-specific molecular interactions
ties are called biofilms, and the bacterial species between adhesins on the cell and receptors on the
and all their genes in a biofilm community consti- surface [7]. Streptococci contain antigen I/II
tute a microbiome. Biofilms can be found on abi- receptors for salivary agglutinin glycoprotein,
otic surfaces such as the tooth, dental implants, which allow them to bind to salivary pellicle,
and dental restorations, as well as biotic environ- dentin, and collagen as well as to Actinomyces
naeslundii [8]. Veillonella and Streptococcus,
two of the earliest and most abundant genera to
P.S. Kumar (*) colonize oral biofilms, share a nutritional syntro-
College of Dentistry, The Ohio State University,
Columbus, OH, USA phy, in that the Veillonella utilize the lactate that
e-mail: [email protected] is produced by the Streptococcus as a food source

DOI 10.1007/978-3-319-53737-5_2
10 P.S. Kumar
[9]. Also, Streptococcus sanguinis and S. oralis and surface proteins known as MSCRAMMs
exhibit synergy in degrading mucins [10], as do (microbial surface components recognizing
Streptococcus mitis, S. gordonii, Streptococcus adhesive matrix molecules) target fibronectin,
cristatus, and A. naeslundii [11], thereby allow- collagen, and host extracellular matrix (ECM)
ing efficient utilization of host glycopolysaccha- [14]. Gram-negative bacteria use pili, autotrans-
rides for nutrition. porters, and ECM-binding proteins for adhesion
The innate immune responses of the host also [15]. Coadhesion allows for co-localization of
play an important role in determining the bacteria metabolic and structural partners within the
that colonize each individual. The sulcular epi- plaque biofilm. Together, this phase of coaggre-
thelium provides physical, chemical, and immu- gation and coadhesion contributes to microbial
nological barriers against bacterial invasion and, succession within the biofilm [8].
in doing so, determines the community structure During the next phase, the biofilm matures
of the subgingival biofilm. Chemical mediators through development of an ECM. This inter-
of innate immunity include antimicrobial pep- bacterial matrix is highly variable; when gram-
tides (AMPs). Defensins (alpha and beta) are positive bacteria predominate, it is very fibrillar
probably the most well-studied AMPs. Early due to the presence of dextrans and levans. On the
colonizers have been shown to upregulate AMPs other hand, the gram-negative matrix is very regu-
but not cytokines and demonstrate a tolerance to lar and contains trilaminar vesicles, which are filled
these protein molecules [12]. In contrast to these with endotoxins and proteolytic enzymes and are
pioneer organisms, the “orange complex” probably involved in adherence. ECM not only
(described later in this chapter) intermediate col- reinforces the physical structure of the biofilm but
onizers induce high levels of both AMPs and also creates fluid and communication channels and
IL-8 and are highly susceptible to both peptide- chemo-osmotic and oxygen gradients. Together
mediated and phagocyte-mediated killing. The with the proximity of specific organisms, this struc-
immunological barriers in the sulcus are provided tural configuration triggers specific gene expres-
by neutrophils, T cells, dendritic cells, macro- sion patterns and inter- bacterial interactions.
phages, and mast cells, which reside in the sulcu- During this maturation phase, elaborate food chains
lar epithelium and underlying lamina propria. are established, and cell-to-cell communication,
Complement, which bridges innate and adaptive quorum sensing, and DNA transfer abilities (com-
immune responses, also plays a major role in petency) are enhanced. The final phase of biofilm
shaping this indigenous microbiome. development is when planktonic cells or coaggre-
Colonization sequence: Bacterial acquisition gates detach from the parent biofilm and metasta-
into the subgingival crevice occurs in a series of size to other sites.
well-orchestrated, hierarchical events, begin- Benefits of biofilm lifestyle: The biofilm life-
ning with conditioning of the tooth surface with style changes the phenotype of certain organisms
salivary proteins. Coaggregation and coadhe- and gene expression patterns. Thus, the func-
sion are two important mechanisms that play a tional potential of a biofilm is not merely the sum
role in this colonization. Coaggregation is of the potentials encoded in the species in a
defined as the attachment of genetically distinct planktonic state.
bacteria through specific molecular interactions.
Coadhesion is the attachment of planktonic cells • The biofilm provides a secure means of attach-
or coaggregates in suspension to already adher- ing to the tooth or mucosal surface and protec-
ent cells or onto a virgin surface. Weak, long tion from friction and shearing forces. This
range, van der Waals-type forces create revers- allows for colonization by species that lack
ible attachment between bacteria and the tooth attachment abilities as well as those that have
surface. These forces allow the development of fastidious growth requirements. For example,
adhesion-mediated attachment to occur. In the presence of Streptococcus cristatus (a
gram-positive bacteria, adhesin I/II, pilus [13], facultative aerobe) promotes colonization and
2 Diversity of Oral Biofilms in Periodontal Health and Disease 11
survival of Fusobacterium nucleatum, an obli- • Antibiotic resistance in dense biofilms. Biofilm

gate anaerobe, since S. cristatus uses up the bacteria are 1000–1500 times more resistant
oxygen in the environment. Thus, the biofilm than planktonic cells [21]. Several reasons
environment creates more “generalists” have been attributed to this incredible resis-
(organisms that require few host-associated tance. The primary reason is that antibiotics
benefits and so can occupy a wide range of that target DNA require actively dividing cells
habitats, e.g., Streptococcus, Veillonella, for efficacy; and biofilm existence drastically
Fusobacterium, Neisseria, and Prevotella) out reduces cell turnover rate [22]. These slow
of species that would normally behave as growers express non-specific defense mecha-
“specialist” (species that are confined to a sin- nisms and make more exo-polymers which
gle or narrow range of habitats). retard diffusion by size selection or by binding
• The biofilm provides a barrier against environ- to the charged antimicrobial agent (diffusion-
mental changes. The presence of a robust inter- reaction theory). This ion-exchange mecha-
cellular matrix creates a diffusion gradient, nism prevents highly charged molecules from
with minimal diffusion occurring in the interior reaching deeper zones. The biofilm is also rich
regions. This creates a buffered region within in extracellular enzymes (beta-lactamases,
the biofilm that is not influenced by pH changes formaldehyde dehydrogenase, formaldehyde
induced by food and lifestyle (brushing, mouth- lyase), which inactivate antibiotics [23].
wash, carbonated and sugar-rich diet, etc.). Importantly, the biofilm lifestyle changes the
This also is a mechanism for the antibiotic and bacterial phenotype, in that these bacteria
antimicrobial resistance that has been observed express different genes and may demonstrate
in biofilms. modification of drug targets. Also, there may
• Transfer of genetic material is crucial to main- be a subpopulation of “persister” organisms
taining genetic diversity. Apart from accep- within the biofilm, which are specialized survi-
tance of DNA (competence), genetic material vor cells that neither grow nor die in the pres-
can be transferred through conjugation, trans- ence of microbicidal agents [24].
formation, plasmid transfer, and transposon
transfer. Gene transfer has been demonstrated Benefits of hosting an indigenous microbi-
as a mechanism of antibiotic resistance among ome: Biofilm existence also benefits the human
commensal species like Streptococcus and host, since bacteria in a biofilm play an impor-
Neisseria [16]. Quorum sensing is another ben- tant role in preventing exogenous colonization.
efit conferred on a species through biofilm liv- This colonization resistance may occur by means
ing [17, 18]. Quorum sensing is regulation of of effective competition for nutrients and attach-
expression of specific genes through accumula- ment sites, the production of inhibitory factors,
tion of signaling compounds that mediate inter- and creation of unfavorable growth conditions
cellular communication and is typically by resident microflora [7]. Thus, by using very
mediated through competence-stimulating pep- specific mechanisms of aggregation and signal-
tide (CSP) in streptococci and autoinducers ing, as well as by partitioning the available
(AI) 1 and 2 in many other species [19, 20]. The resources within the biofilm, a health-compati-
LuxS gene encodes for AI, which is secreted by ble community can prevent colonization by
both gram-negative and gram-positive organ- pathogens. This phenomenon, called niche satu-
isms. AI-1 and AI-2 turn on in response to cell ration, has been observed in several biofilm sys-
density. Commensal bacteria produce and tems in the human body, e.g., the gastrointestinal
respond to low levels of AI-2, while pathogens tract, vagina, etc. Recent evidence indicates that
produce AI-2 in high levels. Thus, the levels of bacteria in dental plaque also play an important
AI-2 may encourage growth of beneficial spe- role in maintaining a healthy biofilm by prevent-
cies and may determine switch from commen- ing adhesion of pathogenic species [25]. There is
sal to pathogenic community. also evidence to indicate that periodontitis is
12 P.S. Kumar
associated with loss of beneficial bacteria, for tion and homeostasis to prevention of pathogen
example, species belonging to Veillonella and expansion. The microflora tends to remain stable
Streptococcus within the biofilm [26–28]. over time (“microbial homeostasis”), which
Evidence has shown some commensal oral results from a dynamic balance of microbial
bacteria have antagonistic activity against peri- interactions, including commensalism, symbio-
odontopathogens [29]. Specific examples of bac- sis, synergism, and antagonism. This stability
terial antagonism by means of producing develops a sense of “familiarity” in the host
metabolites in the oral cavity include hydrogen immune system and establishes a benchmark for
peroxide production by streptococcal species to “normalcy.” The immune system recognizes
inhibit growth of periodontopathogens [30] and deviations in community membership (loss or
lactic acid production to prevent Pseudomonas gain of species) or community structure (change
aeruginosa incorporation into the biofilm [31]. in relative abundances of species) and responds
Evidence has shown streptococci exhibit antago- to it with an upregulation of immune-
nistic properties toward certain staphylococci in inflammatory responses, in an effort to neutralize
the oral cavity as well [32]. Some indigenous these deviations from the norm (Fig. 2.1).
microbiota take colonization resistance a step Bacterial diversity in health: In examining
farther by producing specific antibiotics, such as bacterial diversity in health, it is important to
bacteriocin production in strains of Streptococcus remember that the state of health persists through
salivarius, which act on specific pathogens to three dentitions states: primary, mixed, and per-
prevent their colonization of the community [33]. manent. Relatively few studies have examined the
Hillman and Socransky also demonstrated that alteration of microbial profile in a changing denti-
plaque from periodontally healthy individuals tion through primary, mixed, and permanent
was capable of inhibiting growth of certain peri- stages of dentition. The studies, methodologies,
odontal pathogens [30]. It is also being recog- and findings are summarized in Table 2.1. The
nized that these bacteria provide immense health subgingival plaques of children aged 4–5 years
benefits to the host, ranging from immune educa- with mixed dentition tend to have a multiform
Fig. 2.1 Circular
maximum likelihood
phylogenetic tree at
level of genus. The inner
band shows genera
colored by phylum or
class, the next band
shows significant mean
differences between
healthy controls and
deep pockets (colored
green for genera higher
in healthy pockets and
red for genera higher in
disease pockets), and the
outer band shows overall
relative abundance.
Figure is published in
Griffen et al. ISME J
2012; 6(6):1176–85.
doi: 10.1038/
ismej.2011.191
Table 2.1 Evolving concepts in the bacterial etiology of periodontal diseases

Premise Limitations
Based on Koch discovering a single organism as the Organisms seen in both health and disease
etiological factor of tuberculosis
Concurrent with advances in microscopy Older individuals had more extensive and severe
periodontitis
Fusiforms, spirochaetes, streptococci, amoeba seen in No single organism could be identified (poor resolution
mouth of identification methods)
All bacteria have the potential to cause disease Plaque levels did not correlate with extent and severity of
attachment loss in chronic periodontitis
Disease occurs when the host cannot neutralize bacterial Minimal plaque formation and severe attachment loss
products or toxins characteristically seen in aggressive (juvenile)
periodontitis
Successful prevention and treatment dependent on Plaque control did not prevent continued attachment loss
wholesale plaque removal in recurrent and refractory periodontitis
Microscopic and culture-based studies demonstrating No single organism or consortia could be associated with
that microbial profiles of heathy subjects different from disease initiation or progression
subjects with disease
Specific bacteria associated with disease progression and Several putative periodontal pathogens (e.g., P. gingivalis,
recurrence T. forsythia) identified in healthy sites and individuals
A. actinomycetemcomitans recognized as the principal Subjects with localized aggressive periodontitis did not
pathogen in localized aggressive periodontitis always demonstrate A. actinomycetemcomitans
Local factors determine the composition of the Periodontitis is a multifactorial disease, and the effects of
microbiome environmental shift cannot be established as a single
factor in altering microbial ecology
Change in environment plays a critical role in altering Unlike dental caries, the effect of environmental changes
ratio of beneficial bacteria and pathogens on a single species is not easy to establish
Specific bacteria modulate the host response to improve Keystone species are not definitively identified
their survival
These bacteria, known as keystone species, promote Emerging evidence indicates that disease sites are
growth of other species (accessory pathogens) functionally similar and different species contribute to
similar functions
Keystone species do not have to be in large numbers to Functional genes, rather than species, may be hallmarks
effect their changes of disease and health
gram-negative, anaerobic bacterial composition, host-compatible organisms [38]. These findings

some of which are suspected periodontal patho- are in agreement with the results of other culture
gens [34, 35]. It has been reported from culture studies that examine the prevalence of subgingi-
studies that Gemella morbillorum and val species in sites of gingival health and other
Peptostreptococcus magnus are statistically sig- clinical conditions. Ximenez-Fyvie et al. reported
nificantly more frequently detected in incisors, a significantly larger proportion of Actinomyces
while P. micros, S. intermedius, B. forsythus, species occurred in subjects that were periodon-
Fusobacterium nucleatum, Prevotella loescheii, tally healthy [39]. Molecular studies using
P. melaninogenica, and Selenomonas sputigena culture-independent approaches (e.g., 16S rRNA
are more frequently detected in molars. amplification, FISH) have demonstrated consid-
Periodontal pathogens can be detected in children erable diversity of subgingival microflora in
under 3 years old [36] as well as in young, peri- health [21]. Veillonella sp. oral clone X042, a
odontitis-free children at a carrier state with no gram-negative bacterium, was found to be the
signs of destructive periodontitis [37]. Several most common bacteria detected in a study using
investigators have found, using culturing and 16S cloning and sequencing in a study of adults
DNA-DNA checkerboard, that Actinomyces, who were periodontally healthy [27]. In another
streptococci, and Veillonella may be indigenous, study, Kumar et al. [26] found higher proportions
14 P.S. Kumar
of Streptococcus, Abiotrophia, Gemella, and Royal Society of London. However, for several
Veillonella in plaque of periodontally healthy centuries plaque was thought of as an amorphous
adults. Aas et al. [4] reported that in periodontally collection of bacteria. In 1882, Koch published a
healthy adults (age range 23–55), the most com- treatise describing a specific bacterium as the
mon bacteria found by 16sRNA gene and PCR etiological agent of tuberculosis along with crite-
detection were Gemella, Granulicatella, ria for establishing an organism as the etiological
Streptococcus, and Veillonella in a variety of sites agent of a disease [42]. This began the search for
sampled, including the tongue, buccal fold, hard species involved in the etiology of several other
and soft palate, labial gingiva and tonsils of soft communicable diseases. The time period from
tissue surfaces, and supra- and subgingival plaque. 1880 to 1930 became known as the golden age of
Ledder et al. [40] reported incidence rates of 52 microbiology. During this period, many microbial
bacteria in healthy and diseased adults (mean age pathogens were linked to specific infections in
40.1 years, range 20–55 years) using multiplex the body. This led oral health researchers to seek
PCR analysis. Bacteria that were found with the out specific pathogens for the etiologic agent of
highest incidence were Pseudomonas sp. (56%), dental caries and periodontal disease, and the age
Streptococcus mitis (50%), Streptococcus sangui- of the specific plaque hypothesis was born.
nis (44%), and Neisseria sp. (44%). Additionally, Several possible agents were isolated; however,
four bacteria were associated exclusively in no specific bacterium could be identified as the
healthy patients at an incidence rate of 17% and causative agent. Further, no organism could be
were non-detectable in diseased subjects: isolated only from diseased individuals; on the
Staphylococcus sp. (AB167056), Sphingobium contrary, organisms found in disease were also
yanoikuyae (AJ627009), Corynebacterium found in health.
matruchotii (X82065), and Streptococcus mutans Another important evidence came from epide-
(AE014854). The association of C. matruchotii miological data indicating that the prevalence of
with healthy gingiva has been previously reported periodontal disease was higher in older individu-
[41]. Ledder et al. was unable to correlate biodi- als [43]. While it was later realized that this was
versity with either health or disease, and no sig- because of the cumulative destruction from the
nificant differences were found between the disease, at the time it suggested that the presence
groups (p > 0.05). All of these findings support of plaque, and not necessarily specific organisms,
the hypothesis that bacteria play an important role resulted in disease. As a result, the 1930s brought
in immune education; the host immune system a new view—the non-specific plaque hypothesis
recognizes a biofilm composed of a specific ratio [44]. This new hypothesis evolved around the
of organisms as “friend.” Periodontal health is the idea that all plaque was considered pathogenic
result of this immune tolerance. When the propor- and diseases associated with plaque arose from
tions of organisms within a community change, “elaboration of noxious products by the entire
this balance is upset and leads to an immuno- plaque flora” [44]. Large amounts of plaque
inflammatory response, which results in disease. would lead to production of toxins that could
overwhelm the host defenses and result in dis-
ease. This hypothesis led to wholesale removal of
2.3 acterial Diversity in
B plaque by surgical or nonsurgical methods and
Periodontal Disease: rigorous homecare as therapeutic options for
Historical Perspective periodontal disease.
The 1960s brought another change with many
The presence of bacteria in dental plaque has advances in bacterial detection and characteriza-
been known since 1683, when Antonie van tion. An important discovery was that mutans
Leeuwenhoek first described his observations of streptococci (S. mutans and S. sobrinus) were the
“animalcules” in dental plaque in letters to the primary pathogens in the etiology of dental caries
[45]. Evidence also suggested that subgingival system [48, 49]. These species do not have to be
plaque behaved differently when certain species in high abundances to effect this change. This
were present [46]. Also, longitudinal studies modulation not only allows these organisms to
revealed that many individuals with significant manipulate resources to survive in the environ-
amounts of plaque accumulation never developed ment, it also encourages the growth of other
destructive disease [47]. Further, within any indi- selected members of the indigenous microbiome,
vidual, the disease was seen to be site specific, which now act as accessory pathogens. This
that is, only certain teeth demonstrated large polymicrobial synergy that is orchestrated by
amounts of destruction and these sites were seen species such as P. gingivalis leads to the develop-
in close juxtaposition to normal sites. The devel- ment of a dysbiotic microbiome, which results in
opment of better techniques for microbial charac- disease.
terization demonstrated significant differences in The identification of specific causative spe-
the microbial profiles of periodontal health and cies, or periodontopathogens, has been hampered
disease. Thus, the specific plaque hypothesis was by some of the unique features of periodontal dis-
again adopted, as it was believed specific eases. The foremost of these is that disease occurs
microbes were responsible for disease and once in a site already colonized by an indigenous
identified would allow for targeted treatment of microbial community. Thus, it is difficult to dif-
the disease. This concept gained acceptance ferentiate between species that cause the disease
when Aggregatibacter (formerly Actinobacillus) and species that are present as a consequence of
actinomycetemcomitans was recognized as the the disease. For example, it has been shown that
predominant pathogen in localized aggressive Capnocytophaga spp. are seen in high levels
periodontitis. prior to the onset of gingivitis, while Prevotella
Investigations on the influence of the oral spp. are detected in areas with established gingi-
environment on the structure and composition of vitis. Thus, Capnocytophaga is more likely an
microbial communities led to the development of etiological agent, and Prevotella species are pres-
the ecological plaque hypothesis [7]. This ent as a consequence of the disease process [50].
hypothesis suggests that the resident microflora Colonization by exogenous pathogens is thought
undergoes a transformation from a commensal to to contribute to the episodic nature of disease
a pathogenic population due to environmental progression [51], i.e., the fact that not all sites
perturbations, for example, pH, oxygen tension, with baseline attachment loss demonstrate the
flow of gingival crevicular fluid, and presence of same rate of disease progression or disease activ-
blood and blood products. While the ecological ity at the same time points.
hypothesis is similar to the specific plaque
hypothesis in recognizing the varying pathogenic
potentials of individual bacterial species, it main- 2.4 Specific Microorganisms
tains that perturbation in the microbial homeosta- Associated with Periodontal
sis is the result of environmental shifts. Thus, Health and Disease
treatment of disease does not only require target-
ing specific species, it is also important to alter Different periodontal diseases have fairly unique
the environment from one that promotes patho- profiles of associated bacteria. This, along with
gen enrichment to one that is compatible with the fact that disease occurs in sporadic bursts in
commensal growth. the mouth, strengthens the evidence for the role
A more recent line of thinking has resulted in of specific microorganisms in disease causation
the polymicrobial synergy and microbial dysbio- and progression.
sis theory [48]. According to this, certain species Periodontal health: Bacteria that are associ-
(called keystone pathogens) modulate the host ated with periodontal health include primary or
immune response and, therefore, the local eco- early colonizers such as Streptococcus sanguinis,
16 P.S. Kumar
Streptococcus mitis, Gemella spp., Atopobium spp., Gingivitis: Gram-positive species, for example,
Fusobacterium nucleatum, and Capnocytophaga Streptococcus spp., Actinomyces viscosus, and
spp. [41, 52, 53]. Species belonging to the genera Parvimonas micra (formerly Peptostreptococcus
Veillonella, Streptococcus, and Capnocytophaga micros), as well as gram-negative species such as
are thought to be beneficial to the host [53]. Campylobacter gracilis, F. nucleatum, Prevotella
Molecular analysis has shown the presence of cer- intermedia, and Veillonella, have been associated
tain uncultivated species such as Bacteroides oral with gingivitis [55–57]. Pregnancy-associated gin-
clone BU063 strongly associated with periodontal givitis, however, has a microflora predominated by
health [54] (Fig. 2.2). P. intermedia [58].
Fig. 2.2 Graph of core genes grouped into higher order shown here belonged to the core microbiome (80% or
functions. Circles are sized by relative abundances of more of healthy individuals). The green bars represent the
genes contributing to each function (a). Core metabolic relative abundances of the species in all samples (c).
pathways in the health-associated microbiome. The lines Figure is published in Dabdoub et al. Sci Rep 2016; 6:
are sized by log fold abundances (b). A selected group of 38993. doi: 10.1038/srep38993
species that contributed to these functions. The species
Chronic periodontitis: The bacterial progressing periodontitis. Nevertheless, available

file of chronic periodontitis has been explored evidence suggests that the bacterial profile of
in cross-sectional and longitudinal studies. The generalized aggressive periodontitis is not sig-
effect of various treatment methods on chang- nificantly different from that of chronic peri-
ing the microbial ecology has also been investi- odontitis [70–72].
gated. P. gingivalis, T. forsythia, P. intermedia, Necrotizing ulcerative gingivitis: The bacte-
Campylobacter rectus, Eikenella corrodens, F. rial flora of necrotizing ulcerative gingivitis has
nucleatum, A. actinomycetemcomitans, P. micros, been demonstrated to be composed, for the
and Treponema spp. have been most commonly most part, of fusobacteria and spirochetes.
found. P. gingivalis, T. forsythia, P. intermedia, Recent studies have isolated previously unsus-
C. rectus, and F. nucleatum are found in higher pected spirochetes, e.g., Treponema putidum, a
levels in sites with active disease or with pro- proteolytic treponeme, from lesions of necro-
gressing disease [59–62]. Clinical resolution of tizing ulcerative gingivitis [73]. Other bacteria
disease is also associated with a decrease in the reported in these lesions include Rothia dento-
levels of these species. More recent molecular cariosa, Treponema spp., Achromobacter spp.,
approaches have found uncultivated bacterial spe- Propionibacterium acnes, Capnocytophaga
cies, such as Desulfobulbus sp. oral clone R004, spp., and P. intermedia [52].
Deferribacteres sp. oral clones BH017 and D084, Periodontal abscess: A periodontal abscess is
and Bacteroides sp. oral clone AU126, to be sig- a localized purulent infection within the tissues
nificantly associated with periodontitis [41, 63]. adjacent to the periodontal pocket. F. nucleatum,
Localized aggressive periodontitis (LAP): P. intermedia, P. micra, T. forsythia, C. rectus,
Studies have implicated A. actinomycetemcomi- and P. gingivalis have been recovered from these
tans as an important organism in the etiology of lesions [74, 75].
LAP [64–66]. This species has been found as
the predominant cultivable species in as many
as 90% of sites with LAP. However, it should be Conclusion
noted that not all studies support the association Bacteria are acquired in subgingival bio-
of A. actinomycetemcomitans in aggressive films in a sequential manner to form orga-
periodontitis. This organism is not always found nized, cooperating communities called
in disease sites; further, it has been found in biofilms. These biofilms are composed
healthy children, suggesting that it is a member largely of organisms with low inflamma-
of the healthy microbial flora [67]. Other spe- tory potential, and they play an important
cies such as P. gingivalis, E. corrodens, and C. role in preventing expansion of inflammo-
rectus have also been found in high levels in philic organisms (or pathogens). They also
certain cases of LAP [68]. Viruses such as educate the immune system to recognize
Epstein-Barr virus (EBV-1) and human cyto- “friend and foe.” This immune tolerance is
megalovirus (HCMV) have also been associated the basis for health and is dependent on
with this disease [69]. continuous crosstalk between a stable
Generalized aggressive periodontitis (GAP): microbiome and the host immune system.
The microbial etiology of generalized aggressive Changes in the local environment (short
periodontitis is not as well defined as other forms term, such as food intake, or long term,
of periodontal diseases due to multiple transfor- such as smoking) can alter these ecosys-
mations in disease nomenclature. The disease tems, resulting in dysbiosis, which forms
now encompasses entities such has periodonto- the etiologic basis for all oral diseases.
sis, prepubertal periodontitis, and rapidly pro-
18 P.S. Kumar
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Subgingival Biofilms as Etiological
Factors of Periodontal Disease
3
Thomas Thurnheer, Kai Bao,
and Georgios N. Belibasakis
3.1 efinition of Subgingival

D signals, or protection from harmful conditions
Plaque as a Biofilm [6–8]. Living in a biofilm represents a universal
survival strategy of microorganisms on our
A biofilm is a structured community of microbial planet. It allows microorganisms to colonize new
cells embedded in a self-produced (hydrated) ecological niches and survive in hostile environ-
matrix extracellular polymeric substance (EPS) ments thereby adopting biofilm structure in
and adherent to an inert or living surface, as response to environmental conditions [9, 10].
defined by Costerton [1] and modified in 2012 by The dense and perplexed structure of a biofilm
IUPAC [2]. Microbial cells growing in a biofilm not only hampers diffusion of molecules, but it
differ physiologically from planktonic cells of also forms a barrier against the host’s defense
the same organism, which are swimming or float- mechanisms such as antibodies, lysozyme, or
ing single cells in a liquid medium. Although the against other antimicrobial agents.
fact that microorganisms are able to grow The formation of a biofilm takes place in five
attached to solid surfaces was reported already in sequential stages, namely, initial attachment, irre-
1936 by Zobell [3], it took more than 40 years versible attachment, maturation I, maturation II,
until it was recognized that in nature most bacte- and dispersion [11, 12]. During initial attach-
ria grow in biofilms attached to a surface rather ment, free-floating microorganisms attach to a
than growing planktonically [1, 4]. A cell switch- surface. While still not fully understood, it is
ing to the biofilm mode of growth undergoes a thought that the first colonists of a biofilm adhere
phenotypic shift in behavior with many genes to the surface initially through weak, reversible
being differentially regulated [5]. Biofilms may adhesion via van der Waals forces and hydropho-
be formed in response to factors such as recogni- bic effects [13]. If the colonists are not immedi-
tion of attachment sites on a surface, nutritional ately separated from the surface, they can anchor
themselves more permanently using cell adhe-
T. Thurnheer (*) • K. Bao sion structures such as pili (irreversible attach-
Division of Oral Microbiology and Immunology, ment). Some species are not able to attach to a
Clinic of Preventive Dentistry, Periodontology and surface on their own but are instead able to anchor
Cariology, Center of Dental Medicine, themselves to the matrix or directly to earlier
University of Zürich, Zürich, Switzerland
e-mail: [email protected] colonists. It is during this colonization (matura-
tion I) that the cells are able to communicate via
G.N. Belibasakis
Department of Dental Medicine, Karolinska Institute, quorum sensing (QS) using small diffusible
Stockholm, Sweden signal molecules [14–16]. Once colonization has

DOI 10.1007/978-3-319-53737-5_3
22 T. Thurnheer et al.
begun, the biofilm grows through a combination ronmental conditions actually define which of the
of cell division and recruitment (maturation II). microorganisms are best fit to grow as a biofilm,
Extracellular polymeric matrices consisting within a given site. Throughout the mass of the
mainly of polysaccharides, proteins, nucleic biofilm, “microgradients” for physicochemical
acids, and lipids typically enclose bacterial bio- parameters are established, including tempera-
films [17]. The EPS matrix is an important key to ture, redox potential, oxygen partial pressure,
the evolutionary success of biofilms. One reason pH, and diffusion of nutrients. Hence, the bacte-
is that it traps extracellular enzymes and keeps ria most well adapted to these conditions are
them in close proximity to the cells. Thus, the eventually going to survive and grow.
matrix represents an external digestion system The “ecological plaque hypothesis” summa-
and allows for stable synergistic microconsortia rizes the current concept on the relationship
of different species [17]. The final stage of bio- between oral bacteria, or biofilms, and the devel-
film formation is known as dispersion and is the opment of common oral infectious diseases, such
stage in which the biofilm is established and may as dental caries and periodontal disease [22].
only change in shape and size. Dispersal of cells This hypothesis proposes that under normal con-
from the biofilm colony is an essential stage of ditions, the oral bacteria and the host tissues are
the biofilm life cycle. Dispersal enables biofilms in a dynamic health-compatible balance. The
to spread and colonize new surfaces [18]. microenvironment may undergo local changes,
Enzymes that degrade the extracellular matrix, causing a breakage in this homeostatic balance
such as dispersin B and deoxyribonuclease, may and subsequently shifts in the biofilm’s microbial
play a role in biofilm dispersal [19, 20]. Biofilm composition. Under the newly established condi-
matrix-degrading enzymes may therefore be use- tions, quiescent opportunistic pathogens can now
ful as anti-biofilm agents [21]. Biofilms are become prolific and more virulent, leading to oral
formed on various surfaces, e.g., on boat hulls, disease. Microbes typically associated with the
water pipelines, artificial heart valves, or on teeth, disease may still be present at a healthy site,
the latter commonly known as dental plaque. albeit at too low numbers and proportions to be
deleterious.
3.2 Oral Biofilms:

Microenvironment and 3.2.1 S
ubgingival Biofilms and
Etiological Role in Oral Their Association with
Disease Periodontitis
Biofilms on the tooth surface can grow under The dental plaque that grows on the tooth surface
both aerobic and anaerobic conditions, depend- in the abstinence of oral hygiene is indeed a poly-
ing on the concentration and partial pressure of microbial oral biofilm, comprised of hundreds of
the available oxygen. Hence, both aerobic and different microbial species. A biofilm growing on
anaerobic microorganisms can be encountered in the surface of the dental enamel and above the
these biofilms. The rich diversity of the oral free gingival margin is termed supragingival,
microbiota allows for ample combinations of dif- whereas the one that grows on the dental cemen-
ferent microorganisms to be present within a bio- tum surface underneath this margin is termed
film and define its composition. This microbial subgingival. The development of periodontitis,
affluence can result in very complex microbial which is the primary cause for tooth loss in
combinations that vary between individuals or adults, is associated with the formation of sub-
even between sites of the same individual. gingival biofilms within the periodontal pocket, a
However, this should not be perceived as a ran- clinical sign of the progressing disease. The
dom colonization event, as the local microenvi- mechanisms of pathogenesis of periodontitis are
3 Subgingival Biofilms as Etiological Factors of Periodontal Disease 23
centered around the inflammation caused to the motile bacterial types. However, the gradual con-
periodontal tissues by subgingival biofilms. version to gingivitis and subsequently to peri-
Before describing in more detail the composition odontitis is marked by an increase in fusiforms,
of subgingival biofilms, it should be noted that long filaments, and coiled and motile bacterial
species characterized as “common periodontal types (such as spirochetes).
pathogens” have been isolated and detected more The three red complex species according to
frequently and in higher numbers in periodontal the classification by Socransky (Porphyromonas
disease than health [23]. gingivalis, Tannerella forsythia, Treponema den-
ticola) [27, 30] are the most extensively studied
and discussed putative periodontal pathogens to
3.2.2 C
omposition and Structure of date (Fig. 3.1). All three are highly proteolytic
Subgingival Biofilms: General anaerobes and highly associated statistically with
Concepts deeper periodontal pockets and higher prevalence
in periodontal disease. Additional bacterial spe-
The first microorganisms that colonize the oral cav- cies present in subgingival biofilms and closely
ity are called pioneer species. The predominant pio- associated with periodontal disease are Prevotella
neer organisms in the mouth are streptococci, in intermedia, Eikenella corrodens, Campylobacter
particular, S. salivarius, S. mitis, and S. oralis [24]. A rectus, Fusobacterium nucleatum, and
key element for initial colonization and subsequent Aggregatibacter actinomycetemcomitans [27].
biofilm formation is communication among micro- Otherwise, the presence of Streptococcus sangui-
organisms [25]. By and by the environment changes nis, Streptococcus mitis, and various Actinomyces
due to metabolic activity of the pioneer organisms spp. in the biofilms is associated with periodontal
affecting, e.g., pH, redox potential, or nutrient sup- health rather than disease [31–33]. Recent studies
ply, enabling colonization of other microorganisms. using cutting-edge molecular detection method-
This succession eventually leads to a stable situation ologies are pointing to specific “microbial signa-
with increased species diversity [26]. tures” in subgingival biofilms that can distinguish
The microenvironment of the periodontal between periodontal health and disease [34].
pocket is an anaerobic one, with high protein These will be elaborated in more detail in the fol-
amounts, due to the constant presence of the lowing section of this chapter.
inflammatory exudate of the tissue in periodontal The microbial vitality varies throughout the
disease (gingival crevicular fluid). Therefore, it is biofilm as confocal laser scanning microscopy
rational that the types of bacteria favored to colo- (CLSM) analyses of live/dead stained biofilms
nize, grow, and form subgingival biofilms in this prove, with the most viable bacteria present in
milieu are anaerobic and proteolytic. It is now the central part of biofilms [35]. Regarding indi-
well established that the switch from periodontal vidual bacterial localization within subgingival
health to periodontal disease is associated with biofilms, in situ studies demonstrated that puta-
the conversion of a Gram-positive aerobic and tive periodontal pathogens are located at the
nonmotile microbes to Gram-negative, anaero- outer extremity of the biofilms, in close proxim-
bic, and motile ones [23, 27]. The changes in the ity to the gingival tissue and cells of the immune
composition of biofilms during the conversion system (e.g., neutrophils). Regarding the struc-
from health to disease have also been described ture of the biofilms, lactobacilli are centrally
according to the shapes of the observed bacteria located within the bacterial aggregates, and
(i.e., morphotypes), using dark field microscopy streptococci together with Candida albicans
techniques [28, 29]. These have documented that yeast form corncob structures within the bio-
biofilms sampled at periodontally healthy sites films. Moreover, putative periodontal pathogens
are primarily colonized by cocci and a few rods, may already colonize mature biofilms and form
but almost no fusiform, coiled (i.e., screwlike), or microcolonies therein [36].
Fig. 3.1 Scanning electron microscope images of the forsythia, formerly Bacteroides forsythus, belonging also
three members of the “red complex” bacteria, to the phylum Bacteroidetes, is a Gram-negative, obli-
Porphyromonas gingivalis (a), from the phylum gately anaerobic, nonmotile fusiform rod; (c) Treponema
Bacteroidetes, is a nonmotile, asaccharolytic, Gram- denticola, a member of the phylum Spirochaetes, is a
negative, obligately anaerobic rod, forming black- Gram-negative, obligate anaerobic, motile, spiral-shaped,
pigmented colonies on blood agar plates; (b) Tannerella and highly proteolytic spirochete bacterium
3.2.3 Subgingival Biofilm RNA-based phylogenetic methods, which origi-

Composition as Revealed by nated from the 1980s [38], such as the “checker-
Metagenomics board” DNA-DNA hybridization [39], polymerase
chain reaction (PCR) [40], and microarray chip
Our established knowledge on the microbial [41]. These ever-evolving molecular methods are
composition of the subgingival biofilms and the rapidly increasing our understanding of the bio-
association of certain bacteria with periodontitis film composition. Recently, researchers started
comes mostly from studies using conventional to shear entire DNA samples by the “shotgun”
bacterial cultures. However, most microorgan- approach, followed by the “next-generation
isms of the oral cavity cannot be cultured in the sequencing” (NGS) technologies [42]. This “shot-
laboratory [37]. Therefore, culture-independent gun” approach could clearly distinguish closely
detection methods applied in oral samples allow related subgingival species using complete ref-
for the discovery even of species that we are not erence genomes provided from National Center
yet able to cultivate, which are crucial in under- for Biotechnology Information (NCBI), Human
standing the composition of oral biofilms. Microbiome Project (HMP), or Human Oral
Many of these commonly used molecular Microbiome Database (HOMD). Using combined
methods were advent of different 16S ribosomal “shotgun” and 16S rRNA sequencing for analyz-
ing subgingival biofilms, one study predicted peri- than 774 genera identified in dental biofilms, Tsai
odontal disease cases with an accuracy of 81.1% et al. distinguished eight and six of those to be
and revealed the potential of different microbial significantly enriched in healthy individuals and
taxa for monitoring disease progression [43]. severe chronic periodontitis patients, respectively
However, all these techniques are not without limi- [52]. Ge and his colleagues also found that bacte-
tations. DNA-DNA checkerboard hybridization or rial abundances were significantly altered
microarray chips normally can only provide ana- between shallow and deep sites of periodontal
lytical input for a limited set of species, methods patients [53]. Species diversities between healthy
requiring PCR amplification can be biased due to and periodontal individual were also reported in
different chemistry settings [44], and NGS-based other different studies [54]. Porphyromonas,
technologies highly rely on the available databases Tannerella, Treponema, and Filifactor numbers
and computational abilities. were found to decrease after treated by antimi-
We now know that the microbiota constitut- crobials and root planning [55]. However, another
ing the subgingival biofilm is far more complex metagenomic study showed that antibiotics have
than initially thought based only on bacterial great influence on the subgingival biofilm com-
culture-dependent methods. A recent study has position 3 months after therapy, but these changes
estimated that as many as 19,000 different bac- do not remain effective as long as 6 months [56].
terial taxa can be identified in the human oral Interrelationships between periodontal infections
cavity [45], a number which is far higher than with other diseases or conditions were also
previously reported results (around 700 species) revealed by metagenomics. A recent study has
using culture- based or cloning methods [46]. showed higher level of Fusobacteriaceae and
Contemporary research in periodontal microbiol- lower level of Prevotellaceae in healthy elder
ogy is now stepping into the fast-evolving field individuals, compared to matched individuals
of metagenomics. By definition, metagenomics is with dementia [57]. Further studies have reported
the study of genetic material recovered directly that both smoking and pregnancy are also affect-
from environmental samples using large-data ing the overall microbial compositions of subgin-
analysis methods. Complex subgingival biofilms gival biofilms [58–60].
obtained from periodontal pockets may well be In summary, metagenomics and the associated
considered as such environmental samples. technologies for microbial detection have proved
Using metagenomics for analyzing subgin- to be a powerful tool for the understanding of the
gival biofilm samples, researchers were able composition of subgingival biofilms, providing
to identify less-known uncultivable microbes higher accuracy and efficiency compared to pre-
associated with periodontal disease, such as vious approaches.
Peptostreptococcus stomatis, Filifactor alo-
cis, Desulfobulbus, Dialister, Megasphaera,
Synergistetes, Deferribacteres, and TM7 [38, 3.3 In Vitro Modeling
39, 47–49]. Furthermore, a systematic review of Subgingival Biofilms
of the literature highlighted at least 17 species
or groups of microbes, not previously consid- Artificial subgingival biofilms can be generated
ered to be associated with periodontal disease in the microbiology laboratory in order to study
[44]. In recent years, the contribution of phages their behavior in vitro and deduce conclusions for
in the formation of subgingival biofilm was also their behavior in vivo. That can be clinically very
brought into light with the help of metagenomic useful, for instance, in understanding the role of
technologies [42, 50, 51]. the different biofilm bacteria in the pathogenesis
Distribution of subgingival species present in of the disease but also for testing the antimicro-
different conditions or niches is another major bial efficiency of different agents, prior to being
advance brought in by metagenomics. From more applied for patient treatment. According to our
a b
Fig. 3.2 Confocal laser scanning microscopy images nucleatum in particular appears red in (a) and (b), whereas
(CLSM) of a fluorescence in situ hybridization (FISH)- streptococci appear blue in (a) and Porphyromonas gingi-
stained supragingival (a) and subgingival (b) biofilm, valis appears blue in (b). The biofilm base in the cross
respectively. Bacteria appear green and Fusobacterium section is directed toward the top view. Scale bars: 20 μm
earlier studies [61], there are several require- subgingival model [66–71]. The ten bacteria used
ments that should be met by a good model sys- in that model were selected according to pub-
tem. Most importantly, results generated by the lished observations concerning biofilm formation
model system should be reproducible. Due to the and periodontal disease. An aim was to incorpo-
enormous microbial complexity of periodontal rate the main disease-associated, “red complex”
disease, there are several obstacles to overcome species [27]. To facilitate their incorporation,
in order to successfully build a model system. other species were selected with the goal to pro-
The overwhelming amount of different species as vide a suitable matrix in terms of attachment
well as the diversity of their appearance is a limit- receptors [32] and redox potential, while further
ing factor that restricts the prospect of matching nutritional conditions still remained to be opti-
the in vivo situation accurately with a model mized. In Fig. 3.2, CLSM images of the suprag-
system. ingival and the subgingival model are shown. The
The Zürich biofilm model has been estab- model system proved to produce stable and
lished more than a decade ago and was designed reproducible biofilms, which in proximity to cul-
as a fully defined, in vitro batch model system tured human epithelial cells induced cellular
first as supragingival model consisting of six oral apoptosis [66], and a number of histopathological
microorganism characteristics for supragingival [70, 72] and protein changes known to be associ-
plaque [62–65], which later was extended to a ated with periodontal diseases [73].
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Bacterial Virulence Factors that
Contribute to Periodontal
4
Pathogenesis
Anders Johansson and Gunnar Dahlén
4.1 Overview P. gingivalis is frequently detected in periodontal

pockets of individuals with the chronic forms of
In this chapter, the role of different microbial viru- the disease (Fig. 4.2). However, the role of these
lence factors in relation to the pathogenesis of two bacteria in periodontal breakdown is still not
periodontal diseases is addressed. These factors entirely clear.
are molecules produced by pathogens and contrib-
ute to their pathogenicity by promoting coloniza-
tion and affecting host response. The importance 4.2 athogenesis of Periodontal
P
of different virulence factors in the life of the oral Disease
biofilm and the interplay with the host’s response
is exemplified here by two of the major, and Periodontitis is a microbe-induced inflammatory
most well studied, periodontal pathogens, disease that affects the tooth-supporting tissues,
Porphyromonas gingivalis and Aggregatibacter bone and connective tissues. The commensal oral
actinomycetemcomitans. Both of these microbes microbiota directly colonizes the surfaces of the
have great genetic intraspecies diversity and oral mucosa and the teeth or the microbiota colo-
express a number of different virulence factors, nizes an already established biofilm. These sur-
which have the capacity to cause imbalance in the faces of the oral cavity provide specific receptors
host’s response. A. actinomycetemcomitans is the for ligand binding and glycolipid structures,
major pathogen in aggressive forms of periodonti- which serve as targets for more unspecific bacte-
tis (Fig. 4.1) that affect young individuals, while rial adhesion. The role of specific pathogens for
this degenerative disease is gradually being eluci-
dated, but it is generally accepted that it accounts
for the combination of the total bacterial load, the
release of specific virulence factors, and the
effects on the host’s response. Therefore, the
A. Johansson (*) periodontal bacterial species that are associated
Division of Molecular Periodontology, Department of with periodontal disease could be described as
Odontology, Umeå University, Umeå, Sweden “opportunist pathogens.” In a clinically healthy
e-mail: [email protected]
situation, there is a balance between the peri-
G. Dahlén odontal microbiota in a biofilm and the host
Department of Oral Microbiology and Immunology,
Institute of Odontology, Sahlgrenska Academy, response of the gingiva. That balance results in a
University of Gothenburg, Göteborg, Sweden protective immune response from the host that

DOI 10.1007/978-3-319-53737-5_4
32 A. Johansson and G. Dahlén
Fig. 4.1 Clinical presentation of localized aggressive with 4–8 mm periodontal crevices with bleeding on prob-
periodontitis. A 16-year-old female presenting with radio- ing in the affected region. Microbiological analysis, by
graphic alveolar bone loss associated with bony defects cultivation technique, confirmed the presence of high lev-
(marked with arrows) and probing attachment loss at the els (7.7 × 106) and proportions (92%) of A. actinomy-
lower incisors. The clinical presentation shows sparse cetemcomitans in the sampled lesion (32 m). By courtesy
plaque accumulation and localized gingival inflammation of Dr. Carola Höglund Åberg
Fig. 4.2 Clinical presentation of chronic periodontitis. A graphs that almost all teeth show bone loss of >7 mm. By
56-year-old male patient with severe periodontitis with courtesy of Dr. Giovanni Serino
pathological pocketing around most teeth. Note on radio-
4 Bacterial Virulence Factors that Contribute to Periodontal Pathogenesis 33
Fig. 4.3 Pathogenesis Environmental and acquired risk

of periodontitis factors
according to the
classical model by Page
and Kornman [1]. LPS
lipopolysaccharides, Microbial Host
MMP’s matrix Challenge lmmuno- Connective Clinical signs
lnflam- tissue and of disease
metalloproteinases, initiation and
Growth matory bone
PMN’s and response Metabolism Progression
polymorphonuclear Dysbiosis
neutrophils
Genetic risk factors
does not affect the equilibrium in the tissue systemic diseases such as diabetes) may further
regeneration. The amount and composition of the modulate the severity of that response. There is
molecules that are released from the oral biofilm also reason to believe that certain bacteria (peri-
determine the effect on the host’s response. odontopathogens) associated with the periodon-
Invasiveness of bacteria or bacterial products fur- tal disease progression play an active role in the
ther contributes to a rapid disease progression. modulation of the immuno-inflammatory
The tooth supporting, or else periodontal, tis- response through their virulence factors, making
sues—connective tissue and bone—are under the periodontal process more chronically destruc-
constant renewal through processes involving tive, according to the recent “keystone” patho-
death, proliferation, and differentiation of cells, gens hypothesis [4, 5].
as well degradation and production of matrix Hence, this chapter goes on to describe the role
molecules. The tissues constantly fluctuate of bacterial virulence factors in the development
between destructive phases and healing or tissue of periodontitis, and elaborates how they partici-
repair (granulation tissue formation). Loss of pate in the initiation and modulation of the host’s
periodontal ligament and bone resorption are responses in the periodontal tissues. This is
irreversible processes that lead to a net loss of described with special reference to the two most
attachment, epithelial down growth, and pocket recognized periodontopathogens, Aggregatibacter
formation. The pathogenic process follows the actinomycetemcomitans and Porphyromonas gin-
outline that was proposed by Page and Kornman givalis, which have distinctly different strategies
[1], and it is still the model or hypothesis that is for their establishment, survival, growth, and
believed to be correct today [2, 3], see Fig. 4.3. pathogenic role in periodontitis.
It is well established that the microbial chal-
lenge induces and maintains the immune-
inflammatory response of the host. However, the 4.3 Periodontal Bacteria
specific role of each bacterial species in the dis- and Their Virulence Factors
ease progress and the change in connective tissue
and bone metabolism is less clear. There is rea- Infection occurs when the virulence, the number,
son to believe that the magnitude of the inflam- and the exposed time supersede the local and
matory response is dictated by genetic risk factors general host’s defense, and that leads to a patho-
in the individual host, and this causes some indi- logical reaction in the host’s tissues [6]. The viru-
viduals to be much more susceptible than others lence can be divided into three parts: the ability to
to progression of the disease. Environmental fac- establish (colonization/infectivity), the ability to
tors (e.g., smoking) or acquired risk factors (e.g., invade (invasivity), and the ability to cause tissue
damage (pathogenicity). Virulence means the even motile (Treponema spp., Campylobacter
degree of pathogenicity of a microorganism as spp., Selenomonas spp.), and the motile ones
indicated by the severity of the disease that is normally do not colonize smooth surfaces. They
produced and the ability to invade the tissues of are rather dependent on the co-adhesion with
the host [7]. Thus, virulence is a microbial prop- other species already established in a biofilm.
erty that can only be expressed in a susceptible Since most of the recognized periodontopatho-
host. Hence, virulence is not an independent gens are microaerophilic or strict anaerobic, they
microbial property, because it cannot be defined need an environment with low redox potential
independently of a host. Logically, the depen- (Eh) to survive and establish. In healthy individ-
dence of virulence factors on virulence implies uals, most periodontopathogens are outcom-
that the definition of a virulence factor requires a peted, unless these bacteria find niches that fulfill
functional definition for microbial virulence [8]. the requirement of mechanical forces (retention)
Factors involved in adhesion and persistence. and anaerobic conditions. Such conditions are
The bacterial colonization of the oral cavity found on the dorsum of the tongue and around
takes place at birth and continues throughout the and between the teeth where these bacteria may
whole life of the host. The colonization is mainly persist without being eliminated by the salivary
orchestrated by the host and its surface recep- flow in periodontal healthy individuals. Adhesins
tors. Thus, the microbial adhesins that specifi- may also be important factors in the initial step
cally fit to the host’s receptors determine the of intracellular invasion (see below) in cells such
selection of microorganisms and thereby consti- as epithelial cells, fibroblasts, and leukocytes.
tute and build the commensal microbiota living Microbial metabolic activity and growth
in harmony and balance (microbial homeostasis) (Fig. 4.4). Another mechanism by which the peri-
with the host [9–12]. Periodontopathogens need odontal bacteria can evade the natural elimina-
to compete with the commensal primary colo- tion and cleaning forces is by increased growth.
nizers and pioneer species in order to establish, The gingival pocket is a perfect niche for anaero-
grow, and cause pathology. Most periodontal bic, fastidious, low adherent, motile bacteria such
bacteria are less efficient in adhesion, some are as the red complex bacteria (P. gingivalis,
Fig. 4.4 Microbial
challenge and host
defense. Factors
involved in host–parasite
interaction in
periodontitis
Tannerella forsythia, Treponema denticola, individuals with chronic gingivitis or periodonti-

according to [13]) and the orange complex bacte- tis. Note that the etiology of periodontitis always
ria (Fusobacterium spp., Prevotella spp., starts with an intact periodontium, where the pro-
Campylobacter spp., anaerobic streptococci, and teolytic anaerobes have few advantages as com-
more). The simultaneous inflammatory response pared with the unique milieu in the inflamed
that is elicited in the gingival connective tissue deepening periodontal pocket. The role of this
and subsequent deepening of the gingival pocket group of Gram-negative anaerobes is discussed
gives these bacteria important advantages over more in detail below, exemplified by P.
the primary tooth colonizers such as the saccha- gingivalis.
rolytic, facultative streptococci and Actinomyces Evading the host’s defense. The host reacts
spp. [9, 14]. The gingival pocket offers the peri- extensively to the microbial challenge by employ-
odontal bacteria mechanical persistence (including the inflammatory reaction and assembling
ing motile bacteria), anaerobiosis, and nutrients defense factors such as neutrophils, complement
including specific growth factors such as hemin, activation and antibodies in the exudate and gin-
vitamins, and hormones and other serum factors gival pocket. Early studies [16] showed that 95%
in the gingival exudate [15]. The main nutrient of the leukocytes moving by chemotaxis into the
factors in the exudate are proteins, peptides, and gingival pockets are neutrophils and the rest are
amino acids, which are the targets for the strongly macrophages. In the pockets, neutrophils that are
proteolytic red and orange complex bacteria. loaded with phagocytized bacteria are constantly
Simultaneously, many of these periodontal bacte- removed out from the pockets. However, some
ria produce various virulence factors that may bacteria have specific means to escape phagocy-
help them to evade or escape the host’s defense tosis (leukotoxin) or survive or overcome (cap-
system (see below). The increasing subgingival sule) intracellular killing once they have been
bacterial growth and changed ecology (dysbio- phagocytized [17, 18].
sis) are the major factors that enable a simultane- Leukotoxin is a well-known virulence factor,
ous development and increase of the inflammatory which is found in several pathogenic bacteria
reaction. The higher metabolic activity produces such as the PV-leucocidin in Staphylococcus
toxic metabolites such as short carboxylic acids aureus [19] and Fusobacterium necrophorum
(butyric acid, propionic acid, valeric acid, capro- [20]. This explains the invasive character and
nic acid, and phenylacetic acid), ammonia, and abscess-promoting character of these pathogens.
hydrogen sulfide [14]. These small molecules are Therefore, the leukotoxin-producing microor-
produced in high amounts during growth, ganism, A. actinomycetemcomitans, among the
penetrate through the pocket epithelium, enter periodontal bacteria, has been extensively stud-
the connective tissues, initiate, and maintain the ied due to this virulence factor [21, 22]. The leu-
inflammatory reaction. Subsequently, the kotoxin explains the specific ability of this
periodontal bacteria begin to multiply. The vast bacterium to survive or escape the host’s defense
majority of the periodontal bacteria are proteo- (neutrophils), but it does not explain the peri-
lytic and Gram-negative, and during growth they odontal breakdown that is associated with this
release proteolytic enzymes and endotoxins bacterium in aggressive forms of periodontitis in
(lipopolysaccharides, LPS), which interact with children and young individuals. The specific role
the tissues and the host’s response. This increase of A. actinomycetemcomitans and its virulence
in metabolic activity, increase in bacterial num- factors are extensively discussed below in a sepa-
ber, and adaptation of new bacterial species lead rate section.
to a continuously changing ecology, which lasts Capsule formation is a well-known virulence
until a balance (homeostasis) is created between factor of many pathogenic microorganisms. The
the bacterial community and the host. Such capsule is produced to escape phagocytosis and
homeostasis may continue for long periods, or intracellular killing by neutrophils and macro-
even lifelong for low-periodontitis susceptible phages. The inability of the host’s defense to kill
the pathogens may result in spread of infections The intracellular route has gained more atten-
and to complications such as sepsis [18]. tion since it was noticed that viable and non-
Proteolytic enzymes may split or degrade the keratinized epithelial cells contain bacteria.
host’s defense molecules such as immunoglobu- Buccal epithelial cells regularly contain bacterial
lins and complement [23]. cells, mainly streptococci [32]. Interestingly, P.
Cell and tissue invasion. Invasion is often gingivalis and other periodontal bacteria have
regarded as a key event when colonization is also been shown in such cells [33]. T. forsythia,
transformed into infection. Invasion has also Prevotella intermedia, and C. rectus have been
been claimed to be a key event in periodontitis identified inside crevicular epithelial cells [34]
[24–26]. Invasion has clearly taken place if the in vivo, and this uptake is mediated by receptor
epithelial barrier (junctional epithelium) is dis- interaction between the bacteria and the epithe-
rupted in case of ulceration. In acute necrotizing lial cells. This is a sophisticated way for bacteria
ulcerative gingivitis (ANUG) spirochetes are to escape the host defense factors. On the other
shown to invade the underlying connective tissue hand, the desquamation process constantly
by motility (Listgarten [27, 28]). A similar pic- detaches and removes epithelial cells and their
ture is also found in peri-implantitis where the content through the gingival pocket and out into
epithelium does not cover the connective tissues the saliva. Interestingly, P. gingivalis has been
of the apical part of the peri-implant pocket [29]. shown to invade within 15 min, it may replicate
The periodontal abscess is another example of within 4 h [35], it may be transferred to underly-
invasion into the tissues by bacterial growth, and ing epithelial cells, and enter the subepithelial
the abscess can sometimes even lead to fistula connective tissue [25], before the surface cell
formation. Invasion in these examples is due to becomes detached.
bacterial multiplication and expansion, and it is Modulation of the host’s response. Most atten-
associated with heavy neutrophil attraction and tion for immune modulation has been directed
suppuration and sometimes symptoms, which all toward the endotoxin/lipopolysaccharides that
are characteristic of an acute infection. In the are released after autolysis of Gram-negative
chronic forms (chronic gingivitis, chronic peri- bacteria. The LPS builds up the outer membrane
odontitis) it is more controversial whether inva- of all Gram-negative bacteria and consists of a
sion has taken place. However, it is likely that toxic Lipid A, a core polysaccharide, and a poly-
invasion is an important event at least in the saccharide chain of repeating sugar subunits. The
aggressive form of periodontitis [25, 26]. latter is an important antigen (O-antigen) that has
There are two routes that have been discussed been used for classification of several enteric
for bacterial invasion in periodontitis [25, 30]. genera. The Lipid A is biologically very active,
An intercellular route is suggested to take place and it interacts with most humoral systems (com-
by motility, e.g., spirochetes [30]. The junc- plement and coagulation) and with inflammatory
tional epithelium is non-keratinized and thin B and T lymphocytes [36]. The activation of vari-
(4–5 cells thick), and in a state of inflammation, ous inflammatory cells makes the LPS a primary
the cells are not tightly joined in order to facili- candidate for immune modulation of the gingival
tate gingival exudate and migration of PMN inflammation. Since all Gram-negative bacteria
cells and macrophages through the gingival bar- release LPS, they may all be involved in the peri-
rier. It is hypothesized that this intercellular pas- odontal inflammation process. It is important to
sage also allows motile bacteria such as emphasize that LPS and the Lipid A structure are
Treponema and Campylobacter species to pen- strongly hydrophobic and form free membrane
etrate the barrier. Treponema spp. and P. gingi- structures or outer membrane vesicles [36].
valis produce specific gingipains that can These aggregates may penetrate into tissues and
degrade epithelial junctional proteins interact with most cells through the interaction of
(E-cadherin and occludin), and this impairs the the Lipid A part, which integrates with cell mem-
junction-related structures [31]. branes and activates the cell [36]. The toxicity of
LPS varies greatly among various different bac- bind to other microbial cells (autoaggregation,
teria. The toxicity is primarily dependent on the coaggregation) and to lectins and other food
Lipid A, but it is modulated by the structure of components, e.g., polyglucan production from
the core polysaccharide and the length of the sugars increases the binding capacity of several
O-antigen chain. The impact of the structure for streptococci. This exceptionally strong binding
its modulating ability on the host’s response is capacity is far greater than that of other microor-
extensively studied for P. gingivalis [37]. There is ganisms that might be transmitted and incorpo-
also an increasing interest for outer membrane rated in the microbiota. They have a very broad
vesicles formed by P. gingivalis and their interac- range of biochemical activity. These activities
tion with the host. Outer membrane vesicles are include both saccharolytic and proteolytic metab-
LPS-membrane structures enriched with pro- olism and the use of glycoproteins from saliva
teins, largely gingipains, and those vesicles con- during periods of starvation to produce both acid
tribute to P. gingivalis–host interaction and and alkaline to regulate the ecology. The
pathogenicity [38]. Streptococci produce bacteriocins, which control
the microbiota and block the space and receptors
for more pathogenic bacteria. In addition, the
4.4 Transmission Pattern Streptococci are generally well adapted and tol-
and Colonization erant to environmental changes [15]. The final
selection of the pioneer species early in life is pri-
All microorganisms, despite the fact that they marily determined by the host (the newborn
may occur as resident, transient, or pathogenic child) through its oral receptor pattern and envi-
microbiota, are transmitted from the external ronment. The best-suited strains for this selection
world. According to Marsh & Devine [15], the come from the mother and other family members
establishment of a microbial community passes and explain why certain microorganisms occur in
ecological stages: some families but not in others. Others that are
Transmission—Acquisition/colonization— well adapted to the oral cavity and teeth are
Pioneer species—Microbial succession— Actinomyces spp., Neisseria, and Haemophilus
Increasing species diversity—Climax community. species, while others are outnumbered (coli-
All stages are affected by environmental condi- forms, staphylococci, and more) and are kept in
tions or modifications. The colonization, succes- the transient microbiota. Environmental factors
sion, and diversity, which lead to a climax modify the prerequisites for acquisition and colo-
community, may differ greatly in composition nization so that low pH (caused by early sugar
and microbial activity between sites, individuals, intake, e.g., breast-feeding and bottle-feeding)
and populations. Factors involved are host recep- lower the pH to an increasing number of low
tors for attachments, pH and redox potential, (compared to streptococci) adherent microorgan-
nutrients, and metabolic breakdown products. isms such as acidophilic lactobacilli and Candida
These factors differ greatly by age, from the new- spp. The comparatively high Eh in newborns and
born child, through childhood, adolescence, and children also gives an advantage to the aerobic,
into adulthood. facultative, and oxygen-tolerant microorganisms.
Streptococci are far and away the most supe- The more strict anaerobic bacteria are suppressed
rior microorganisms for colonization of the oral even if they are exposed to the child, but they
cavity [39]. They are the outstanding pioneer spe- might occasionally find a hidden place to survive.
cies in all oral niches including mucosal surfaces The number of Fusobacterium, Veillonella, and
and tooth surfaces after tooth eruption. They are Prevotella can be frequently found in 3-month-
extremely well adapted to the oral cavity by a old children although in low number [40, 41].
multitude of receptor interactions with host cells, Although strictly anaerobic, these are also adher-
salivary glycoproteins, and other salivary and ent, saccharolytic, acid-tolerant, and can survive
serum components (agglutination). Streptococci in the oral cavity of babies. Other so-called
p eriodontopathogens such as P. gingivalis, T. for- induce an inflammatory reaction in the gingival

sythia, Campylobacter, and Treponema species tissues, and cause progression of attachment loss
are hardly detected even with very sensitive and pocketing. The colonization of the gingival
methods. It is generally believed that they do not area translates the bacteria from a resident stage
occur until later in life (adolescence) when most to infection (see below). The production of spe-
permanent teeth are fully erupted, some gingivi- cific virulence factors and increased metabolic
tis is regularly present, and hormonal changes activity makes the bacteria more aggressive and
have taken place. These conditions present suffi- causes severe environmental changes. The
cient pH and Eh environments for survival and increasing inflammation and exudation promote
growth of these more fastidious proteolytic and those bacteria with more proteolytic activity in
non-saccharolytic bacteria [42]. They are low contrast to those with a saccharolytic metabolism
adherent and need mechanical retention, which that have no advantage in the subgingival area
develops on the dorsum of the tongue and due to lack of sugars. Facultative bacteria have no
between the teeth and the subgingival dental advantage of the lowered Eh and a slightly alka-
plaque when teeth are fully erupted and consti- line pH. Other less aggressive subtypes may col-
tute a niche were they reside (non-adherent sub- onize and compete more efficiently. Few studies
gingival plaque) to minimize the risk for have tried to evaluate whether one individual or
elimination. They require hemin or other serum even one site may harbor several genotypes of the
products for growth, which are hardly available same species. However, in a recent study, up to
until inflammation occurs and they constitute an four different genotypes were found in one deep
increasing proportion of the subgingival plaque. periodontal site in Thai adults with periodontitis
A special attention in the colonization pattern [46]. It is therefore reasonable to argue that there
has been paid to the periodontal bacterium, A. is a dynamic change between genotypes resulting
actinomycetemcomitans. It has been associated in one dominating genotype at various time
with the localized aggressive forms of periodon- points.
titis in young individuals (earlier called localized Less is known about the P. gingivalis geno-
juvenile periodontitis). This bacterium is found type distribution, and it is generally believed that
to colonize in a family pattern, and the same phe- P. gingivalis has a much more clonal distribution
notype is frequently found in mother and child between individuals than A. actinomycetemcomi-
pairs [43]. This bacterium has generally good tans [47]. Thus, adults living together (periodon-
adherent abilities, but it has strict specificities for titis cases and their spouses) frequently share the
host cells (at least humans and old world mon- same genotype. This indicates that transmission
keys) [44]. This bacterial species is facultative also continues between adults, and the climax
anaerobic, tolerant to oxygen stress (catalase community has no upper age limit and may con-
positive), moderately saccharolytic, and acid- tinue throughout the entire life of the host.
tolerant, which makes it suitable for early coloni-
zation in the oral cavity in children. This
bacterium does not seem to be dependent upon 4.5 Colonization and Infection
other bacteria, and some antagonism between A.
actinomycetemcomitans and some streptococcal Virulence factors are usually genetically con-
species has been reported [45]. Under most conserved, but it must be emphasized that the varia-
ditions, A. actinomycetemcomitans, like most tion between genotypes of the same species may
other bacteria, are controlled by the host and the be significant [48]. In addition, the phenotypic
resident microbiota (streptococci). This bacte- expression of virulence factors in vivo may
rium occurs in several genetic and phenotypic strongly differ due to environmental conditions.
subgroups (and clones) and aggressive variants Even if the genetic basis for the virulence factors
(see below) may colonize early in life. If not is present, their expression and function may be
properly controlled, they increase in number, up- or downregulated when they are most/least
needed [49, 50]. The expression of virulence fac- composition in most anaerobic infections is char-
tors in a polymicrobial community or infection is acterized by its heterogeneity, and the infecting
even more complex due to the dependence of combinations seem to occur at random or as
microbial interactions, which normally control teams (combinations), which include a number of
the microbial homeostasis. Under certain circum- bacteria supporting each other [60, 61]. That
stances, the microorganisms may evade the inter- does not necessarily exclude that some species
action with other microorganisms by activating more frequently occur in these polymicrobial
specific genes. This can suppress the controlling infections and that some teams have a greater
mechanisms in the community, increase their degree of infectiosity. The so-called red complex
metabolic activity, and cause rapid growth that bacteria (P. gingivalis, T. forsythia, and T. denti-
leads to dysbiosis or overgrowth. This uncon- cola) are statistically more associated with pro-
trolled overgrowth of certain microorganisms is gressive periodontitis than other bacteria, and the
the basis for the “Burst theory” proposed by orange complex bacteria occur less frequently in
Socransky [51, 52]. Changes in the microbial the same sites [13]. A number of studies have
environment (nutrients, pH, anaerobiosis, bacte- reported other bacterial constellations, which
riocins, etc.) may result in dysbiosis. also correlate to the deep periodontal pockets and
Communication occurs between the bacterial disease progression [62, 63]. A recent review
cells in the biofilm (quorum sensing) [53]. Small [64] listed 139 species that could be classified as
peptides (signaling molecules) are produced to potential periodontopathogens. However, it is
activate certain genes in target bacteria. The role unwise to evaluate a single bacterial species inde-
of viruses in the pathology of periodontitis has pendently without concomitantly evaluating
been little investigated. However, it is clear that other bacteria, their functions in the microbial
viruses such as Herpes simplex virus can directly community, and the infectious process [14, 65].
cause gingival inflammation [54] or can damage The etiological role of each single bacterial spe-
the gingiva indirectly by genetic exchange of cies in the initiation and progression of periodon-
DNA or plasmids by transduction [55]. titis is still largely a matter of hypotheses [66].
From a microbiological point of view, it is There is one exception to the view of peri-
pertinent to have a holistic approach to periodon- odontitis as a polymicrobial and predominantly
tal infections and consider them not essentially anaerobic infection with a low specificity, and
different from other polymicrobial infections in that is A. actinomycetemcomitans. This bacte-
the body. Characteristically most polymicrobial rium does not fit into the complex system of the
infections such as the periodontal, endodontic, Socransky model [13] and constitutes its own
and other subepithelial oral infections are pre- factor using factor analysis [63]. Especially in
dominantly anaerobic [56]. Single anaerobic spe- young individuals, A. actinomycetemcomitans
cies may lack essential virulence factors to occurs independently of other bacterial species.
establish and infect as a monoinfection, and This form of periodontitis resembles a more spe-
therefore need cooperative mechanisms with cific type of infection and thus needs to be
other bacteria to formulate a prerequisite envi- described separately (See below).
ronment for survival and growth. Numerous ani-
mal experiments have shown the necessity of
cooperation with facultatives to reduce the oxy- 4.6 he Role of Aggregatibacter
T
gen tension low enough for growth, invasion, and Actinomycetemcomitans
infection [57, 58]. Only few isolates of certain
anaerobes have been shown to cause experimen- A. actinomycetemcomitans (Fig. 4.5) is a faculta-
tal infections in monoculture. The isolates P. gin- tive anaerobic Gram-negative bacterium associ-
givalis W83 and W50 have such a capacity and ated with periodontitis, and it expresses a number
thus have been used in numerous experimental of potential virulence factors [67]. This bacte-
in vitro models and in animals [5, 59]. Microbial rium is strongly associated with the localized
cetemcomitans in individuals with an IL-6 poly-

Pg morphism results in enhanced expression of this
Pg ↓
↓ cytokine. It was later shown that the growth of A.
actinomycetemcomitans can be stimulated by
Pg
ligand binding to a specific cytokine receptor on
↓
the outer membrane, the IL-1β-binding protein,
Aa which has a high affinity for IL-6 [77]. This dis-
covery might be involved in the findings that
Aa
show a significantly greater risk for developing
periodontitis in individual’s positive IL-1 poly-
morphic sites [78].
A great genetic diversity has been shown
Fig. 4.5 Colonies of A. actinomycetemcomitans and P. within the A. actinomycetemcomitans species
gingivalis from a clinical subgingival sample cultured on with six distinct different serotypes and a large
a blood agar plate for 5 days under anaerobic condition proportion of variable genes within the pan
genome of this bacterium [79, 80]. Moreover, a
aggressive form of periodontitis, but its relation number of mutations within the core genome of A.
to other forms of periodontitis is still not fully actinomycetemcomitans contribute to further dif-
understood [68, 69]. However, instead of con- ferences, where the highly virulent JP2 genotype
necting the presence of A. actinomycetemcomi- has attracted most of the attention [73]. This gen-
tans with a certain diagnosis, it is obvious that otype has a 540-base pair deletion in the promoter
this bacterium plays a role in the early phase of of the gene operon responsible for expression of a
the disease [70, 71]. One hypothesis is that A. leukotoxin [81]. This genotype of the bacterium is
actinomycetemcomitans colonizes the oral highly leukotoxic and has a certain strong associ-
mucosa, and when it is translocated to the gingi- ation with disease risk in the carrier, as compared
val margin, it can initiate disease. The presence with other genotypes with a full-length leukotoxin
of this bacterium on an individual basis has been promoter [70, 73]. In addition, highly virulent
shown to be a strong risk marker for initiation of genotypes of A. actinomycetemcomitans have
the disease [70, 72, 73]. Results from a recent also been detected with a 640-base pair deletion
study showed that children (6–12 years) of par- or with an insertion in the leukotoxin promoter
ents with aggressive periodontitis have increased [82, 83]. The predictable enhanced disease risk in
frequencies and quantities of A. actinomycetem- individuals colonized with this bacterium is a
comitans as compared with children with peri- unique property among the various periodonto-
odontally healthy parents [74]. The vertical pathogens [84].
transmission pattern of this bacterium indicates a A. actinomycetemcomitans expresses a num-
strong role as an etiologic factor in this disease, ber of different virulence factors, including
even though none of the children had been diag- adhesins and exotoxins [67]. The different adhes-
nosed for aggressive periodontitis already at this ins of the bacterium allow colonization of the
early age. The facultative anaerobic property of oral mucosa, the tooth surface and the bacterial
A. actinomycetemcomitans promotes coloniza- biofilm, as well as promoting epithelial invasion
tion at an early age in individuals with a healthy [85]. Once adhered to the oral surface, the ability
periodontium and limited accumulation of sub- to produce virulence factors determines the role
gingival microbiota [75]. The importance of of the bacterium in the pathogenesis of periodon-
interplay between host-genetic factors and the titis [86]. Several different virulence factors of A.
bacteria has been nicely described and named as actinomycetemcomitans have been described and
infectogenomics [76]. In that study it was shown have been nicely summarized by Henderson and
that a significant association between increased coworkers [67]. Longitudinal studies have shown
presence and concentrations of A. actinomy- the highest odds ratio for disease onset or disease
progression among carriers of the highly leuko- cleavage [97]. A subunit of Cdt has homology
toxic JP2 genotype of the bacterium, which indi- with human DNaseI, and the Cdt subunit is sug-
cates an important role of the leukotoxin in the gested to be responsible for the Cdt-induced
pathogenesis of periodontitis [70, 87]. Recently, DNA break [98]. This DNA break results in
a subpopulation within the serotype b of A. acti- growth arrest of periodontal fibroblasts and
nomycetemcomitans, with an intact leukotoxin lymphocytes [99, 100]. In addition, the Cdt-
promoter, but with enhanced leukotoxin produc- intoxicated fibroblasts and lymphocytes increase
tion, has been identified [88]. This genotype of the expression of a receptor activator for
the bacterium has also been associated with a sig- NF-kappaB ligand (RANKL), a key component
nificantly increased odds ratio for disease proin osteoclast differentiation [101, 102]. About
gression, which further strengthens a role of 80% of A. actinomycetemcomitans isolates have
leukotoxin in the disease progression. The leuko- an intact operon for Cdt expression, which is a
toxin promotes resistance to phagocytic killing prerequisite for expression of an active Cdt
and affects our defense cells by inducing release toxin [103, 104]. Despite these potent virulence
of tissue-degrading enzymes and pro-mechanisms of Cdt, results from a longitudinal
inflammatory cytokines before the challenged study, which examined carriers of either Cdt-
cells die [21]. These events are all cellular and positive or Cdt-negative bacteria, failed to show
molecular mechanisms that are involved in the a significant difference in disease progression
homeostasis of tissue remodeling [3, 66]. between the two groups [104].
Macrophages exposed to leukotoxin have been A third exotoxin that is produced by A. actino-
shown to release substantial amounts of the pro- mycetemcomitans is the CagE molecule, a highly
inflammatory cytokine interleukin (IL)-1β that conserved protein of 38.6 kDa [105]. Its ability to
activates bone resorption in a mouse calvarial cause apoptosis in human epithelial cells and its
model [89]. The immunogenic properties of the reactivity to serum immunoglobulin from indi-
leukotoxin are obvious due to the high concentra- viduals with periodontal disease indicate a pos-
tion of neutralizing antibodies in the peripheral sible role in the pathogenicity of the disease
circulation of the A. actinomycetemcomitans car- [106]. However, a correlation between the ability
riers [90]. The leukotoxin has been shown to be to express CagE and initiation and progression of
released from the bacterial outer membrane in periodontitis has not yet been shown.
the presence of serum proteins, and the toxin is Lipopolysaccharide (LPS) is a well-characterized
protected from degradation by the protease inhib- pathogen-associated molecular structure, which is
itors present in this mixture [91, 92]. In addition, found in the outer membrane of most of the Gram-
leukotoxin has also been identified in vesicles negative bacteria, including A. actinomycetem-
secreted from the bacteria, which might promote comitans [107]. It can initiate a strong immune
systemic distribution of the toxin [93]. These response and serves as an early warning signal of
properties might contribute to the immunogenic bacterial infection. LPS is initially released from
potential of the protein, even though a protective bacterial membranes and outer membrane vesi-
role of specific antibodies in the periodontal cles that interact with host proteins, and LPS
pocket is questioned [94]. Leukotoxin is a large results in increased expression of pro-inflamma-
pore-forming protein that belongs to the Repeat tory cytokines. Different serotypes of A. actino-
in ToXin (RTX) family of bacterial proteins that mycetemcomitans have been described based on
are produced by a number of Gram-negative the LPS-O-polysaccharide antigenicity and host
pathogens [95]. cells, which react differently to LPS from various
A second exotoxin that is produced by A. serotypes [108, 109]. However, independent of
actinomycetemcomitans is the cytolethal dis- serotype, LPS from this bacterium activates
tending toxin (Cdt) [96]. This toxin affects all expression of pro- inflammatory cytokines that
eukaryotic cells by entering the nuclei of the tar- can be activated and released upon exposure to
get cell and causing double stranded DNA the exotoxins [110, 111]. In addition, the p resence
of A. actinomycetemcomitans upregulates expres- while Type-II fimbriae are associated with

sion of inflammasome components in human increased pro-inflammatory and invasive activi-
macrophages, which indicates synergistic effects ties in macrophages.
between the two exotoxins on their pro-inflam- Six capsular serotypes have been identified
matory response to their host [112]. among periodontal P. gingivalis isolates [120,
121]. The capsule of P. gingivalis leads to a
reduction in the host inflammatory response,
4.7 he Role of Porphyromonas
T evasion of phagocytosis, and increased viru-
Gingivalis lence [18] as compared with nonencapsulated
strains. Two capsular types, K1 and K6, seem
P. gingivalis (Fig. 4.5) is a Gram-negative oral to include isolates with a higher virulence than
anaerobe that is involved in the pathogenesis of the other capsular types [121]. Non-capsulated
periodontitis [113]. This bacterium is one of the strains adhere significantly more than their
major colonizers in deep periodontal pockets, capsulated variants to pocket epithelial cells
and it provides a strict anaerobic ecological [122].
niche that promotes the bacterium’s growth. P. The gingipains are trypsin-like cysteine pro-
gingivalis has been described as a “keystone teases and are considered to be the major viru-
pathogen” [4]. A microorganism that supports lence factor of P. gingivalis [114]. The direct
and stabilizes the dysbiotic microbiota associ- and indirect activities of gingipains are impor-
ated with a disease state was their criterion for a tant in every stage of infection, such as attach-
species to be named as a keystone pathogen. P. ment, colonization, invasion, acquisition of
gingivalis can locally invade periodontal tissues nutrients, evasion of host’s defenses, and dis-
and evade the host defense mechanisms [114]. It semination [123]. The gingipains consist of
utilizes its major virulence factors, lipopolysac- three enzymes: RgpA and RgpB that degrade
charide, capsule, gingipains, and fimbriae, to proteins with arginine in the p1 position, and
establish the infection by interacting with the Kgp that degrades proteins with lysine in the
host. Although it has a high number of potent p1 position [124]. Gingipains also digest a
virulence factors, it has not yet been shown that broad spectrum of host proteins, some of which
its presence in a healthy periodontium predicts are completely degraded, and this provides
an increased risk of disease onset. This indicates peptides for P. gingivalis growth and metabo-
that this bacterium is probably a late colonizer lism. Some host proteins are only partially
that invades already diseased tissues and contrib- degraded, which might lead to dysregulation of
utes to an increased progression of periodontal the host’s defensive inflammatory reactions
breakdown [115]. and the failure of the host to eliminate P. gingi-
Early analyses on the restriction fragment valis [4, 123]. The reduced condition in the
polymorphisms of the fimbrillin locus, fimA, of P. periodontal pocket promotes activation of
gingivalis, revealed a substantial genetic varia- these cysteine proteases [124]. In addition, the
tion [116]. Later Amano et al. [117] showed that gingipains have also been shown to have the
some fimA genotypes (fimA II and IV) are more capacity to release A. actinomycetemcomitans
prevalent in periodontitis than fimA type I, which from the microbial biofilm and neutralize its
is most prevalent in healthy periodontal tissue. leukotoxin by proteolytic degradation [125,
Fimbriated P. gingivalis are more efficient than 126]. Gingipain inhibitors have been proposed
fimbria-deficient P. gingivalis to enter human to be a future tool for therapeutic strategies
dendritic cells in vitro [118]. A Type-I fimbriae P. against periodontal diseases [127].
gingivalis strain induces more bone loss than P. gingivalis expresses a peptidyl-arginine
Type-II P. gingivalis in a mouse model [119], deiminase (PAD), an enzyme that converts
arginine within a peptide (peptidylarginine) [134]. The outcome of TLR2 activation in

into peptidylcitrulline [128]. This bacterium is response to distinct microbial molecules may be
the only prokaryote that has been reported to influenced by differential TLR2 association with
possess PAD. Citrullination by human PADs is accessory receptors [134]. These activations
an important mechanism in normal physiology involve interactions that have been shown to
and inflammation [129]. Systemic autoanti- inhibit pro-inflammatory and antimicrobial host
bodies to citrulline are strongly associated responses. The various effects reported by P. gin-
with the preclinical phase of rheumatoid givalis on osteoclast differentiation might be a
arthritis [130]. The role of P. gingivalis PAD result of its complex interaction with the TLRs
in the pathogenesis of periodontitis is not [135, 136].
known, but it may be involved in the associa-
tion reported between periodontitis and rheu-
matoid arthritis [131]. However, it has been 4.8 Concluding Remarks
shown in a mouse model that there is a PAD
dependent synergistic effect between these We have described the virulence of two major
two diseases [132, 133]. periodontopathogens, A. actinomycetemcomitans
In line with reports from other Gram-negative and P. gingivalis, which have different strategies
bacteria, P. gingivalis expresses LPS that induces for their pathogenicity in periodontitis in humans
a strong pro-inflammatory response [107]. The (Table 4.1). This is due to completely different
most well characterized pathway for host-cell spectra of virulence factors expressed during
activation by LPS is through interaction with the transmission, colonization, growth, and partici-
LPS binding protein (LPB) followed by binding pation of the microbial challenge to the gingival/
to soluble or membrane-bound CD14 molecules periodontal tissues and the evasion of host
that activate the transmembrane toll-like receptor defense factors assembled in the immune-
(TLR) 4. These interactions activate intracellular inflammatory host response. It is concluded that
signaling, which results in enhanced expression A. actinomycetemcomitans fulfills the pathogenic
of proteins involved in the pro-inflammatory role according to the “specific plaque hypothe-
response. Interestingly, LPS from P. gingivalis sis,” while P. gingivalis fulfills its role according
has also been shown to act as a TLR 2 agonist to the keystone hypothesis for periodontitis.
Table 4.1 Comparison of virulence pattern for the two periopathogens A. actinomycetemcomitans and P. gingivalis
A. actinomycetemcomitans P. gingivalis
Colonization Vertical transmission from parents and older Horizontal transmission and prefer an
siblings. Colonizes mucosal and tooth established microbial biofilm or gingival
surfaces in early childhood pockets, which means a preference of older
individuals
Translocation Colonizes gingival crevice and become a risk Colonizes gingival pockets and established
marker for tissue degradation microbial biofilms
Inflammation Activate inflammation through LPS and Activate inflammation through LPS and
exotoxins proteolysis
Degradation Localized, rapid, and deep degradation of the General and slow degradation of the tooth-
tooth-supporting tissues supporting tissues
Stability Decreases in proportion in an established Increases in proportion due to their ability to
lesion due to the proteolytic environment that detach other bacteria from the biofilm and the
degrades the major virulence factors, adhesins advantage of the more anaerobic ecological
and exotoxins niche
Clinical Relevance Summary Points gingivalis to become reestablished in

• Periodontitis is a microbe-induced the periodontal pocket by keeping the
inflammatory disease that affects the gingiva healthy with a minimum of
tooth-supporting tissues. inflammation.
• A shift from a symbiotic microbiome to
dysbiosis develops concomitantly with
disease progression.
• A number of microorganisms have been References
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Active Matrix Metalloproteinase-8:
Contributor to Periodontitis and
5
a Missing Link Between Genetics,
Dentistry, and Medicine
Timo Sorsa, Anna Maria Heikkinen,
Jussi Leppilahti, Taina Tervahartiala,
Solomon Nwhator, Nilminie Rathnayake,
Päivi Mäntylä, Dirk-Rolf Gieselmann,
and Lutz Netuschil
5.1 Introduction fibroblasts, cementoblasts, and epithelial cells,

bone cells) triggered to express and release pro-
Periodontitis is one of the most common inflammatory cytokines, reactive oxygen species,
infection-induced inflammatory tissue destruc- and matrix metalloproteinases (MMPs) [2–5].
tive diseases globally. According to the epidemi- MMPs are genetically distinct but structurally
ological studies in Western countries, about 30% related proteinases that can degrade not only
of populations are affected by periodontitis [1]. almost all extracellular matrix proteins but also
The pathogenesis of periodontitis can be briefly non-matrix bioactive molecules such as growth
described and summarized as follows: the dental factors, serpins, insulin receptor, apolipoprotein-
plaque, a bacterial biofilm, induces an inflamma- 1, complement components, and pro- and anti-
tory and immune responses in the adjacent gingi- inflammatory cytokines and chemokines [2–5].
val and periodontal or peri-implant tissues. The Thus, MMPs can modify immune responses
cells of the immune and inflammatory system are [2–5]. The active form of catalytically competent
together with resident gingival cells (including matrix metalloproteinase-8 (aMMP-8; neutrophil
T. Sorsa (*) S. Nwhator

Department of Oral and Maxillofacial Diseases, Department of Preventive and Community Dentistry,
University of Helsinki and Helsinki University Faculty of Dentistry College of Health Sciences,
Central Hospital, Helsinki, Finland Obafemi Awolowo University, Ile-Ife, Nigeria
Division of Periodontology, Department of Dental N. Rathnayake

Medicine Karolinska Institutet, Division of Periodontology, Department of Dental
Huddinge, Sweden Medicine Karolinska Institutet, Huddinge, Sweden
e-mail: [email protected] D.-R. Gieselmann
Dentognostics, Jena, Germany
A.M. Heikkinen • J. Leppilahti • T. Tervahartiala
P. Mäntylä L. Netuschil
Department of Oral and Maxillofacial Diseases, Department of Periodontology, Faculty of Medicine
University of Helsinki and Helsinki University Carl Gustav Carus, Technische Universität Dresden,
Central Hospital, Helsinki, Finland Dresden, Germany

DOI 10.1007/978-3-319-53737-5_5
52 T. Sorsa et al.
collagenase or collagenase-2) is the predomi- after scaling/root planning (SRP), cleaning/depu-

nant MMP in periodontitis-affected gingiva, ration, and adjunctive medications, i.e., host-
gingival crevicular fluid (GCF), peri-implant sul- modulating subantimicrobial dose doxycycline
cular fluid (PISF), saliva, and mouthrinse [2–5]. or antimicrobial drugs [2–5].
MMP-8 cleaves preferably and efficiently the Consequently, aMMP-8 can be utilized as a
interstitial collagens, mainly type I fibers of the diagnostic biomarker for tissue-destructive and
gingival and periodontal tissues, leading to irre- progressive periodontal and peri-implant inflam-
versible soft and hard tissue destruction, i.e., the mation related to active periodotal degeneration
development of periodontal pockets and attach- (APD), especially reflecting and indicating sur-
ment loss, and eventually leading to tooth loss rogate and pathologically excessive collagenoly-
[2–5]. Physiological levels of MMP-8 in peri- sis [2–5]. These outcomes (i)–(iv) of aMMP-8
odontal tissue also participate to protective and have not only been evidenced in GCF and PISF
anti-inflammatory resolution of infection-induced but also in saliva and mouthrinse [2–17].
tissue destruction [6, 7].
aMMP-8 is released mainly by degranulating
neutrophils and lesser extent by resident non- 5.2 CF Monitoring of
G
neutrophil lineage mesenchymal cells (fibro- Periodontitis by aMMP-8
blasts, epithelial cells, endothelial cells, plasma
cells, cementoblasts, bone cells, etc.) present in Furthermore, Lee et al. [12] have shown that
the the gingival and periodontal as well as peri- the GCF collagenolytic activity of MMP-8 is a
implant tissues of the GCF, PISF, saliva, and prognostic parameter; “active collagenase
mouthrinse [2–5]. Significant and increasing activity” was found to be sixfold elevated in
amount of international publications have repeat- cases with progressive loss of connective tis-
edly and consistently evidenced that the levels of sues [2–5]. In addition, Leppilahti et al. [13–
aMMP-8 in GCF and PISF correlate with the 15] have recently and repeatedly demonstrated
pathology of the periodontium, i.e., with health, using MMP-8 immunofluorometric assay
gingivitis, and periodontitis as well as peri- (IFMA) that GCF aMMP-8 can predict peri-
implant mucositis and peri-implantitis [2–5]. odontal treatment and medication outcomes site
Moreover, the outcome and reflection of success- specifically (Fig. 5.1) [13–15]. Sorsa et al. [10]
ful periodontal treatment can conveniently be were able to differentiate between “stable sites”
monitored by oral fluid (GCF, PISF, saliva, and without periodontal breakdown combined with
mouthrinse) aMMP-8 analysis [2–5]. Overall, low GCF aMMP-8 levels and “unstable sites”
these publications demonstrate clearly that (i) showing tissue breakdown as well as concomi-
oral fluid (GCF, PISF, saliva, and mouthrinse) tant pathologically elevated GCF aMMP-8.
aMMP-8 values are low in case of a healthy gin- Overall, aMMP-8 in GCF has been shown to be
giva and peri-implant tissue, (ii) are more low in periodontal health and increase with
increased in gingivitis and peri-mucositis, (iii) advancing and ongoing periodontal inflamma-
are most increased and pathologically elevated in tion and high oral inflammatory burden associ-
the untreated periodontitis and peri-implantitis, ated with APD [5]. These GCF aMMP-8 studies
and (iv), in periodontitis and peri-implantitis, have been described more in details by recent
reduce to lower values after successful periodon- reviews [2–5].
tal and peri-implant treatments, for example,
5 Active Matrix Metalloproteinase-8 53
GCF MMP-8 level Periodontal site level MMP-8 diagnostics
Active disease Monitoring during MMP-8 levels High risk of

the maintenance continuously compromised outcome
at baseline period above the cutoff LR: 4.5 (2.1-10)*
Treatment prognosis
at baseline
MMP-8 above the

cutoff High-
responding
Moderate risk of pattern
compromised outcome
LR: 1.5 (1.1-2)*
Low-
responding
pattern
Quiescent
period of Range of
desease physiological
at baseline fluctuation
Time
Baseline Maintenance period
Fig. 5.1 *LR = likelihood ratios (95% CI). Presented [13, 14]. Reproduction published with permission of
likelihood ratios are combined measures of both smokers American Academy of Periodontology
and nonsmokers reported originally by Leppilahti et al.
5.3 aliva and Mouthrinse

S show the history of the already existing or expe-
Monitoring of Periodontitis rienced periodontal pockets and attachment loss
by aMMP-8 [17] and cannot be conveniently used as prognos-
tic factors. Moreover, after successful periodon-
aMMP-8 quantifications in addition to GCF and tal treatment, the periodontal pockets can still
PISF have also been made from salivary or in exist, but they show significantly reduced
mouthrinse samples [5]. Various international aMMP-8 concentrations [3–16]. Bleeding on
research groups have published unequivocally probing (BOP) as well is known to be relevant
and repeatedly that salivary and mouthrinse parameter in some way to predict further peri-
aMMP-8 is significantly elevated in periodonti- odontal tissue loss [18, 19]. Absence of BOP is
tis, even in adolescents with initial periodontitis, regarded to indicate and reflect periodontal health
patients relative to the healthy controls [2–7, 11]. [18, 19]. With this background, we describe the
Also a relationship between salivary aMMP-8 development of the straightforward chairside and
concentration and the oral inflammatory status quantitative aMMP-8 oral fluid assay technolo-
and burden, i.e., active periodotal degeneration gies to be utilized as periodontal and peri-implant
(APD), has been established [5–7, 11, 12, 16]. diagnostic tools to distinguish conveniently peri-
No clinical standard exists for a direct com- odontal and peri-implant health and disease [2–
parison of aMMP-8 data with conventional peri- 16]. Overall, these salivary and mouthrinse
odontal parameters. Probing pocket depth (PPD), aMMP-8 studies have been described in detail in
clinical attachment level (CAL), and X-rays only recent papers [2–17].
54 T. Sorsa et al.
5.4 Chairside Monitoring phase of periodontitis, i.e., active periodotal degen-

of Periodontitis by aMMP-8 eration (APD), and peri- implantitis sites and
patients at risk and intervene therapeutically before
Diagnostic parameters like PPD and CAL, as the periodontal and peri-implant tissues show fur-
usually utilized by clinical periodontitis diagnos- ther breakdown, i.e., active periodotal degeneration
tics, measure mainly the attachment loss that has (APD) [5, 16, 20] (Fig. 5.2, Table 5.1). Thus, oral
already occurred [18, 19]. Until now, no prognos- fluid PoC-aMMP-8 chairside test can be utilized in
tic or predictive test and especially no chairside/ a preventive manner identifying elevated initial and
point-of-care test exists that could predict a future early periodontitis risk, i.e., development of APD,
progressive phase during the course of periodon- and during maintenance [5, 16, 20–22] (Table 5.1,
tal disease [2–5, 8–16]. Fig. 5.2). The PerioSafe® aMMP-8 mouthrinse test
There have been attempts to develop chairside has been introduced and validated independently in
systems to be utilized as adjunctive for the quan- Nigeria, Finland, the USA, Turkey, Holland, and
tification to aMMP-8 from GCF, PISF, saliva, and Germany and repeated and consistently found to
mouthrinse. In this regard, it has been possible to exert excellent performance to differentiate peri-
measure and diagnose aMMP-8 to GCF, PISF, odontal health and disease (Table 5.1) [5, 16, 20–
saliva, mouthrinse, and/or serum a laboratory test 22]. The aMMP-8 test can be used alone and/or in
via an aMMP-8-specific immunofluorometric combination with other potential biomarkers such
assay (IFMA) or ELISA [2–5]. In fact, such sys- as interleukin-1β (IL-1β) and Porphyromonas gin-
tem has recently been commercialized to be used givalis especially in epidemiological studies [23,
as a diagnostic aid for the dental and medical pro- 24].
fessionals [4, 5, 16, 20–22]. The mouthrinse collection is a noninvasive
Recently lateral-flow point-of-care (PoC)/ approach and not causing any discomfort for the
chairside tests (PerioSafe®, ImplantSafe®), dis- patient. Due to the simple testing (5–7 min) not
covered in Finland and further developed in necessarily need for dental equipment for knowl-
Germany, have been developed based on the edge exists (Fig. 5.2, Table 5.1) [5, 16, 20–22]. It
before-mentioned technologies and monoclonal is performed with purified water that does not
antibodies [4, 16]. The immunoassay test harm the patient. All further and additional pro-
(PerioSafe®) can be administered to patients cessing, analysis, and measurements of potential
according to the manufacturer’s instructions, and other diagnostic components are performed in
which include a 30 s prerinse with tap water, fol- the laboratory and do not represent an additive
lowed by a 30 s wait, and then rinsing with the burden or discomfort for the patient. The
test liquid (aqua purificata) for 30 s, and then PerioSafe® aMMP-8 test has recently been recom-
patients pour the mouthrinse into the a little col- mended by the German Society of Periodontology
lection cup accompanied by the test kit. Three and German Society of Oral and Maxillofacial
milliliters of the rinse is to be drawn up into a Surgery.
syringe, and then a filter is placed on the syringe It can be concluded that no extra risks exist or
through which a maximum of four drops are troubles when the diagnostic tests are performed
placed in a lateral-flow immunoassay system (Fig. 5.2) [5, 16, 20–22]. Due to the prognostic
which showed one single blue line for negative and predictive chairside aMMP-8 test, patients
results (no risk) and two blue lines for positive would benefit from early, fast, exact/precise, con-
results (increased risk). The test resembles a venient, and inexpensive disease diagnosis and
pregnancy test [2, 5]. The result was read as a detection of current and/or future disease pro-
color change within 5–6 min. Even a thin second gression [5, 16, 20–22]. Finally, this approach
line indicates increased risk for periodontitis and would indicate an enough early therapeutic inter-
peri-implantitis (Fig. 5.2) [5, 16, 20–22]. vention according to Axelsson and Lindhe [23]
Due to the positive results with the chairside and or Reinhardt et al. [24] before an irreversible
test, the clinician can identify and predict an active tissue destruction or APD occurs, respectively
> 25 ng/ml < 25 ng/ml
Fig. 5.2 Equipment of PerioSafe® aMMP-8 immunologi- that tested patient is positive and at risk for periodontitis.
cal chairside/point-of-care lateral-flow test, including When one line is detected (B), patient is negative and
syringe with filter, test liquid (aqua purificata), lateral- under control and out of periodontitis risk as well as under
flow immunoassay sticks, test liquid/mouthrinse collec- good maintenance. Arrows indicate the lines resulting
tion cup. When aMMP-8 in the collected mouthrinse is from immunoreactions. In fact, the test resembles closely
>25 ng/mL (cutoff), two lines are detected (A) indicating a typical pregnancy test [2–5]
Table 5.1 The sensitivity and specificity of the PerioSafe® aMMP-8 chairside test according to at least one, two, or
more than two ≥4-mm-deep pockets and at least one caries lesion n Finnish adolescents. Original published by
Heikkinen et al. [16]. Reproduction published with permission of American Academy of Periodontology
At least one At least two More than two At least one caries
Clinical parameters ≥4-mm-deep pockets ≥4-mm-deep pockets ≥4-mm-deep pockets lesion
The sensitivity of 48.3% 63.6% 76.5% 76.5%
aMMP-8 chairside test
The specificity of 100% 100% 96.8% 96.8%
aMMP-8 chairside test
p-value <0.0001 <0.0001 <0.0001 <0.001
False positive 0 0 1 5
[25, 26]. When the test is negative, the patient is Undiagnosed and untreated APD, i.e., active
under control and out of risk (Fig. 5.2, Table 5.1). progressive periodontitis, gradually leads to miss-
The aMMP-8 test (PerioSafe®) can be utilized in ing teeth and tooth loss mainly by the uncontrolled
diagnostics and maintenance and eventually also proteolytic action of aMMP-8 [2–5]. Missing
in self-diagnostics [5, 6, 20–22]. teeth or tooth loss has been repeatedly regarded
56 T. Sorsa et al.
as surrogated markers of experimented or ongo-

ing periodontitis. Significant causation has been MMP-8 (aMMP-8) is important and useful
repeatedly presented between missing teeth and in these regards. aMMP-8 can be utilized
circulatory mortality as well as all-cause mortal- alone or together with other potential bio-
ity. In fact, missing teeth predict incident cardio- markers such as interleukin-1β (IL-1β) and
vascular events, diabetes, and death [27]. Porphyromonas gingivalis to calculate
Regarding interdisciplinary medical and dental MMP-8 cumulative risk score (CRS) of the
cooperation, MMP-8 PerioSafe® test eventually subject level as a successful diagnostic
represents the missing link between genetics, tool, especially in large-scale public and/or
medicine, and dentistry [4, 5, 22, 28]. Thus, with epidemiological health surveys, in which
PerioSafe®, a MMP-8 test medical professionals the complete periodontal examination is
(gynecologists, rheumatologists, diabetologists, not always possible to conduct. PerioSafe
cardiologists, surgeons, nurses, and other experts) test can identify genetically predisposed
can detect and identify in 5–6 min the patient adolescents for initial/early periodontitis
exposed in the long run to fatal systemic risks and thus eventually act as a “gene” test.
from APD and/or peri-implantitis [4, 5, 22]. Inexpensive, easy/practical to use, quick
Patients with repeatedly elevated levels of oral (5–7 min), predictive chairside oral fluid
fluid aMMP-8, >25 ng/mL, thus PerioSafe® posi- PoC aMMP-8 tests, such as PerioSafe® and
tive, as indicated by the two lines (Fig. 5.2), ImplantSafe®, are currently commercially
should be referred to periodontist or dentist/ available for the routine use by dental and
hygienist for treatments/interventions [2, 26] to medical professionals in fact linking these
reduce the oral fluid aMMP-8 levels to be and stay disciplines; even the self-test done by the
<25 ng/mL, PerioSafe®, thus negative, as indi- patients is possible.
cated by one line (Fig. 5.2). Furthermore,
PerioSafe® identifies genetically predisposed ado-
lescents for initial/early periodontitis [28]. Thus,
it can also be regarded as a “gene test” [28]. References
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Inflammatory Pathways of Bone
Resorption in Periodontitis
6
Franco Cavalla, Claudia C. Biguetti,
Thiago P. Garlet, Ana Paula F. Trombone,
and Gustavo P. Garlet
6.1 Introduction determine the best course of action and to pro-

vide the best possible care. In spite of the aston-
The most common periodontal diseases in ishing amount of time and resources devoted to
humans are periodontitis and gingivitis. These unveil the intricate details of periodontitis’ patho-
diseases share dental biofilm as their etiologic genesis invested in the last four decades, we are
factor and periodontal inflammation as their main still far from a comprehensive explanatory model,
feature. Although the diagnosis, treatment, and and, more importantly, of new therapeutic tools
prognosis of the vast majority of the periodontitis to take better care of the most susceptible patients.
cases pose no special challenges to the trained That said, new developments in the field of osteo-
periodontist, there are extreme clinical pheno- immunology that will be discussed later in this
types at both ends of the susceptibility chain that chapter provide us with an appealing pathway to
are disturbingly difficult to identify with the clas- follow in our search for novel diagnostic tools,
sical periodontal diagnose tools. When a patient therapies, and clinical interventions.
presents a differential clinical phenotype, partic-
ularly of extreme susceptibility to alveolar bone
destruction, the periodontist often found himself/ 6.2 eneralities of Inflammation
G
herself lacking of the conceptual framework to
in Periodontitis
Pathogenesis
F. Cavalla
Periodontitis’ hallmark feature is the inflamma-
OSTEOimmunolgy Lab, Department of Biological
Sciences, School of Dentistry of Bauru, University of tory resorption of tooth-supporting alveolar bone
Sao Paulo (FOB/USP), Bauru, Brazil due to uncontrolled host response to periodontal
Departamento de Odontología Conservadora, infection. Even though the periodontal infection
Facultad de Odontología de la, Universidad de Chile, is essential to trigger the host’s immune and
Santiago, Chile inflammatory response, the destructive events
C.C. Biguetti • T.P. Garlet • G.P. Garlet (*) that lead to the irreversible disease phenotype of
OSTEOimmunolgy Lab, Department of Biological periodontitis are the result of the uncoupling of
Sciences, School of Dentistry of Bauru, University of
the soft and mineralized tissue turnover mecha-
Sao Paulo (FOB/USP), Bauru, Brazil
e-mail: [email protected] nisms caused by the persistence of a chronic and
exacerbated inflammatory immune response.
A.P.F. Trombone
Universidade do Sagrado Coração (USC), Although the primary role of the immune sys-
Bauru, Brazil tem is to provide the defense of the host against

DOI 10.1007/978-3-319-53737-5_6
60 F. Cavalla et al.
potentially hazardous microorganisms, the per- but we are still only scraping the surface of the
sistence of immune responses due to their inabil- pathogenic mechanisms that tilt the balance from
ity to eliminate the microbial antigens causes the health to disease in the periodontal environment.
alterations in the tissue’s metabolism that eventu- From a clinician’s perspective, the understand-
ally results in its irreversible destruction. The ing of the pathogenic mechanisms of periodontal
anatomic particularities of periodontium and the disease is an invaluable asset in the establishment
capacity of periodontal bacteria to form a biofilm of a successful treatment plan, general and indi-
structure favor the establishment of a long-last- vidual teeth prognosis, and follow-up strategies.
ing and non-resolving chronic inflammation that In addition, the knowledge of the general mecha-
acquires a destructive nature. The bacterial bio- nisms of periodontitis’ pathogenesis helps the
film adheres to the tooth surface and confers a clinician to identify putative susceptible subjects
series of advantages to the microbes, including and implement individualized therapeutic mea-
the isolation and protection from host defensive sures to better their long-term prognosis. Least,
mechanism, ultimately impairing the eradication but not less important, is the perspective of the
of the infectious focus. development of new therapeutic approaches using
It is noteworthy that in periodontal health the the knowledge gained from the applied research,
immune and inflammatory response is still pres- which include the appealing possibility of using
ent. While the exact nature of the host response immune modulatory drugs. These drugs will be
associated with periodontal health remains to be specifically targeted to the periodontal tissues and
established, such state is considered an equilib- aimed to abolish the deleterious effects of inflam-
rium state where a low intensity response is mation in the alveolar bone without dampening
enough to limit the invasion and multiplication of the ability of the host to deal with potentially
periodontal microorganisms without inflicting hazardous periodontal microorganisms. As pre-
damage to the host tissues as a collateral damage. viously stated, although periodontitis is an infec-
This balance is finely tuned and dependent on a tion, the pathologic tissue changes characteristic
series of active modulatory processes, which can of the disease (particularly bone resorption) are a
be tilted in each direction (exacerbated suppres- consequence of the sustained inflammation of the
sion or activation) by intrinsic or environmental periodontal tissues.
factors. The active suppression of the immune Inflammation can be defined as a protective
response, without complete abolition of its infec- response intended to eliminate injurious stimuli
tion control mechanisms, is supposed to be the that causes tissue damage, as well as removing
cornerstone in the maintenance of stable healthy the injured tissue resulting from the original
periodontal tissues. insult. Inflammation can be triggered by physical
In this scenario, this chapter focuses in the role or chemical trauma or by foreign bodies, includ-
of the inflammatory and immunological events that ing microbes. Even though inflammation is
lead to the pathologic destruction of the alveolar intended to eliminate harmful stimuli, the inflam-
bone, and consequently of the periodontal attach- matory reaction has the potential to cause wide-
ment during the progression of periodontitis. spread tissue damage, since the same mechanisms
intended to kill infecting microbes and clear the
tissue from injured and dead cells have the poten-
6.2.1 F
rom Health to Inflammatory tial to damage normal tissue.
Immune Response: How It is fundamental to understand that the distinc-
Homeostasis Evolves to tion between inflammation and immune response
Disease is only didactic and that both processes not only
occur simultaneously, but also are functionally
After more than four decades of incessant research integrated. Indeed, the main purpose of inflamma-
in the immune aspects of the pathogenesis of tion is to facilitate the access of immune cells and
periodontitis many lessons have been learned, defense molecules from the circulation to the
6 Inflammatory Pathways of Bone Resorption in Periodontitis 61
damaged or infected tissues. Thus, most inflam- Conceptually, inflammation is categorized in

matory events are related to vascular and perivas- two types: acute and chronic. Acute inflammation
cular events, including increased blood flow, is by definition a short duration response, ranging
alterations on the vasculature to facilitate immune from minutes to a few days, and it is character-
cells transmigration, and increased extracellular ized by profuse exudate and the predominance of
matrix metabolism. These molecular and cellular neutrophilic leukocyte infiltration. With the per-
processes translate into the clinical signs of sistence of the inflammatory stimuli, acute
inflammation (a.k.a. “cardinal signs”), as classi- inflammation evolves to chronic inflammation is
cally described by Rudolph Carl Virchow in the a long-lasting response, ranging from days to
late nineteenth century: redness, heat, swelling, years, characterized by the tissue infiltration of
pain, and loss of function. In the particular case of mixed populations of leukocytes and monocytes/
periodontal diseases, these first inflammatory and macrophages following specific chemotactic gra-
immune phenomena translate in the clinical entity dients, and tissue degeneration including vascu-
known as gingivitis. lar proliferation, parenchymal involution, and
In the sequence, we will approach inflamma- fibrosis [7]. The events of acute and chronic
tion and immune response as intermingled and inflammation are analog and complementary to
mutually dependent occurrences in order to bet- events of innate and adaptive response. The first
ter picture the actual cellular and molecular phe- vascular and molecular occurrences of inflamma-
nomena that lie beneath the clinical signs and tion favor the migration of the cell populations’
symptoms of periodontitis, which ultimately characteristic of the innate immunity, namely
explain the pathologic irreversible destruction of neutrophils and macrophages, which possess
alveolar bone. We will focus in bone destruction, very effective but unspecific microbe clearance
since it is the defining occurrence of periodonti- machineries. When innate immune responses
tis, even though the same inflammatory immune cannot clear the invading microorganisms, the
events depicted in this chapter could be consid- inflammation becomes chronic and the character-
ered as responsible for the destruction of the istic effector cells of the adaptive immune
remaining of the tooth-support apparatus. response invade the tissue, predominantly acti-
In normal healthy conditions, the gingival tis- vated lymphocytes with highly selective and spe-
sues are capable of coping with the presence of cific microbe killing capabilities [2, 8]. As in any
bacteria due to various innate immune defense biological process, these rigid definitions do not
mechanisms, among them: the flushing action of capture the changing dynamics of inflammation
gingival crevicular fluid and saliva, the rapid epi- and immune response, where long-lasting chronic
thelial turnover, the secretion of immune active inflammatory processes are periodically broken
peptides, a permanent influx of innate immune up by acute inflammatory bursts [9, 10].
response cells to the periodontal tissue and the The inflammatory immune response is trig-
transmigration of neutrophils into the sulcus, and gered by the interaction of resident cells with the
the action of saliva agglutinins and antibodies, bacterial biofilm attached to the tooth surface. The
which are supposed to limit the potential of the particularities of the periodontal anatomy and
bacterial biofilm to grow and invade [1–4]. Indeed, periodontal tissue architecture are intimately asso-
in a distinctive fashion in comparison with other ciated with the natural course and p athophysiology
body tissues, in the healthy periodontium exist a of the inflammatory processes that leads to peri-
constant subclinical inflammation. When the bal- odontitis [5, 11]. Bacterial biofilm attaches to the
ance between the infection control mechanism and tooth surface, making impossible for the immune
the subgingival biofilm is lost, the mandatory first system to eradicate the infecting microorganisms
step into the pathologic chain of events leading to efficiently. The attached biofilm acts as a perma-
periodontitis is gingival inflammation, an acute nent reservoir of microorganism and their prod-
inflammatory response of the gingiva to the micro- ucts, perpetuating the insult to the periodontal
bial insult that is clinically evident [5, 6]. tissues [12].
The junctional epithelium is the first peri- family (TLR), composed of at least nine isoforms
odontal structure to face the bacterial challenge. named TLR1 to TLR9. The signaling pathway is
This is a highly specialized and unique tissue, initiated by the binding of the pathogen-derived
characterized by the capability to attach directly ligands to membrane-bounded TLRs, leading to
to the mineralized surface of the tooth by the for- the dimerization of the receptor. The dimeriza-
mation of an extremely organized structure tion of the TLRs triggers the recruitment of vari-
known as the internal base membrane. The junc- ous protein kinases in the cytoplasmic end of the
tional epithelium is more permeable than most receptors, ultimately causing the activation of
epitheliums, mainly because of the incomplete proinflammatory transcription factors (such as
maturation of its supra basal layer, by its unusu- NFκB and AP-1) [22, 23].
ally high proliferation rate, by the relatively Following the initial stimuli, classic inflam-
scarce presence of desmosome junctions between matory pathways are deflagrated, where the
cells, and by the absence of keratohyalin [13, 14]. cyclooxygenase pathway oxygenates arachidonic
After surpassing the epithelial barrier, infecting acid producing a variety of proinflammatory
microorganisms gain access to the subjacent gin- molecules, such as prostaglandin E-2 (PGE-2),
gival connective tissue, being gingival fibroblasts prostacyclin, and leukotriene A-4. The enzyme
the predominant cell type in this compartment. cyclooxygenase 1 (COX-1) is constitutively
Despite the fact that fibroblast are not profes- expressed, while COX-2 is induced under proin-
sional immune cells and have a limited output of flammatory conditions and responsible for the
signaling molecules available, such cell type can amplification of the inflammation [7, 24]. Several
be also active in the triggering of initial inflam- in vivo studies have demonstrated that the inhibi-
matory events [15]. tion of COX-2 activity significantly reduces the
Extensive evidence indicates that the first alveolar bone loss in experimental periodontitis
immune modulatory events in this process are [25, 26]. In parallel with the activation of classic
orchestrated by the keratinocytes of the junc- mediators of the vascular events of inflammation,
tional epithelia and fibroblast of the periodontal TLR recognition of microbial ligands results in
connective tissue [16–20]. These cells are capa- the secretion of important molecular mediators of
ble of “sensing” the environment and differen- inflammation, including inflammatory cytokines
tially react to diverse bacterial antigens and other (such as TNFα and IL-1β) and chemokines (such
signals, responding with the secretion of media- as CXCL8/IL-8, CCL2, CCL3, and CCL5),
tors that will mediate the vascular and cellular which along the mediators that target the blood
events of inflammation. Indeed, the resident peri- vessels, will orchestrate the inflammatory cell
odontal cells (epithelial cells and gingival fibro- influx into the inflaming tissue [1, 27].
blast) directly interact with the microbes or its At this step, to make room for the profuse
products produce and secrete molecular signals inflammatory cell infiltration the perivascular
to trigger inflammation and chemoattract immune extracellular matrix is degraded by enzymes such
cells [14, 15]. The host cells recognize the as matrix metalloproteinases (MMPs), which also
microbes by the interaction of pathogen- will degrade the extracellular matrix along the
associated molecular patterns (PAMPs) with way of the cells from the vessel until the inflam-
PAMP-receptors constitutively expressed in the matory focus, being this way determined by gra-
cell membrane of most cells. Those PAMPs are dients of chemoattractant molecules [4, 28].
signature molecules preferentially associated In this environment, it is mandatory to con-
with pathogens and absent from host’s cells, such sider that gingival fibroblasts are responsible
as lipid polysaccharide (LPS), a main component for the periodontal tissue turnover, doubling as
of Gram-negative cell wall, and lipoteichoic acid, the main source of collagen fibers and of MMPs
a major constituent of the cell wall of Gram- within the connective tissue. However, under
positive bacteria [21]. The archetypal membrane- the influence of the copious inflammatory sig-
bound PAMP-receptors are the Toll-like receptor nals, gingival fibroblast and periodontal liga-
ment fibroblasts are directly responsible for the activated by the interaction of their own TLRs
destruction and disorganization of the fibrous with PAMPs. After their activation, the neutro-
component of the extracellular matrix of peri- phils produce and secrete molecular mediators to
odontal tissue by increasing the local production amplify the inflammation; also, their phagocytic
and activity of MMPs [29]. While the extracellu- activity is enhanced, as well as the production of
lar matrix degradation has a physiological value, reactive oxygen species (ROS) used to kill the
since it is necessary to facilitate the transmigra- phagocyted microorganism. These ROS affect
tion and permanence of immune cells within the the oxidative status of the periodontal tissue,
tissue during the immune response, the problem activating and sustaining a pervasive inflamma-
arises when the inflammatory/immune response tory response that amplifies the initial signaling,
turn out to be indefinite and the tissue homeo- and when uncontrolled promotes the spreading
stasis becomes permanently affected, leading to of the inflammation to the surrounding tissues
the pathological irreversible destruction of teeth- [15, 35, 36].
supportive tissues. While neutrophils exert a very important anti-
At this stage, the first clinical signs of gingi- infective role in the periodontal environment, it is
vitis become evident. Namely, redness, swelling also important to consider that most of the peri-
and loss of texture of the free gingiva, provoked odontal microorganisms have evolved complex
bleeding (at the clinical examination with a blunt and elegant strategies of immune evasion and
instrument or during normal oral hygiene pro- have the capacity to use the immune/inflamma-
cedures), and eventual suppuration. When the tory response to their own benefit, modulating the
inflammation persists, the disease progresses into periodontal microenvironment to adjust it to their
periodontitis, characterized by the widespread specific metabolic needs [37]. For example, the
destruction of the extracellular matrix and the recognized periodontopathogen Porphyromonas
collagenous fibers that compose the connec- gingivalis is capable of downregulating the initial
tive attachment apparatus, the apical migration immune response of periodontium resident cells,
of the junctional epithelium, and the pathologi- completely abolishing the secretion of the neutro-
cal resorption of the alveolar bone and radicu- phil-attractant chemokine IL-8/CXCL8 by a gin-
lar cementum. In the periodontal examination, gipain-dependent mechanism [19, 38], facilitating
these histopathological changes are evidenced as its survival and growth within the periodontal
increased probing depth usually accompanied by connective tissue. Interestingly, despite the initial
all the clinical signs of gingivitis [30]. dampening of inflammatory cell migration over
time will ultimately result in an exacerbated, but
inefficient in microbial control terms, host
6.2.2 T
he Parallels Between Acute response. Indeed, as previously mentioned, in the
Inflammation and Innate development of periodontitis, infecting microor-
Immune Response ganism resists eradication in part due to the par-
ticular anatomy of the periodontal sulcus/pocket
At the early host response phase, the predomi- and in part due to their proprietary virulence and
nant immune cell type in the periodontium is the evasion mechanism along the additional protec-
polymorph nuclear neutrophil [6, 31]. Even in tion conferred by the biofilm structure, eliciting a
healthy conditions, a stable influx of neutrophils sustained chronic inflammatory response.
(3 × 105 cells/min) permeates the connective tis- At this point, neutrophils may have or have
sue in transit to the sulcus though the junctional not succeeded in eliminating the agent that elic-
epithelium, attracted by a constant gradient of ited the inflammatory response. If not, additional
IL-8/CXCL8 permanently secreted from kerati- cell types with different antimicrobial strategies
nocytes and gingival fibroblast in response to the and weapons may be able to achieve the task, but
recognition of PAMPs by TLRs [32–34]. Once even if neutrophils are successful in eliminating
inside the tissue, neutrophil leukocytes become infecting agents, additional cell recruitment is
required to repair the damage (even if minimal) In summary, the acute inflammatory response
inflicted at the response site. To achieve this in the early stages of periodontitis development
objective, a special polarized subpopulation of begins when bacteria and their products gain
macrophages with regulatory and reparative access to the gingival connective tissue through-
functions may be recruited to the damaged tissue. out the junctional epithelium, whose loose inter-
This distinctive subpopulation of macrophages cellular junctions do not provide an impermeable
possesses the capacity to potentiate the repair, physical barrier. Proinflammatory molecular sig-
and in some instances, the fibrosis of the tissue as nals emanated from the epithelial and connective
required [39]. tissue trigger the first inflammatory events,
Additionally, monocytes also play a relevant increasing the blood flow and permeability of the
role during the early stages of the acute inflam- subepithelial gingival plexus and recruiting large
matory process and initial innate immune amounts of leukocytes to the site, particularly
response, and readily infiltrate the periodontal neutrophils and macrophages.
tissue in the first hours post infection [12, 40]. In the coming sections we will describe the
Monocytes are attracted from the circulation to chronic inflammatory events that later will be
the periodontal tissues following gradients of responsible for the bulk of the tissue destruction
chemokines, such as monocyte chemoattractant in periodontitis. Briefly, we can summarize the
protein 1 (MCP-1, a.k.a. CCL2), which is the process as a continued response driven by the
master regulator of monocyte/macrophage mobi- impossibility of eliminating the microbes exclu-
lization [41–43]. Some indirect evidence links sively with the recruitment of neutrophils and
the infiltration of macrophages into the periodon- macrophages. In turn, other leukocyte subsets are
tium with the severity of the periodontitis, since recruited and join the inflammatory infiltrate, and
MCP-1/CCL2 levels appear augmented in the the host response acquires a chronic nature that
gingival crevicular fluid of periodontitis patients will result in the dampening of the normal
and its levels seem to correlate with the severity homeostatic balance of the periodontal tissues.
of the disease [44].
The macrophages are able to phagocyte the
invading microorganism into phagocytic vesicles 6.2.3 Osteoimmunology
(a.k.a. phagosomes), but require additional cyto- and the Molecular Connection
kine signaling to promote the fusion of phago-
somes and lysosomes, where the microbicidal Indeed, when the response becomes chronic,
mechanisms take place to kill effectively the adaptive immune cells invade the tissue and the
ingested microbes. The principal molecular inflammatory reaction becomes firmly estab-
mechanisms of microbe killing and digestion are lished, flooding the periodontium with additional
the conversion of molecular oxygen into reactive bioactive proinflammatory molecular signals
oxygen species (ROS), the production of highly (cytokines, chemokines, enzymes, ROS, bacte-
reactive nitrogen species, such as nitric oxide rial products and metabolites, etc.) [1, 2, 4, 28].
(NO), and the action of several proteolytic The accumulation of these molecular signals in
enzymes [45, 46]. It is noteworthy that all these the tissue facilitates the spreading of the inflam-
microbicidal mechanisms, when exacerbated and mation to the underlying bone and tamper with
uncontrolled, are partially responsible for the the bone homeostasis signaling system, tilting
amplification of the inflammatory response and the balance of bone metabolism favoring resorp-
the degradation of the host’s tissues. Indeed, acti- tion over formation. More than four decades ago,
vated macrophages are an important cellular Page and Schroeder postulated that the presence
source of MMPs and greatly contribute to the of bacterial plaque closer than 2.5 mm from the
intensification of the degradation of the collage- alveolar bone could trigger its inflammatory
nous matrix in the connective periodontal tissue resorption [31, 49]. Nowadays we know that the
[47, 48]. pathological mechanism underlying this observa-
tion is the disruption of the balance of bone through calcium-dependent activation of the tran-
metabolism by inflammatory and immune molec- scription of NFATc1 gene [55]. The membrane-
ular mediators, which mark the transition from bound form is characteristically expressed in the
gingivitis to periodontitis. surface of osteoblasts (a.k.a. RANKL isoform 1
The understanding of the molecular basis of and 2) and the soluble form (a.k.a. RANKL iso-
the interplay between inflammatory immune form 3) is the secreted product of various cell
responses and the bone tissue is the keystone of types, including B-cells and T-cells [56, 57].
“osteoimmunology,” a new and evolving field RANKL is the master activator of osteoclasts
that studies the shared components and mecha- and the molecular signal directly responsible
nisms between the immune and bone systems. To for bone resorption. RANKL interacts with its
understand such connection, we must remember cognate receptor RANK in the surface of osteo-
that bone is a highly specialized mineralized con- clast and osteoclasts’ precursors, triggering their
nective tissue, characterized by constant renewal recruitment to the bone surface, cell fusion, and
dependent on the coupling of bone formative and activation. The secreted form of RANKL is the
resorptive processes. This dynamic behavior of molecular signal that couples immune response
bone enhances its adaptive capacity to functional and bone metabolism. Numerous animal model
demands and increases its healing potential after experiments have demonstrated that alveolar
an injury. The balance between bone resorption bone resorption could be prevented by the selec-
and apposition is governed by a unified molecu- tive inhibition of the RANKL/RANK axis [58,
lar signaling system and is dependent on the 59]. Osteoprotegerin (OPG) is a soluble protein
effector functions of specialized bone cells. upregulated in inflammatory conditions, with the
The modulatory processes leading to bone capacity to block RANKL’s biological functions
resorption in periodontitis are very complex and by competitive inhibition, acting as a decoy recep-
intricate, and will be described along this entire tor, limiting the availability of RANKL able to
chapter, including the interaction of different spe- bind to RANK. The quotient or ratio of RANKL
cies of bacteria and their subproducts with ele- to OPG determines if at any given moment the
ments of the innate and adaptive immune systems conditions are favorable for bone apposition or
[50–52]. Fortunately, despite the astonishing bone resorption. A high ratio of RANKL/OPG
complexity of the input signals, the bone has a creates the conditions favorable to bone resorp-
very straightforward transduction system and a tion, while a low RANKL/OPG ratio favors bone
limited number of molecular signals and cells are apposition [60, 61]. During bacteria-induced
directly involved in controlling the balance inflammation in experimental periodontitis, it has
between bone apposition and bone resorption. been demonstrated a net increase in the RANKL/
This is a common finding among complex bio- OPG ratio, leading to osteoclast genesis and bone
logical systems, which are capable of “sensing” a resorption [62]. Analog evidence is also available
vast variety of inputs, but where the effector in human periodontitis [28], and in the closely
mechanisms are governed by a much more lim- related condition of pathologic bone resorption
ited number of “key effector signals.” In the spe- of periapical bone as a consequence of end-
cific case of alveolar bone, the system that odontic infection [63, 64]. Indeed, the key event
controls the bone metabolism balance comprises in the inflammatory alveolar bone resorption in
the RANKL/OPG/RANK triad, secreted and rec- periodontitis is the manifold increase in the tis-
ognized by the specialized bone effector cells sue levels of RANKL unaccompanied by an
osteoblasts and osteoclasts [53, 54]. equivalent increase in OPG levels. The resultant
RANKL (receptor activator of nuclear factor augmented RANKL/OPG ratio drives the recruit-
κB ligand) is a cytokine, member of the TNF fam- ment of osteoclast’s monocyte precursors, their
ily that can be membrane bounded or secreted, fusion, and later activation. The uncoupling of
and stimulates osteoclasts’ differentiation, cell bone metabolism due to the imbalance between
fusion, and activation leading to bone resorption bone apposition and resorption is the result of the
uncontrolled interplay of the immune system and adaptive immunity cells. Indeed, after the initial
the bone metabolism throughout their common acute inflammation and innate immune response
molecular mediators. had taken place, the selective migration of spe-
Recently, it was reported that RANKL levels cific T lymphocyte subsets drives the transition to
in gingival crevicular were increased fourfold in the adaptive immune response [1, 4]. These two
chronic periodontitis patients compared to processes are analogous and in a certain way cor-
healthy controls, while OPG levels were equiva- respondent to the acute and chronic phases of the
lent in both groups, causing an increased inflammatory response.
RANKL/OPG ratio in the patient’s group. In this second stage of the immune response,
Interestingly, RANKL/OPG ratios levels were T CD4+ lymphocytes (a.k.a. T helper or Th) play
unaffected by the periodontal treatment and a critical role in orchestrating the host’s response.
remained augmented for at least 6 weeks post- T helper lymphocytes secrete bioactive signaling
treatment, despite the notorious clinical improve- molecules, namely cytokines, as their main effec-
ment and normalization of inflammatory tor mechanism, fine-tuning almost every aspect
parameters of the subjects [65]. This could be the of the inflammatory/immune response.
reflection of a long-lasting unbalance of bone Depending on a series of environmental and
metabolism, spanning beyond the resolution of host’s intrinsic factors, Th lymphocytes could
the inflammation in the immediate posttreatment differentiate into distinct subtypes or lineages,
period. Alternatively, this could be the result of each one with a characteristic subset of cytokines
an inherent predisposition to increased secretion in its secretion pattern. The differential lineage
of RANKL, which could be the underlying commitment depends of the predominant cyto-
molecular basis for increased susceptibility to kine present in the environment during the pro-
bone resorption. cess of antigen presentation by antigen-presenting
cells (APC) in the regional lymph node. During
this process, APC capture antigens in the periph-
6.2.4 Chronic Inflammation eral tissues and migrate to the regional lymph
and Adaptive Immune node, where they encounter a naïve T cell with a
Response in Periodontitis specific receptor for the antigen. After the spe-
cific interaction of APC-T CD4+ cell, the naïve
The activation of adaptive immunity has a great lymphocyte becomes activated and committed,
influence in the bone loss associated with peri- suffers clonal expansion, and massively migrates
odontitis, since numerous evidence points to B into the peripheral tissue to perform their effector
and T lymphocytes as the main cellular sources functions. It is during this stage that the different
of soluble RANKL during periodontal inflamma- lineages of Th lymphocytes emerge and differen-
tion [66]. Experimental evidence from SCID tiate, dictating the curse of the following steps of
mice (Severe Combined Immune Deficient, lack- the disease [1, 10].
ing both T and B lymphocytes), demonstrated the The relative predominance of a subpopulation
importance of adaptive immunity effector cells in of Th lymphocytes over the rest determines that
bone loss in periodontitis. When SCID mice the immune response variates from a controlled
where challenged with Porphyromonas gingiva- self-contained process with low potential to
lis, they demonstrated significantly lesser bone destroy the periodontal tissues to an exaggerated
resorption than the control wild-type mice [67]. reaction with high destructive potential. The
As previously mentioned, with the impossibil- knowledge of the existence and regulatory func-
ity of clearing the insulting microbes by means of tions of the different Th subpopulations has been
the recruitment of neutrophils and macrophages, one of the most dramatic revolutions in the
other leukocyte subsets are subsequently enrolled immunology field of the past three decades and
to join the inflammatory infiltrate, and the host has affected profoundly our understanding of the
response acquires a chronic nature and mobilizes immune processes underling the clinical course
of periodontal disease beyond the classical pro- sion potential of periodontal pathogens.
versus anti-inflammatory perspective [1, 8, 68]. Experimental evidence in IFN-γ deficient mice
In the late eighties, the first two Th lympho- demonstrates that although they develop a less
cyte subtypes where described first in mice and severe phenotype of alveolar bone destruction
then in humans and named Th1 and Th2 [69, 70]. following the infection with Aggregatibacter
Th1 cells were characterized as responsible to actinomycetemcomitans, they are also more
mediate the immune responses against intracellu- prone to suffer widespread infection, with lethal
lar pathogens. Naïve Th cells committed to Th1 consequences in some cases. The IFN-γ deficient
lineage when the antigen presentation occurred in mice demonstrated reduced levels of many
an IL-12 enriched environment, and the stabiliza- inflammatory cytokines and chemokines, as well
tion of the Th1 phenotype was dependent on the as significantly reduced numbers of infiltrating
transcription of the key transcription factor T-bet macrophage and markers of macrophage activa-
[71]. The characteristic cytokine product of Th1 tion in the periodontal tissue [78].
committed lymphocytes is IFN-γ, which is the The Th1 lineage is also responsible for the
cytokine responsible for the classical pathway of secretion of various cytokines that support, main-
macrophage’s activation, and fundamentally tain, and amplify the inflammatory response,
involved in the stimulation of the eradication of directly or indirectly favoring the recruitment
phagocyted pathogens [72]. Conversely, Th naïve and permanence of immune cells within the peri-
cells committed to a Th2 phenotype when the odontal tissues. Both prototypical inflammatory
antigen presentation occurred in an IL-4 enriched cytokines, IL-1β and TNFα are characteristic
environment, being the stabilization of the pheno- secreted products of Th1 lymphocytes. TNFα
type dependent on the transcription of the key and IL-1β produce vasodilatation, stimulate the
transcription factor GATA-3. Th2 cells secrete activation of endothelial cells to increase immune
IL-4, IL-5, IL-9, IL-13, and IL-25, among others cell recruitment, increase the chemokine produc-
cytokines. The committed Th2 cells favor the tion in most cell types, participate in neutrophil
effector mechanisms involved in the eradication activation, and stimulate the secretion and tissue
of extracellular pathogens, mainly stimulating the activation of MMPs, among other functions.
secretion of antibodies by activated plasma cells Even though neither IL-1β nor TNFα is directly
[1, 73]. As in many biological processes, the lin- involved in the stimulation of bone resorption,
eage commitment to the Th1 or Th2 phenotypes is they indirectly favor the destruction of bone by
an excluding and self-amplifying event. Once a stimulating the sustained inflammation of peri-
naïve Th cell is committed to one of the subtypes, odontal tissue [79].
the secreted cytokines will serve as an autocrine Alternatively, some evidence supports the
stimulus to maintain the commitment, as a posi- hypothesis that periodontitis pathogenesis is more
tive feedback amplification signal, and as a para- related to the differentiation and effector functions
crine stimulus to prevent the commitment of other of the Th2 lymphocyte lineage. This idea is sup-
naïve cells to alternative lineages [74]. ported by the fact that Th lymphocytes isolated
The pivotal role of the Th1 subtype in the from inflamed gingival tissues predominantly pro-
establishment and progression of periodontitis duce IL-4 over IFN-γ after non-antigen-specific
was supported by extensive evidence indicating stimulation [80], and by the classical histological
increased levels of IFN-γ in the tissues of peri- description of the advanced lesion of periodontitis
odontitis patients [75] and corroborated by as a “B-cell type of lesion” [31].
in vitro evidence of the augment of IFN-γ protein Th2 lymphocytes are the main cellular source
levels and transcription during the progression of of the cytokine IL-4, doubling as an inducer of
experimental periodontitis [76, 77]. Th2 polarization, as well as an effector molecular
It is noteworthy that the role of the Th1 lin- signal. Among its more important functions, it
eage (and its signature cytokine product: IFN-γ) promotes the class switching to IgE secretion in
is of paramount importance in limiting the inva- B-cells, and favors the alternative activation of
macrophages in an IFN-γ-independent pathway. Simultaneous analysis of multiple cytokines

It is noteworthy that both effector functions limit in periapical osteolytic lesions demonstrated that
the capacity of the immune response to control a complex network of cytokines drives the evolu-
the periodontal infection, which is predominantly tion of the bone resorption (active lesion) or
mediated by intracellular pathogens [81, 82]. IgE arrest the progression of the lesion (inactive
is the isotype class of antibodies best suited to lesion). Characteristic Th1 and Th17 cytokine
combat large extracellular parasites (such as hel- products, such as TNF-α, IL-21, IL-17, and IFN-
minths) and in not pertinent to fight periodontal γ, appear strongly associated with the bone
pathogens. Further, the alternative activation of resorptive process, while the stabilization of the
macrophages inhibits their microbicide func- lesion seems associated with the expression of a
tions, suppresses the production of iNOS and different subset of cytokines: IL-10, IL-9, IL-4,
consequently of nitric oxide (NO), and stimulates and IL-22 [89]. The latter are among the signa-
the secretion of IL-10 and TGFβ, which in turn ture cytokine products of a distinct Th lineage
can downregulate pro-inflammatory and Th1 with immune suppressive properties, known as T
responses [83]. It is for these reasons that some regulatory cells (Tregs).
authors consider that the Th2 polarization in peri-
odontitis may represent an impaired adaptive
immune response, in which IL-4 inhibits the 6.2.5 A
daptive Immune Responses
more effective Th1 polarization. This may be due May be Also Protective: A Role
to inherent host characteristics, or may be also for Regulatory T Cells (Tregs)
caused by evasion strategies triggered by highly
evolved periodontopathic bacteria [84, 85]. Tregs are Th lymphocytes associated with the
In the beginning of the twenty-first century, a secretion of anti-inflammatory cytokines and
new subset of T helper cells characterized by the reparative molecular signals, such as IL-10 and
secretion of IL-17 was described, and named TGFβ. Their lineage commitment depends on the
Th17 accordingly. The polarization of these cells expression of the transcription factor FoxP3, and
was driven by IL-23 and was dependent on the they characteristically express the surface mark-
transcription of the transcription factor RORγT ers CTLA4, CD103, and CD45RO. The secretion
[86]. Along with IL-17, Th17 cells also secrete of IL-10 and the contact inhibition of lymphocyte
the cytokines IL-21 and IL-22. Shortly after the activation by the CTLA4 co-receptor are the sig-
discovery of Th17 cells, IL-17 was found to be nature effector mechanisms of Tregs, leading to
highly expressed in osteolytic lesions, such as an active suppression of the immune response
periodontitis and periapical lesions [87]. and a return to tissue homeostasis. Experimental
Cytokines such as IL-23, TGF-β, IL-17, IL-6, in vivo data associates the presence of Tregs with
and IL-1β are highly expressed in inflamed peri- the attenuation of osteolytic progression in peri-
odontal tissues, providing the necessary signals odontal lesions [90]. Recently, the mechanisms
to drive the polarization of Th cell to the Th17 responsible for Tregs’ migration to periodontal
lineage. Interestingly, secreted RANKL is a char- tissues were unraveled, being the IL-4/CCL22/
acteristic cytokine product of Th17 cells, which CCR4 axis responsible for the chemoattraction of
in cooperation with its other proinflammatory these cells to the periodontium. The mechanism
cytokine products are capable of tilting the bone can be mimicked by the injection of CCL22-
metabolism favoring resorption over apposition. releasing particles, which result in a therapeutic
Additionally, Th17 cells provide the necessary arrest of bone resorptive activity [90–92].
proinflammatory signals to upregulate the expres- While the mechanisms of Tregs chemoattrac-
sion, secretion, and activation of MMPs, generat- tion have been discovered, the issue of their origin
ing an amplification loop of inflammatory and and generation remains an unsolved question.
pro-resorptive mediators [88]. Interestingly, Tregs have been described as c entral
elements in the determination of a host–microbe cytokines, endogenous pro-resolving lipid medi-

homeostasis in several tissues. ators (resolvins), and growth factors.
Recently, periodontitis has been characterized Resolvins are endogenous lipid mediators that
as a disease of dysbiosis, where the pathologic modulate cellular fate and inflammation. They
process is initiated by the disruption of the nor- are biosynthesized during the resolution phase
mally balanced equilibrium between the host and of acute inflammation, and are capable of induc-
the resident microbiome. In this context, some ing cessation of leukocyte recruitment, reversing
keystone pathogens can produce an alteration in of vascular inflammatory phenomena and cause
the microenvironment that is capable of turning prompt apoptosis of neutrophils [94].
commensal microorganisms into opportunistic Tregs and mesenchymal stem cells (MSC)
pathogens, triggering the disease. This framework play a central role in tissue repair, supporting tis-
requires that in healthy conditions the host must sue regeneration by direct control of undesired
be able to tolerate the presence of microorganism immune reactivity and by direct interaction with
without eliciting robust effector responses. This nonimmune tissue cells. Tregs can directly inter-
tolerance is an active process, where the host act with MSC progenitors, favoring their recruit-
recognizes harmless microbial antigens and sup- ment and differentiation in the required cell types
presses the development of exacerbate inflamma- for tissue regeneration [95]. Recent experimen-
tory immune responses against them to preserve tal evidence suggests that MSC and Tregs act in
the functional equilibrium. Tregs are at the center coaction, favoring the establishment of an anti-
of this active tolerance, inducing antigen-specific inflammatory environment that promotes the
hyporesponsiveness and suppressing the initia- repair of bone defects [96].
tion inflammation by a series of specific mecha- Therefore, the sequence of events of adaptive
nisms [8, 93]. immune response that leads to pathologic alveo-
Additionally, Tregs can also regulate the lar bone resorption can be summarized as fol-
duration and intensity of the immune response lows: after the acute inflammation is firmly
against pathogens, limiting the destructive poten- established, adaptive immune cells are recruited
tial of uncontrolled reactions. In this sense, it and infiltrate the periodontium, marking the tran-
is important to highlight that experimental evi- sition to chronic inflammation. Depending on a
dence demonstrates that Treg-mediated immune series of environmental factors and host’s intrin-
regulation does not interfere with the capacity of sic characteristic, different lineages of effector T
the immune response to fight the infection effi- cells might predominate inside the tissue, deter-
ciently, and that the directed recruitment of Tregs mining the clinical outcome of the disease. If
into diseased periodontal tissues is capable of proinflammatory subtypes predominate (Th1 and
restore the homeostatic tissue balance, even in Th17), the tissue destruction and bone resorption
the presence of an infectious burden that usually are favored; conversely, if the anti-inflammatory
results in the development of destructive peri- and pro-reparative lineage predominate (Tregs),
odontitis [90, 91]. These former properties trans- the inflammation is halted and the tissue tends to
form Tregs and their selective recruitment to the regenerate (Fig. 6.1).
periodontium in an attractive therapeutic tool to Consequently, the variation in the pattern of
modulate the immune response, without interfer- Th responses seems to be a critical determinant
ing with the infection control mechanisms while of periodontal bone loss, since such cell types not
avoiding irreversible tissue damage and promot- only regulate the overall immune response pat-
ing repair. tern but also may directly interfere in the
The tissue repair that occurs after the contrac- RANKL/OPG balance [1, 8]. However, we must
tion of immune response is an active process regu- consider that the inflammatory process is a con-
lated and orchestrated by the secretion of specific tinuum and frequent acute bursts of inflammation
molecular mediators, such as anti-inflammatory or variations in the pattern of host response occur,
Gingivitis Periodontitis
Soft tissue Bone Lesion arrest
Clinical signs Redness, Bleeding, Swelling destruction resorption repair
MMPs Rankl
Vascular events
Inflammation
PAMPs PRRs Inflammation Cell downregulation
LPS TLR immune response Chemokines migration Cytokines or resolution
Cellular events
Pro-inflammatory mediators Anti-inflammatory mediators
Fig. 6.1 Pathogenic pathway of periodontal diseases. The tial secretion of cytokines by specific subpopulations of
recognition of PAMPs by PRR triggers the first events of infiltrating leukocytes could lead to self-amplification or
inflammation, which leads to the recruitment of immune modulation of the immune/inflammatory response, deter-
cells to the periodontium. The clinical signs are the expres- mining the outcome of the disease
sion of the underlying inflammatory process. The differen-
even after the establishment of the chronic phase These contradictory clinical phenotypes could
of inflammation. Indeed, the periodontitis pro- represent the reflection of the balance and inter-
gression is classically described to progress in play of several underlying risk determinants and
bursts, where periods of active bone resorption environmental risk factors [99]. While in most
may be followed by periods where the inflamma- cases there is a correlation between the amount of
tion persists, but the bone resorption seems to be dental biofilm and the extent and severity of the
restricted [97, 98]. While this model remains to periodontitis, patients located at both extremes of
be confirmed from a molecular point of view, the susceptibility spectrum exist and are a rou-
accumulating evidence support the idea that vari- tinely encountered in a specialist’s dental office.
ations in the intensity and nature of the host’s Similarly, patients presenting a lack of response
inflammatory response may drive the disease to the classical clinical treatment of periodontitis,
evolution via the control of bone resorption. without any apparent clinical reason to explain
In this context, virtually any factor or event such unresponsiveness, comprise a real and rela-
that could modify the host–microbe interactions tively frequent challenge to clinicians.
and the inflammatory immune response derived The factors that contribute to the differen-
from such interactions would influence the evo- tial susceptibility to inflammatory destruction
lution and outcome of periodontitis. These com- of the alveolar bone and other periodontal tis-
plex interactions explain the tremendous sues are not totally understood. Nevertheless,
variability in susceptibility and clinical pheno- strong evidence points to an increasing number
type of the disease. of modifying factors, both innate (e.g., genetic
variations) and acquired (e.g., microbial fac-
tors, environmental factors, and comorbidities),
6.3 Risk Determinants that can modulate the host–microbe interactions
to Alveolar Bone Resorption in the periodontal environment, tilting the resis-
in Periodontitis tance or susceptibility phenotypes [75, 99–101].
As previously stated, the pathogenic process of
Clinicians often face the disconcerting experience alveolar bone destruction is dynamically modu-
of examining a patient with extensive periodon- lated by the interplay of risk factors affecting
tal destruction, but without any obvious clinical the homeostatic balance of the host and his/her
finding to explain the severity of the disease. capacity to cope with the presence of periodontal
Conversely, the opposed scenario is also possi- microbes (Fig. 6.2).
ble: a patient with widespread inflammation and Indeed, the delicate balance between a pro-
abundant presence of dental biofilm, but with- tective immune response (without bone loss or
out any clinical sign of periodontal destruction. periodontal tissue damage) and an exacerbated
6.3.1 Acquired Risk Factors
6.3.1.1 Tobacco
The latest consensus report from the 11th
European Workshop in Periodontology stab-
lished that the ask, advise, refer (AAR) approach
is the absolute minimum standard of care when
dealing with tobacco smokers in the dental clinic.
The consensus considered the copious evidence
linking tobacco consumption with the occurrence
and severity of alveolar bone loss around teeth
and dental implants, as well as with negative
treatment outcomes [105].
Probably, the most convincing piece of evi-
dence linking tobacco smoking with periodontitis
comes from the longitudinal cohort of Dunedin,
New Zealand. This birth cohort of 1037 partici-
Fig. 6.2 Interaction of risk factors and determinants in
the pathogenesis of periodontitis. Environmental, micro-
pants has been longitudinally followed since
biological, and intrinsic host factors determine the bal- birth in 1972–1973, including a complete dental
ance between the infecting microorganism and the and periodontal examinations at ages 32 and 36,
immune response. In homeostatic conditions the host’s and tobacco smoking determination at ages 15,
immune systems are able to tolerate the presence of
microorganism without triggering a destructive inflamma-
18, 21, 26, 32, and 36. The study demonstrated
tory response. The qualitative changes of the oral micro- that smokers had a 23% greater attachment loss
biota (dysbiosis) are a reflection of the loss of balance in than nonsmokers at age 36, confirming the strong
the host–microbe interaction leading to disease association between chronic smoking and peri-
odontal disease [106].
immune response (with widespread alveolar The mechanistic link between tobacco smok-
bone destruction) can be broken by numer- ing and alveolar bone loss in periodontitis is com-
ous environmental and host’s intrinsic factors. plex and involves alterations in the microbiome,
Among them, changes in the oral hygiene habits, host’s immune response, and metabolism of peri-
placement of deficient dental restorations, anti- odontal tissues. Chronic tobacco consumption
biotics usage, smoking, nutritional alterations, leads to profound changes in the periodontal bio-
hormonal changes, metabolic diseases, acquired film, dramatically increasing the pathogenicity
infections that alters the periodontal microbiota, and disease-initiating capacity of it. Tobacco
trauma, corticosteroids usage, stress, and even smoke is responsible for increasing the ability of
aging [102, 103]. In addition, there is strong P. gingivalis to form biofilms and decreases its
evidence that genetic factors contribute in a sig- potential to elicit an effective host’s immune
nificant extent to determine the susceptibility to response, acting in synergy with its proprietary
inflammatory destruction of the tooth-attachment multiple evasion mechanisms [107]. The chronic
apparatus, determining the quantity and quality use of tobacco also generates extensive deleteri-
of the inflammatory immune response to peri- ous effects in the host’s immune system, both sys-
odontal infection [104]. Once the balance is temically and locally. At the systemic level,
tilted, the host’s response against the periodontal tobacco smoking generates altered tolerance to
microbiota can become “destructive” leading to self-antigens, suppression of the innate immune
the setting up of the clinical signs of periodon- response, and aberrant adaptive immune responses
titis: gingival inflammation, increased probing [108, 109]. At the local level, innate immune cells
depth of the periodontal sulcus, clinical attach- from smokers (i.e., neutrophils and macrophages)
ment loss, and alveolar bone resorption. have impaired phagocytic capacity, chemotactic
deficiencies, and increased proinflammatory periodontitis [114]. In the case of the congenital
capacity [110]. Therefore, the clearance of invad- conditions, the periodontal destruction appears
ing microorganism becomes compromised, lead- very early in life, in some cases affecting both
ing to a sustained chronic and ineffective local deciduous and permanent dentition and leading
inflammation, characterized by the accumulation to premature exfoliation of all teeth [115].
of neutrophils, lymphocytes, and macrophages It is possible to mention the infection by
within the periodontal connective tissue. HTLV-1 virus as an additional example of an
Neutrophils can then be activated to degranulate, acquired disturbance on the immune system that
liberating their rich content of tissue-destructive impacts periodontitis outcome [116]. HTLV-1
proteases. Lymphocytes persist within the tissue, results in an overall deregulation of immune
liberating proinflammatory mediators that amplify response, being associated with a series of patho-
the inflammatory process. Macrophages produce logical conditions. In periodontitis context, it was
and liberate proteolytic enzymes, reactive oxygen demonstrated that even presenting a standard
species, and nitrogen species, further contributing periodontopathogen infection, patients with
to connective tissue degradation [111]. HTLV-1 demonstrated an exacerbated immune
response and increased periodontal destruction.
6.3.1.2 Immune Deficiencies
Obviously, any condition affecting the quality and 6.3.1.3 Metabolic Diseases
effectiveness of the immune response will have a Some metabolic diseases affect the clinical pre-
tremendous impact in the clinical presentation of sentation of periodontitis, correlating with the
periodontitis. The most severe forms of periodonti- presence, extension, and severity of the alveolar
tis are those categorized as “periodontitis as a mani- bone loss [117]. Among them, diabetes mellitus
festation of a systemic diseases,” where the systemic is most widely studied, and a large body of evi-
disease is without exception an immune subduing dence demonstrates that uncontrolled diabetic
condition, including acquired neutropenia, leuke- patients are under increased risk of suffering
mia, familial and cyclic neutropenia, down syn- severe forms of periodontitis, with extensive
drome, leukocyte adhesion deficiency syndrome, alveolar bone loss, and with poor response to
Papillon-Lefevre syndrome, Chediak-Higashi syn- conventional periodontal treatment [118].
drome, histiocytosis, glycogen storage disease, Recent evidence from clinical trials supports
infantile agranulocytosis, Cohen syndrome, Ehler- the notion that diabetic patients exhibit increased
Danlos syndrome, and hypophosphatasia [112]. RANKL/OPG ratios, and that the periodontal
When any of the immune mechanism respon- treatment is capable of diminishing the RANKL/
sible to withhold the dissemination of the infect- OPG in follow-up periods of 3 months [119]. In
ing microorganism becomes compromised, the this respect, the mechanism behind the increased
host can develop extensive periodontal destruc- susceptibility to alveolar bone loss would be the
tion and the possibility of suffering the systemic same as in patients without diabetes, and the
propagation of the infection, with potential severe increased susceptibility would be a consequence
health outcomes. For example, leukocyte adhe- of impaired inflammation control mechanisms.
sion deficiency type I patients, who suffer from a Further, evidence from a recent systematic
mutation affecting the CD18 integrin that disrupts review including 35 parallel randomized clinical
the transmigration of neutrophils, often present a trials (2565 participants) demonstrated that peri-
very severe periodontitis, with extensive bone odontal therapy has a measurable effect in the
loss and a characteristically increased infectious glycemic control of diabetes patients, pointing to
burden within the periodontal tissues [113]. a bidirectional association between periodontitis
In the case of the acquired conditions, the and diabetes mellitus [120]. The linking factor
extensive and rapid alveolar bone loss and peri- behind the bidirectional relationship of diabe-
odontal destruction is the common feature, tes and periodontitis could be the inflammatory
resembling the clinical phenotype of aggressive molecular signals, that emanated from the local
environment of the periodontium could act at dis- enzyme peptidyl arginine deiminase (PAD), which
tant sites affecting the glucose metabolism and catalyzes the conversion of arginine residues to
modify the course of the disease [121]. A recent citrulline. The irreversible citrullination of arginine
systematic review and meta-analysis, including residues changes the structural characteristic of
nine clinical trials, demonstrated that periodontal proteins, transforming them in antigens capable of
treatment significantly reduced circulating levels eliciting an immune response [127]. Citrullinated
of TNFα and C-reactive protein, diminishing the proteins characteristically accumulate in the joints
inflammatory burden in type 2 diabetic patients of RA patients, and specific autoantibodies against
[122]. This evidence supports the mechanistic them are one of the central causes for joint degen-
link of periodontitis and diabetes through inflam- eration and bone destruction during the progression
matory mediators. It is important to consider of the disease [128, 129]. Additionally, the pro-
that the study of the potential interconnection inflammatory molecular signals liberated in the
of multifactorial diseases such as diabetes and chronic immune response of periodontitis and RA
periodontitis is complex due the multitude of could reach the circulation, generating the mutual
variables inherent from both conditions. In this exacerbation and perpetuation of the inflamma-
scenario, data from experimental models is espe- tory response in distant compartments [130]. In
cially important in the confirmation and under- this sense, both diseases will act in synergy in a
standing of the alleged interaction. Interestingly, loop of reciprocal inflammatory amplification. The
experimental models demonstrate that diabetes connection between RA and periodontitis is also
can in fact disrupt host–microbe homeostasis supported by experimental model’s data, which
in periodontal environment, since the spontane- demonstrate that periodontitis and arthritis interac-
ous periodontitis development in diabetic rats tion in mice involves a shared hyper-inflammatory
involves an unrestricted expression of inflamma- genotype and functional immunological interfer-
tory cytokines and tissue destructive factors in ences [131]. In an analog fashion to the scenery
the absence of major changes in commensal oral described for diabetes, RA also seems capable of
microbiota [123]. In other words, the modifica- disrupting the host–microbe homeostasis, lead-
tions in host responsiveness caused by diabetes ing to aberrant responses to microorganism previ-
can trigger a destructive inflammatory response ously recognized as commensal and nonhazardous
against previously well-tolerated microorganism. microbiota [131].
Another metabolic disease strongly associated Another example of the complexity of the
with alveolar bone loss in periodontitis is rheu- immune mechanisms that drive the inflamma-
matoid arthritis (RA). RA patients present the tory destruction of host’s tissues is the evidence
inflammatory degeneration of diarthrodial joints that the high dietary salt intake (characteristic of
with a profuse infiltration of immune cells into the modern western diet) has the potential to induce
synovial lining. As in periodontitis, the character- enhanced macrophage infiltration, increased
istic bone erosions in inflammatory arthritis are inflammatory cytokine secretion, and polariza-
caused by a decontrolled activation of osteoclast tion of immune response towards a Th17 pheno-
due to the inflammatory upregulation of RANKL type. In sum, the high dietary salt intake can be
and the increase of the RANKL/OPG ratio [124]. regarded as an environmental risk factor for the
Epidemiologic data points to a strong correlation development of autoimmune diseases [132–134].
between the occurrence of both diseases, suggest- Along the same lines, recent evidence points to
ing common susceptibility traits and a possible an association between obesity/metabolic syn-
common pathogenesis [125]. The hypothetical drome and exacerbated alveolar bone loss, sup-
pathogenic link could be the capacity of some peri- ported both by epidemiological data and by
odontal pathogens to produce enzymatic changes in experimental in vivo evidence [135–137]. Again,
structural proteins that trigger a humoral response the linking factor between both conditions would
against self-antigens [126]. Specifically, P. gingi- be the exacerbation of inflammation and the mal-
valis is the only known bacteria that produce the function of inflammation control mechanisms.
6.3.2 Innate Risk Determinants associated with an increased risk of periodontitis

in Indians, but not in Chinese [141]. The above
Genetic susceptibility determinants are among example testifies for the difficulty of stablishing
the most studied topics in the periodontal litera- associations between genetic variations and the
ture. Nevertheless, despite the generalized accep- presence and severity of periodontitis, with con-
tance of genetic factors as key regulators of the troversies spanning for decades in the literature
susceptibility to periodontitis, a great controversy and still unresolved.
still persist regarding the specific contribution of A major source of bias in periodontal genetic
particular mutations to the overall clinical pheno- research came from the necessity to select a pri-
type, and more importantly a model integrating ori a gene(s) to study in an association research,
the human genetic diversity into the pathogenesis often based in their putative theoretical involve-
of periodontitis is still lacking. ment in key steps of the immune response or
The first clear evidence that genetic factors periodontal tissue’s metabolism. Nevertheless,
played a central role conferring susceptibility or this approach has proved inconclusive and most
resistance to periodontal destruction came from associations between mutations and the occur-
the elegant work of Michalowicz et al. with adult rence, severity, or extension of periodontitis lack
twins [138, 139]. Despite later controversies, the necessary replication in different populations,
Michalowicz et al. established that genetic fac- as previously exemplified with the case of the
tors accounted for at least a 50% of the clinical IL-1 cluster. Probable causes of these difficulties
variation on the disease phenotype, regulating the are multiple, among them: the complexity of the
susceptibility to suffer bone loss and periodontal immune regulatory mechanisms, overlap and
destruction. Since that seminal work, many mod- redundancy in the functions of many mediators
els and hypothesis have been proposed to explain of inflammation and bone metabolism, the lack
the mechanisms of genetic influence over the of a linear relationship between gene expression
phenotype of periodontitis. and protein production/cell phenotype, and the
The focus of the genetic research in periodon- lack of a comprehensive understanding of the
tal susceptibility have been the molecular media- regulatory pathways that maintain tissue homeo-
tors of inflammation, particularly the mutations stasis. Additionally, the difficulty in the definition
affecting the levels and expression of cytokines of the periodontitis cases and the selection of
recognized as key regulators of the inflammatory suitable controls poses another level of complex-
process. The most studied mutations in periodon- ity over the design of periodontitis genetic asso-
titis are the polymorphic variations of the IL-1 ciation studies [99].
gene cluster and/or its promoter region. In the Even with the inherent problems of the classic
late years of the twentieth century, Kornman periodontal genetic studies, the literature consis-
et al. reported the association between the com- tently demonstrates that mutations altering the
posite polymorphic genotype for IL-1A (−889) host’s capacity to mount and efficient yet con-
and IL-1B (+3953) genes and the severity of peri- trolled immune response, or those related to the
odontitis in nonsmokers, concluding that the exacerbation of the bone and connective tissue
composite polymorphic phenotype contributed turnover, are strongly associated with the pres-
significantly to increased IL-1β levels and aug- ence and clinical phenotype of the disease. As
mented risk of suffering severe forms of peri- examples: the single nucleotide polymorphism
odontitis [140]. Since then, numerous replication (SNP) rs4794067 of the TBX21 gene that
studies in various populations have been con- increases the transcription of T-bet, leading to an
ducted with conflicting results. A recent system- exacerbated inflammatory response, is strongly
atic review and meta-analysis of 20 case–control associated with the occurrence of periodontitis in
studies in Asian subjects, including in excess of a Brazilian population [75]. Further, the SNP
3000 patients and controls, concluded that the rs1800872 in the promoter region of the IL-10
polymorphic form of IL-1B (+3953) was strongly that decreases the transcription of IL-10, leading
to impaired immune regulation, is strongly asso- that has predictive value and can be used in the
ciated with the occurrence and severity of peri- decision-making process during the management
odontitis in the same population [142]. These two of breast cancer cases [144]. An analog tool for the
examples show the value of the classical approach risk assessment of periodontitis, aiding in the
to periodontal genetic studies, but also testify for long-term clinical management of patients, is an
its inherent flaw, where a theoretical framework old longing of the practicing periodontist.
taking into consideration the functional conse-
quences of the studied mutations is a sine qua
non condition. This condition greatly limits the 6.4 uture Perspectives for the
F
validity of the studies, since many genes are not Treatment of Periodontitis:
studied by the lack of a supporting theory linking From the Bench to the
them to the pathogenesis of periodontitis. Dental Chair
To overcome some of these problems, a new
strategy using the results of large genome-wide The accumulate knowledge of the immune mech-
association (GWA) studies has been recently pro- anisms driving the progression of bone loss in
posed. The rationale of this approach is to scan periodontitis has stimulated the development
the whole genome searching for genes or groups of new therapeutic approaches to modulate the
of genes associated with the presence of the dis- immune inflammatory response with the intention
ease (or disease subrogate) without any a priori to limit the adverse consequences of unregulated
selection bias. responses. Theoretically, bioactive molecules
Using the GWA-based selection strategy a with the potential to regulate key points of the
completely new set of genes with strong associa- immune response could be used as coadjutants in
tion to the occurrence of periodontitis have been the treatment of periodontitis, enhancing the clini-
discovered. These previously ignored genes code cal effectiveness of conventional treatment, pre-
for neuropeptides, innate immune response serving the results for longer periods, limiting the
receptors, enzymes linked to cytoskeletal rear- progression of periodontal destruction, and modi-
rangement, intracellular signaling transduction fying the susceptibility to suffer relapses.
molecules, and proteins directing vesicle fusion Various promising efforts in the development
during synapsis, and even some noncoding genes of clinically relevant and safe bioactive molecules
or genes with no known function. The diversity are in different stages of experimental testing,
of the newly discovered periodontitis-associated with the perspective of reaching to the clinical
genes testifies for the power of the GWA practice in the near future. The classic approach
approach, since it allows for the unveiling of dis- to coadjuvant pharmacological treatment in
ease–gene association without the necessity of a periodontitis is the use of anti-infective agents.
previous theoretical framework [143]. Although not immune modulatory agents in a
While the real contribution of genetic determi- strict sense, the use of anti-infective therapy has a
nants to periodontitis risk remains to be estab- pronounced effect in the immune system, decreas-
lished, it is important to consider that extensive ing the number of antigens available to trigger
research efforts in the field aim to provide addi- an immune response and limiting the extent and
tional tools to the clinician in the direct risk assess- duration of it. Characteristically, systemic antibi-
ment, which in turn may influence a series of otics has been used to enhance the results of peri-
clinical decisions. An interesting example of the odontal treatment in severe or high-risk patients,
clinical value of being able to assign and objective nevertheless there is a series of problems associ-
risk value to the presence of certain SNPs, is the ated with the use of these agents, including the
algorithm that forms the PRS index (polygenic appearance of bacterial resistance and the occur-
risk score) for the risk discrimination in breast rence of adverse reactions. Over the years, various
cancer. The PRS index additively combines the controlled-release local anti-infective agents have
risk effect of 77 SNPs, providing a valuable tool been developed in an effort to limit the negative
consequences of the use of systemic antibiotics, capacity to fight the infection. In an analog fash-
but providing their putative benefits. In a system- ion to the trend of using controlled-release local
atic review and meta-analysis including 32 stud- anti-infective drugs, the goal is to develop an
ies (3705 subjects), the use of minocycline gel, immune modulatory agent that could be directly
microencapsulated minocycline, chlorhexidine targeted to the periodontal tissue, preventing the
chips, and doxycycline gel during scaling and potential risks of undesirable immune modulation
root planning (SRP) proved marginally better in other body compartments. It is noteworthy that
than SRP alone, but not to a clinically significant the systemic administration of classic nonsteroid
extent [145]. Another shortcoming of antimicro- anti-inflammatory drugs (NSAID) as adjunctive
bial therapy is that after the completion of the treatment in periodontitis, aimed at reducing the
treatment the risk factors and susceptibility traits levels of arachidonic acid metabolites, has shown
that contributed to the establishment of a patho- no long-term clinically relevant benefits in several
genic microbiota (dysbiosis) remain unaltered, clinical trials, pointing to the need of a very spe-
leading to a recolonization of periodontal sites by cific and focused inhibition of inflammation in
a pathogenic microbiota and to the recurrence of order to attain meaningful clinical benefits.
the disease [146, 147]. Additionally, the potential risks of the chronic use
The new emerging therapeutic approach will of NSAID, such as gastric ulcer and coagulation
use immune modulatory drugs, capable of selec- impairment, seriously limit their usefulness as
tively regulating key points of the immune adjunctive therapy in periodontitis [148].
response, specifically limiting the degree of As depicted in Figs. 6.3 and 6.4, an interesting
inflammation but without dampening the system’s approach would be the use of drugs capable of
Fig. 6.3 Immune and inflammatory response to periodon- appear sequentially determine the outcome of the response.
tal infection. Periodontal pathogens are recognized by the The relative predominance of pro-inflammatory lineages
resident cells, which trigger the first inflammatory response (Th1 and Th17) favors soft tissue destruction and osteoclast
mediated by PAMP/PRR interactions. Infiltrating innate genesis. Conversely, when regulatory lineages predominate
immune cells rapidly reach the infection site and amplify (Tregs and possibly Th2), the inflammation is halted, osteo-
the primary response. The adaptive immune cells that clast genesis is inhibited, and the tissue tends to repair
Fig. 6.4 Modulatory strategies to control the inflamma- metabolites, modulating the inflammatory response.
tory destruction of alveolar bone. Anti-infective therapy Specific antibodies against inflammatory cytokines and
diminishes the presence of the antigen, limiting the receptors (Anakinra, Rilonacept, Canakinumab, met-
response. TLR inhibition offers the possibility of inhibit- RANTES, and Denosumab) could inhibit osteoclast gen-
ing the first events of inflammation, but with the danger of esis directly or indirectly. SDD inhibits soft tissue
facilitating the infiltration of pathogens. Resolvins could destruction and the lysis of the demineralized bone matrix.
exert a modulatory event reversing the vascular events of Selective recruitment of Tregs by CCL22 could fine-tune
inflammation and inducing apoptosis of infiltrating leuko- the inflammatory response, leading to an efficient
cytes. NSAIDs inhibit the production of arachidonic acid response without tooth-attachment loss
inhibiting the first signaling events in the inflam- geting these receptors is prodigious. There is a
matory process, but without compromising the growing body of in vivo evidence demonstrating
later steps of the response. In this sense, Toll-like that the selective inhibition of TLR’s activation or
receptors (TLR) appear as promising therapeutic signal transduction is protective against periodon-
targets. These receptors are fundamentally titis-induced alveolar bone loss. In a murine
involved in the recognition of pathogen- model of Porphyromonas gingivalis-induced peri-
associated molecular patters (PAMPs). TLRs are odontitis, the pharmacological inhibition of GSK3
capable of triggering the first events of the (a key factor in the signal transduction chain
inflammatory cascade, including the upregula- downstream of TLR activation) significantly
tion of inflammatory cytokines and MMPs. decreased the systemic levels of TNFα, IL-1β,
Theoretically, the selective inhibition of one or IL-6, and IL-12 post infection, and suppressed
various TLR subtypes could limit and modulate alveolar bone loss [149]. Further, mice geneti-
the extent of the initial reaction, thus modifying cally deficient (i.e., knock out) for the expression
the nature of the immune response, and prevent- of TLR pathway signaling molecules, such as
ing the occurrence of an uncontrolled inflamma- MyD88, demonstrated resistance to LPS-induced
tion leading to bone metabolism uncoupling and alveolar bone loss, associated with diminished
resorption. Given that TLR are expressed in the levels of inflammatory cytokines expression and
surface of every cell type in the periodontal tissue, osteoclast differentiation markers [150].
the immune modulatory potential for a drug tar- Nevertheless, it is fundamental to bear in mind
that the innate immune events triggered by the bone matrix allowing for bone remodeling.
activation of TLR are of paramount importance Increased collagen lytic activity as a consequence
for the control of infection and that any drug tar- of inflammation is the ultimate responsibility for
geting this receptors will need to be highly selec- net bone loss during the progression of periodon-
tive and tissue specific to avoid the potential titis. Tetracyclines in general and doxycycline in
dangers of the spreading of the periodontal infec- particular have the property of inhibiting the
tion to other body compartments. activity of MMPs independently of their antimi-
Moving down the inflammatory cascade, the crobial action [148, 156, 157]. The inhibition of
next step susceptible of pharmacological modu- MMP using systemic subantimicrobial-dose dox-
lation is the selective inhibition of the effector ycycline (SDD) is a well-established therapeutic
molecules of inflammation. One alternative is the approach and there is a three-decade body of evi-
use of nonsteroidal anti-inflammatory drugs dence supporting its effectivity as an adjunct
(NSAID) to control and modulate the production treatment in chronic periodontitis [158]. The last
of pro-inflammatory molecules, such as prosta- advance in the use of SDD as MMP inhibitors is
glandins derived from arachidonic acid. The clas- the development of sustained release nano-
sical COX-1 and COX-2 inhibitors have the structured films, with the property of delivering a
potential to limit the magnitude of the inflamma- constant dose of doxycycline directly into the
tion. Even though they have proven effective in periodontal tissue for prolonged periods.
diminishing the alveolar bone loss in experimen- Preliminary clinical trials proved that the use of
tal models and marginally effective in clinical the sustained release films improved the clinical
testing [148, 151, 152], their routine use is outcome of periodontal treatment after a follow-
unpractical due to the appearance of unwanted up period of 2 months [159].
side effects in the long-term treatment. Another alternative is the use of highly specific
Other emerging therapeutic approach is the antibodies against the molecular mediators of
use of endogenous pro-resolving lipid mediators inflammation. Although there is not any cytokine
(resolvins) to modulate the inflammatory inhibitor currently being used for the treatment of
response and protect the alveolar bone from periodontitis, several cytokine-inhibiting drugs are
resorption [153]. These lipid mediators are struc- routinely used to prevent the inflammatory bone
turally related to prostaglandin and leukotriene, loss in rheumatoid arthritis (RA) [160]. Given the
but contrary to the pro-inflammatory activities of pathogenic similarities of inflammatory bone loss
the former are capable of inhibit leukocyte in RA and periodontitis, and the possible common
recruitment and promote inflammatory resolu- pathogenic mechanisms among them, it is possible
tion. Evidence form Porphyromonas gingivalis- to hypothesize that such drugs would be also use-
induced periodontitis in vivo experiments ful to prevent alveolar bone loss in periodontitis.
demonstrated that the administration of Resolvin Three approved drugs for the treatment of RA
E1 as monotherapy resulted in complete resolu- reduce the activities of IL-1α/β, diminishing the
tion of the bone lesion and in reduction of sys- inflammation and preventing bone loss. Anakinra
temic markers of inflammation [154]. is a recombinant form of the naturally occurring
An additional possible target of modulation IL-1 receptor antagonist. Anakinra conjugates to
are the enzymes responsible for connective tissue the IL-1 receptor, preventing the activity of
degradation, namely MMPs. Collectively, MMPs IL-1α/β [161]. The soluble IL-1β decoy receptor
are responsible for collagen turnover in all peri- Rilonacept and the neutralizing antibody
odontal connective tissues, including bone [155]. Canakinumab block the biologic actions of IL-1β
After bone demineralization, collagenases and [162]. Taking into consideration the link between
gelatinases degrade the organic component of the inflammation and the increasing ratio of RANKL/
OPG previously discussed, the selective down- associate with increased bacterial load or with
regulation of IL-1α/β could prove valuable as bone impairment in the bacteria clearance mecha-
anti-resorptive drugs. Nevertheless, the infectious nisms, providing an attractive possibility for safe
nature of periodontitis and the risk of impairment local immune modulation, which can be explored
of the pathogen clearance mechanisms pose a seri- as a future approach to enhance the clinical
ous limitation for the use of immune suppressive results of conventional periodontal therapy [91].
drugs in the treatment of periodontitis and the Additionally, approaches based in the induction
potential risks must be carefully weighed. or chemoattraction of Tregs may restore the local
Yet other possible pharmacological target are host–microbe homeostasis, potentially resulting
the cytokines/receptors responsible for the selec- in longer-term effects with increased potential
tive recruitment of osteoclasts precursors to the impact in avoiding subsequent disease events [8].
bone compartment. These chemotactic cytokines Finally, a further possible target of pharmaco-
(a.k.a. chemokines) and their receptors are respon- logic modulation is the RANKL/RANK/OPG
sible for the control of the influx of monocyte/mac- axis. As previously stated, the relative abundance
rophages to the periodontal tissue, and are induced and equilibrium among these molecules is largely
under inflammatory conditions [163]. For example, responsible for the balance between bone apposi-
there is experimental in vivo evidence of the effec- tion and bone resorption. The inflammatory pro-
tiveness of the pharmacologic inhibition of the cess increases the RANKL/OPG ratio, causing
chemokine receptors CCR1 and CCR5 in reduc- bone resorption. Denosumab is a human mono-
ing the alveolar bone loss in a murine model of clonal anti-RANKL antibody approved for use in
Aggregatibacter actinomycetemcomitans-induced osteoporosis patients with high risk of fracture
periodontitis, using the functional competitive [164]. Clinical trials have demonstrated the effi-
inhibitor met-RANTES [163]. In this model, the cacy and safety of the drug in long-term use,
reduced recruitment of osteoclast precursors and opening the possibility for its use in other bone
other inflammatory cells caused a protective effect resorbing pathologies, such as periodontitis [165].
in alveolar bone levels, but proved detrimental to At this time, it is reasonable to suppose that the
the anti-infective response at higher doses. These modulation of the RANKL/RANK/OPG axis to
results highlight the complexity of immune modu- manage periodontitis may become a therapeutic
latory therapies, where there is a thin line between tool available for the clinician in the near future.
bone protective effects and increased risk of sys- Those and other future developments will
temic spreading of the local periodontal infection. contribute to the increase and diversification of
In an interesting approach and proof-of- the therapeutic tools available to the periodontist.
principle in vivo experiment, it was demonstrated The perspective of new therapeutic approaches
that harnessing endogenous Tregs and recruiting allowing for the successful treatment of refrac-
them to specific sites of periodontal inflammation tory cases is a long-standing desire of the practic-
is effective in limiting bone resorption and pro- ing periodontist, but their implementation will
moting the establishment of a regenerative envi- require a thoughtful understanding of the under-
ronment, and associated with diminished markers lying cellular and molecular mechanism of peri-
of inflammation [68]. In this experimental set- odontitis’ pathogenesis, and the accompanying
ting, endogenous Tregs were recruited to the ability of judging the convenience of their use in
periodontal tissue using a controlled-release a particular clinical situation. As the body of
polymeric vehicle designed to create a gradient knowledge of periodontics swiftly increases, the
of CCL22, a known selective chemokine for periodontist must remain up to date to be able to
Tregs. It is noteworthy that the selective recruit- take full advantage of the coming therapeutic
ment of Tregs to the periodontal tissue did not advances in benefit of his/her patients.
with special emphasis on mechanisms of colla-

Clinical Relevance gen degradation in the periodontium and the burst
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Genetic Influences on the
Periodontal Microbial-Host
7
Crosstalk
Luigi Nibali
7.1 Introduction 7.2 Infectogenomics
Previous chapters of this book have discussed In the fourteenth century, one-third to half of the
the importance of subgingival microbial coloni- population living in Europe was exterminated by
zation and of the inflammatory-immune response a mysterious disease which was called ‘the Black
as triggers of periodontal tissue breakdown. In Death’. It is now known that this disease, later
fact, it appears clear that periodontal health or named ‘plague’, was most likely a rodent-
pathology is the result of the interaction between associated, flea-borne zoonosis caused by Gram-
the human host and its invading microbes. Or negative bacterium Yersinia pestis [2].
perhaps we should not define them as ‘invading’, Interestingly, among all people who came into
since it is well known that microbes not only contact with the bacterium, a large proportion
coexist with their human host, but also provide became infected and died, some were ill but man-
multiple vital functions for the survival of the aged to survive and some had no clinical signs of
host itself [1]. Compelling evidence has now infection. It appears plausible that each subject
emerged to suggest that host genetic variants responded to infection with Yersinia pestis as well
have a fundamental effect in regulating the host’s as to other potentially fatal infections in a way
relationships with the microbial ‘guests’ and a that was largely determined by his/her genetic
better knowledge of how these effects are imple- make-up [2, 3]. A more up-to-date example is
mented is crucial in the understanding of disease given by the HIV, the virus responsible for
processes. This chapter will review the evidence AIDS. Following transmission, HIV enters the
on the effect of host genetic factors on periodon- bloodstream and infects helper T cells, macro-
tal microbial colonization and will provide phages and dendritic cells, causing killing of T
examples of how this could have an impact in cells (especially CD4 T cells) by CD8 cytotoxic
clinical practice. lymphocytes, with reduction in CD4 T cell num-
bers and loss of cell-mediated immunity [4]. The
HIV most commonly uses chemokine receptors
CCR5 and/or CXCR4 co-receptor to enter its tar-
get cells. The CCR5 receptor is coded for by the
L. Nibali
Centre for Oral Clinical Research, Institute of CCR5 gene on chromosome 3. A deletion of a
Dentistry, Queen Mary University of London, 32-bp segment in this gene (named CCR5-Δ32)
Turner Street, London E12AD, UK has been discovered to result in a non-functional
e-mail: [email protected];
receptor which prevents this way of HIV R5 entry
[email protected]

DOI 10.1007/978-3-319-53737-5_7
88 L. Nibali
[4]. This genetic variant is rare in Africans/Asians gotic twins frequently have more similar gut micro-
and more common in North European, possibly biomes than non-twin siblings [12]. Furthermore,
due to selective pressure by previous epidemics microbial profiles of faecal samples collected at
[3, 5, 6]. Homozygosity to this gene variant is various times from a given individual are more
characterized by resistance to infection by the similar to each other than to the intestinal microbial
most common strain of HIV, while heterozygosity communities in a different individual [13]. A little
seems to confer partial resistance with slower pro- dip into the human genome can allow a better
gression after onset of AIDS [7]. Based on this understanding of the host- microbial axis. More
principle, CCR5 receptor-antagonist drugs have than 60 million common genetic variants in
been experimented for the treatment of AIDS [8]. 19,000–25,000 genes located in 23 pairs of chro-
The evidence described above is in line with a mosomes are listed in the Single Nucleotide
concept defined ‘infectogenomics’, suggesting Polymorphism Database (dbSNP) by the National
that host genetic factors play a major role in Center for Biotechnology in collaboration with
determining the response to bacterial coloniza- National Human Genome Research Institute [14].
tion [9, 10]. This concept can be extended also to Different individuals are thought to be 99.4% iden-
the presence of common ‘symbiotic’ bacteria and tical in chromosomal structure and 99.9% identical
not just pathogens. In other words, host geno- at sequence level [15]. Functional SNPs are located
types may influence the composition of human in the gene promoter (affecting gene activity) or in
biofilms, including oral biofilms [11]. Therefore, the coding region of the gene (affecting the protein
the composition of microbial biofilms in the produced). The disease-predisposing effects may
human body will be dictated by a combination of be determinant such as for haemophilia A, caused
genetic variants, coupled with environmental by a specific mutation (single gene defect) in the F8
factors. As a result of this, a group of human dis- gene leading to defects in coagulation factor VIII
eases originate from a genetically determined [16]. However, most diseases are characterized by
failure to properly recognize or respond to mem- a complex susceptibility profile, where a variety of
bers of the normal human microbiota [11]. This SNPs contribute to the disease risk. Such SNPs
disease-predisposing effect can potentially may be involved, for example, in microbial recog-
extend not just to microbial diseases, in the tradi- nition (determining aberrant responses to the nor-
tional meaning of the term, such as, for example, mal microbiota), in the inflammatory cascade or in
bacterial vaginosis and periodontitis, but also to DNA repair (associated with a reduction in the abil-
diseases not traditionally considered of microbial ity to repair damaged DNA).
origin. Among them, rheumatoid arthritis, reac-
tive arthritis and even cancer, which could be
influenced by microbial shifts (dysbiosis) even at 7.3.1 Microbial Recognition Genes
distant sites. Hence the concept of genetic dysbi-
osis, which suggests that host genetic variants Following the earlier example of the HIV, it
could be responsible for a range of chronic seems reasonable to believe that genetic variants
human diseases through an effect on dysbiosis of affecting microbial recognition will have a major
microbial biofilms [11]. role in determining the composition of microbial
biofilms. The search for the possible ‘microbial
recognition’ gene affecting microbial c olonization
7.3 vidence for Genetic
E could focus on pattern-recognition receptors
Variants Influencing the (PRRs), which recognize evolutionarily con-
Response to Microbial served constituents of microbes called pathogen-
Challenge associated molecular patterns (PAMPs). PRRs
include normally cell-bound proteins such as
Where is the evidence for the Infectogenomics toll-like receptors (TLRs), RIG-I-like receptors
principles outlined above? Circumstantial evidence (RLRs), NOD-like receptors (NLRs), C-type-
can be derived from studies showing that monozy- lectin like receptors (CLRs), scavenger receptors
7 Genetic Influences on the Periodontal Microbial-Host Crosstalk 89
(SCs), innate DNA receptor proteins termed receptor-mediated pathogen recognition and/or
AIM2-like receptors (ALRs), members of the regulation. While no association between these
complement pathways and peptidoglycan-SNPs and presence of bacterial vaginosis was
recognition proteins (PRPs) and soluble PRRs detected, some of the studied SNPs were associ-
(including collectins, ficolins, pentraxins, galec- ated with carriage of A. vaginae and G. vaginalis
tins, sCD14 and natural IgM). Upon microbial during early pregnancy. The authors suggested
interaction, PRRs activate a series of downstream that some degree of genetic susceptibility involv-
mechanisms through selective cells signalling, ing pathogen recognition may occur, which influ-
leading to the generation of pro- or anti- ences vaginal presence of potential pathogenic
inflammatory proteins. Mutations in the coding microorganism [22]. In a separate study on 238
or promoter regions of PRR genes could result in pregnant women, TLR4 genotypes were associ-
an altered ability to recognize microbial patterns, ated with increases in vaginal pH and in vaginal
affecting the ‘binding/recognition’ process and detection of Gardnerella vaginalis, Prevotella,
its downstream pathways, leading to aberrant Bacteroides and Porphyromonas, suggesting that
response to microbial challenge and shifts in the genetic variants may drive a change in the vagi-
normal biofilm composition [17]. nal environment which favours the growth of
The circumstantial evidence for a role of host pathogenic bacteria [23].
genetic variants in determining microbial coloni-
zation is strengthened by observations on inflam-
matory bowel disease, encompassing Crohn’s 7.3.2 Genes Involved
disease (CD) and ulcerative colitis (UC). Genetic in Inflammatory Pathways
variants in the NOD2 gene, coding for an intra-
cellular pattern recognition receptor able to rec- It is now becoming clear that inflammation can
ognize molecules containing bacterial muramyl have profound effects on microbial communities,
dipeptide [18], are now recognized as increasing causing a progressive decrease in the microbial
the risk of CD [19]. In an experimental ileal diversity through an increased availability of sub-
inflammation model in mice [20], when NOD2- strates for growth of Gram-negative bacteria (e.g.
CD- susceptible animals were subjected to iron and serum, dead or dying cells) and loss of
Toxoplasma gondii-induced ileitis, an increase in niche and substrates for Gram-positive flora (e.g.
inflammation and dysbiosis was noticed (shift mucus, goblet cells) [24]. Therefore, it is conceiv-
from mainly Gram-positive to Gram-negative able that variants in genes directly involved in
bacteria, associated with invasive E. coli) com- inflammatory pathways, could impact the thresh-
pared to non-genetically susceptible animals. old for dysbiosis and the ability to resolve the
Furthermore, genetic variants in the NOD2 and dysbiosis-inflammation cycle generated by an
autophagy-related 16-like 1 protein (ATG16L1) acute trigger [20]. Based on this concept, genetic
have been associated with gut microbiota struc- variants affecting inflammatory responses may be
ture alterations, including decreased major candidates for an effect on microbial bio-
Faecalibacterium levels and increased film composition. The evidence for this comes
Escherichia levels [21]. These results are likely mainly from studies on periodontal disease which
due to an alteration of the inflammatory cascade will be discussed in the next section.
resulting from an aberrant response upon micro-
bial recognition through the NOD2 receptor.
Some evidence exists also for the effect of 7.4 Genetic Effects on Microbial
microbial recognition genes on microbial pres- Colonization: Studies
ence in vaginal biofilms. In a study on the vaginal in Periodontal Disease
microbiota of 144 pregnant women, detection of
A. vaginae and G. vaginalis by PCR was studied Previous chapters of this book described how
in relation to 34 single nucleotide polymorphisms pathogenic pathways leading to periodon-
pertaining to 9 genes involved with Toll-like tal breakdown involve the role of subgingival
90 L. Nibali
microbes, host response and environmental fac- of a case of Localized Aggressive Periodontitis
tors and how important the crosstalk between (LAgP), characterized by a molar-incisor pattern
host and bacteria is. This chapter introduced the of bone and periodontal attachment loss.
concept of ‘Infectogenomics’ to mean the effect The understanding that heritability accounts
of host genetic background on the colonizing for about half of the risk of developing periodon-
microbes, and examples relative to inflamma- titis [27] has led to a flourish of studies trying to
tory bowel disease and bacterial vaginosis have identify inflammatory, metabolic or structural
been provided. In the last 10–15 years, evi- gene polymorphisms which could predispose to
dence for periodontal infectogenomics [25] has periodontal diseases [28]. Most studies focused
also emerged. In particular, it is striking how on selected candidate SNPs, starting from the
the JP2 leukotoxic strain of A. actinomycetem- case-control association study suggesting an
comitans has a strong tropism of for subjects of effect of the Interleukin-1 (IL-1) ‘composite gen-
mainly North African and West African descent, otype’ on disease predisposition [29]. More
increasing the risk of development of Localised recent studies are often using an explorative
Aggressive Periodontitis (LAgP) [26]. Since car- genome-wide approach [30, 31]. However, stud-
riage of this strain does not seem to depend on ies so far failed to reach a consensus after analy-
geographic location but rather on ancestry, it is ses in different populations and settings, with
likely to be linked with heritability and with the promising studies pointing towards the role of
host genetic make-up. Figures 7.1 and 7.2 show SNPs in ANRIL (antisense non-coding RNA in
a typical clinical and radiographic presentation the INK4 locus), COX2 (cyclooxygenase 2),
IL-10 (Interleukin-10) and DEFB1 (β-defensin-1)
and possibly others in disease predisposition [31,
32]. These genes are involved, respectively, in
glucose and fatty acid metabolism regulation
(ANRIL gene) [33], coding for antimicrobial
peptides involved in the epithelial response to
microbial invasion (DEFDB1 gene) [34] and in
the periodontal inflammatory response (COX2
and IL-10 genes) [35, 36].
Sigmund Socransky and Anne Haffajee were
probably the first to investigate the relationships
between SNPs supposed to affect the periodon-
titis trait and presence of subgingival microbes.
Fig. 7.1 Clinical photograph of 15-year-old non-smoker In their 2000 paper, they observed an association
LAgP patient of Afro-Caribbean origin, showing buccal
between IL-1 genotypes and presence of subgin-
migration of the upper right central incisor and generally
good oral hygiene gival microbes [37]. In particular, more IL-1 ‘gen-
otype positive’ subjects [29] exhibited high mean
counts of ‘red’ and ‘orange’ subgingival species
than ‘genotype negative’ subjects. Bacteria found
at higher levels in IL-1 genotype positive subjects
were Bacteroides forsythus, Treponema denti-
cola, the Fusobacterium nucleatum subspecies,
Fusobacterium periodonticum, Campylobacter
gracilis, Campylobacter showae, Streptococcus
constellatus, Streptococcus intermedius,
Streptococcus gordonii and 3 Capnocytophaga
Fig. 7.2 Panoramic radiograph of patient shown in
Fig. 7.1. Please note localized alveolar bone loss affecting species. The differences in bacterial colonization
mainly upper right central incisor and first molars by genotype were mainly visible in deep peri-
odontal pockets (>6 mm). Based on these results, genetic variants and presence of A. actinomy-
the authors postulated that genetic variants might cetemcomitans and of both bacteria concomi-
either directly affect bacterial growth and viru- tantly [43]. To explore this concept further, we
lence or alter the inflammatory milieu, favouring conducted a pilot treatment study on 12 AgP
the growth of specific bacteria. In contrast with patients selected based on their IL6 genotypes
these findings, no associations between IL-1 (‘pro-inflammatory IL6 haplotype positive’ vs.
composite genotypes and subgingival bacteria ‘IL6 haplotype negative’). In this population,
analysed by PCR were detected in a similar study higher A. actinomycetemcomitans counts were
published shortly afterwards [38]. detected subgingivally in IL6 ‘haplotype posi-
Our group has extensively investigated the tive’ subjects before treatment. Despite a reduc-
associations between candidate genetic vari- tion after non-surgical and surgical treatment,
ants affecting the inflammatory response (e.g. these subjects showed a sharp increase in counts
interleukin-1 and interleukin-6 genes) and sub- of A. actinomycetemcomitans again 3 months
gingival detection of periodontopathogenic bac- after periodontal treatment, suggesting a strong
teria by culture and polymerase chain reaction genetic influence on gingival pocket re-coloni-
(PCR). In 45 untreated aggressive periodontitis zation, which was not observed in IL6 ‘haplo-
(AgP) patients from London, IL6 and Fc-γ poly- type negative’ subjects [44].
morphisms were both associated with increased A larger study used a genome-wide approach
odds of detecting A. actinomycetemcomitans, P. in 1020 subjects participating in the
gingivalis and T. forsythensis after adjustment Atherosclerosis Risk In Communities (ARIC)
for age, ethnicity, smoking and disease sever- study to investigate the relationship between
ity [39]. In particular, subjects with supposedly host genotypes and eight periodontal pathogens
pro-inflammatory IL6 genotypes [40, 41] had analysed by checkerboard DNA-DNA hybrid-
increased detection of A. actinomycetemcomi- ization [45]. They detected no genome-wide
tans and P. gingivalis. The study was repeated significant signals, but suggestive evidence
in a rural population living in Andhra Pradesh, (p < 5 × 10−6) of association for 13 genetic loci
India [42]. Subjects had subgingival plaque and ‘red’ and ‘orange’ complex microbiota. The
samples taken and analysed by checkerboard same effect direction was detected in a second
DNA-DNA analysis for 40 periodontal taxa and sample of 123 African-American participants.
had their DNA extracted for IL6 SNP analyses. Interestingly, these authors confirmed the mod-
In this population not exposed to regular den- erate association our group previously reported
tal care and to use of antibiotics, most subjects between IL6 SNPs and high ‘red complex’ colo-
harboured A. actinomycetemcomitans and P. nization. No association was detected between
gingivalis subgingivally, which did not allow any of the identified SNPs with CP diagnosis,
any analysis on bacterial detection by genotype. suggesting once more the examination of bacte-
However, associations between IL6 genotypes rial colonization as a distinct trait to ‘presence of
and elevated counts of A. actinomycetem- disease’ [45]. Recently, in a case-control study
comitans and Capnocytophaga sputigena were analysing polymorphism TBX21-1993T/C
observed, strengthening the previous report. (rs4794067) in healthy (n = 218), chronic peri-
This was further confirmed when a population odontitis (n = 197) and gingivitis patients
of 267 chronic and aggressive periodontitis (n = 193), no associations were detected between
patients was studied. Host DNA samples were genotypes and presence of ‘red complex’ bacte-
extracted from blood samples and analysed for ria [46]. A summary of genetic variants shown to
five IL6 SNPs, while subgingival plaque sam- be associated to detection of subgingival peri-
ples were analysed by PCR for the presence odontal bacteria is provided in Table 7.1. A more
of A. actinomycetemcomitans and P. gingiva- systematic and comprehensive review of the lit-
lis. The study confirmed again the association erature on periodontal infectogenomics has been
between IL6 supposedly ‘pro-inflammatory’ recently published [47].
92 L. Nibali
Table 7.1 Summary of genetic variants shown to be associated with detection of subgingival periodontal bacteria in
some of the studies reviewed in this chapter
Study Population Study design Genetic variant-microbial association
[37] U.S. University-based CP case-control candidate IL-1 genotypes: counts of B. forsythus,
gene association study with T. denticola, F. nucleatum, F.
checkerboard DNA-DNA periodonticum, C. gracilis, C. showae, S.
microbial analysis constellatus, S. intermedius, S. gordonii,
3 Capnocytophaga species
[39] UK University-based AgP case-control candidate IL6 and Fc-γ R genotypes: A.
gene association study with actinomycetemcomitans, P. gingivalis
microbial culture analysis detection
[42] Indian rural village Cross-sectional candidate IL6 genotypes: A.
gene association study with actinomycetemcomitans, C. sputigena
checkerboard DNA-DNA counts
microbial analysis
[43] UK University-based Mixed CP and AgP IL6 genotypes: A.
case-control candidate gene actinomycetemcomitans, P. gingivalis
association study with detection
microbial PCR analysis
[45] U.S. University-based GWAS with checkerboard 13 loci (including KCNK1, FBXO38,
DNA-DNA microbial UHRF2, IL33, RUNX2, TRPS1,
analysis CAMTA1 and VAMP3): suggestive
evidence of associations with ‘red’ and
‘orange’ complex/A.
actinomycetemcomitans
CP chronic periodontitis, AgP aggressive periodontitis
Host genetic variants Host genetic variants

affecting microbial affecting the inflammatory
recognition (e.g. Fc-γ receptor response (e.g. Interleukin-6
polymorphisms) polymorphisms)
Shift in the composition of the subgingival

biofilm (dysbiosis) (e.g. increased growth of
inflammophilic members of the microbial
community)
Behavioural/lifestyle
factors,
environmental
factors,
other pathways of
genetic influence
Potential effects on Potential effects on
disease initiation response to
and progression treatment
Fig. 7.3 Schematic representation of periodontal infectogenomics
Summarizing the findings above, there is now and more in general ‘red complex’ bacteria may
increasing evidence that host genetic variants can be a small representation of changes in the subgin-
have an effect on periodontal pathology by influ- gival biofilm (‘dysbiosis’) (see Fig. 7.3). There is
encing the subgingival bacteria composition. The still a lack of studies investigating whether health-
shifts in A. actinomycetemcomitans, P. gingivalis associated bacteria may be affected by specific
genetic variants. The supposed shift towards a pathogenic pathways and could open new man-
more pathogenic microbiota may occur through agement avenues. The above-mentioned pilot
the effects on microbial recognition and inflam- study in AgP patients selected based on their IL6
mation discussed above. It is conceivable that a haplotypes [44] could, for example, suggest that
more inflamed milieu, characteristic of patients ‘IL6 positive’ subjects may benefit from adjunc-
with ‘pro-inflammatory’ genetic profiles, may tive antimicrobial therapy, as they might be more
favour the growth of bacteria which grow well in likely to have a tendency to re-developing dysbi-
inflamed environments, then shifting the whole otic, disease-associated biofilms also after treat-
microbiota towards a disease-predisposing one. ment. Studying mechanisms of association
between the subgingival biofilm and other bio-
films elsewhere in the body, such as in gastro-
7.5 hallenges and Future
C intestinal tract, vagina and skin, could shed light
Directions into mechanisms of host-bacteria crosstalk.
Periodontal genetic research still has a long way

to go before it identifies clear predisposing host
gene variants in different populations. This is Clinical Relevance
complicated by issues such as sample size, defi- • Host genetic variants seem to play an
nition of health and disease, genetic methodol- important role in determining the compo-
ogy, difficulty at controlling for other predisposing sition of microbial biofilms in the human
factors and epigenetic influences. Recently, gene body, including the dental biofilm.
variants in the IRF5 gene have been associated • Some subjects may be more predisposed
with IBD [48], while SNPs in the DEFDB1 gene to periodontal disease onset and pro-
and PRDM1 gene have been associated with gression through the activity of host
chronic and aggressive periodontitis, respectively genetic variants in the response to the
([31, 49]). These gene polymorphisms appear to microbial challenge.
be able to contribute to a disturbance of the • Knowing which subjects are more pre-
immunological barrier, thus promoting dysbiosis disposed to colonization by specific
of the local microflora, potentially predisposing microbes could affect the clinical man-
to disease. Hence, it would be interesting to focus agement of periodontitis cases.
periodontal infectogenomics research on a vari-
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Antimicrobial Peptides: Roles
in Periodontal Health and Disease
8
Daniel Jönsson
8.1 Antimicrobial Peptides The endogenous cationic AMPs, like cathelicidin

LL-37 and defensins, exert a similar mechanism
Why do cockroaches and rats survive in sewers on prokaryotic cell walls as chlorhexidine, indi-
and other extremely challenging conditions? The cating an importance of maintaining a healthy
answer is that through evolution they have been homeostasis between the oral AMP-profile and
equipped with antimicrobial peptides (AMPs) the oral microbiome to promote and sustain peri-
that protect them from microbiological insult. In odontal health.
the case of cockroaches and rats, the AMPs are The online AMP database APD3 (http://aps.
very potent, allowing for these challenging envi- unmc.edu/AP/) includes 1007 AMP, out of which
ronments [1–3]. AMPs are the most ancient and 112 are human AMPs. It is not within the scope
primitive arm of the human immune system and this chapter to discuss all 112 peptides, but rather
are expressed in mammals, insects, fungus, introduce the reader to human AMPs, particularly
trees—virtually every multicellular organism in the context of the oral milieu and periodontal
that coexist with bacteria, including bacteria. disease.
AMPs cover the outer barriers of our body, such
as epithelium and skin, enabling us to live in
coexistence with what some consider a complex 8.2 Introduction
organ—the microbiome [4]. As we now, through
the technological advancements in microbiology, AMPs were first described more than 90 years ago
start to comprehend the complexity of the micro- [5]. We now know that this diverse group of pep-
biome, we can also appreciate the complexity of tides display their antimicrobial effects through
the AMP-profile. different mechanisms. AMPs are often referred to
AMPs and agents mimicking AMP molecular as the endogenous antibiotics, although they are
properties are widely used in the everyday dental actually more efficient than antibiotics, as they
clinic that practice periodontal treatment and care, clean up after themselves! One of the problems
namely penicillin and chlorhexidine, respectively. with antibiotics, especially when treating sepsis, is
that although the bacteria get lysed the cell wall
can still cause pro-inflammatory and proapoptotic
D. Jönsson
Department of Periodontics, Malmö University, signaling [6]. AMPs, on the other hand, do not
Malmö, Sweden only lyse b acteria, but also neutralize endotoxins,
Folktandvården Skåne, Malmö, Sweden including lipopolysaccharides (LPS) from gram-
e-mail: [email protected] negative bacteria.

DOI 10.1007/978-3-319-53737-5_8
98 D. Jönsson
Perhaps because of the antibiotics reference, The history of research on AMPs has also
there has been a focus on the cell lytic activity of been the story of the research on innate immune
AMPs. The cell lytic action of AMPs are definitely response. Hans Boman studies dating from the
important in the phagosomes of leukocytes and in 50s to his death (passed away 2009) is an expose
the core of inflammation, e.g., in the periodontal of the progress of research in the field of innate
lesion. However, when it comes to the complex immune response and AMPs in particular.
and intricate homeostasis between the microbiome Starting with a continuation on Stephens’ and
and AMPs in the oral cavity, the neutralizing Marshall’s work were Boman and colleagues
effects of AMPs on bacterial endotoxins may be vaccinated flies by an injection of harmless bac-
more important, as it occurs at lower concentra- teria which protected against Pseudomonas aeru-
tions of AMPs typically associated with health. ginosa, indicating a cell-free defense [11]. Due to
The concentration of the AMP LL-37 found in the work of Boman’s group we now have a much
GCF in periodontal health is sufficient to reverse more full picture of the innate immune response,
LPS-induced production of pro- inflammatory particularly the cell-free epithelial response [12,
cytokines [7]. The homeostasis between bacteria 13]. Boman’s group also published an important
and the inflammatory response in the gingival sul- paper on the LL-37 and periodontal disease,
cus is always ongoing, including in clinically as reporting that lack of the cathelicidin LL-37 may
well as histologically healthy tissues. be the reason why Kostmann patients get peri-
odontal disease [14].
The arginine-rich cationic peptides that later
8.3 History of AMPs were named defensins and cathelicidin were first
described to possess antibacterial properties
Sir Alexander Fleming, Nobel Prize awardee in against both gram-positive and gram-negative
1945, discovered AMPs in 1922. He published bacteria by Zeya and Spitznagel in 1963. The
his findings in the paper “On remarkable bacte- molecules extracted were from guinea pigs poly-
riolytic element found in tissues and secretions” morphonuclear leukocytes. In 1984 Selsted et al.
[5], in which he recognized the antimicrobial purified the molecules from rabbit granulocytes
capacity of endogenous nasal secretions. He and named them defensins [15]. The same
named the activity lysozyme, as it lysed the bac- research group later identified defensins in
terial lawns on a culture-dish. Six years later, Sir humans [16].
Fleming made the discovery that would start a A recent game changer in AMP-research is
new era in medicine and save billions of lives that beta-defensins can unmask potent antimicro-
when he described the antimicrobial capacity of bial activity by structural changes [17]. This sug-
Penicillium notatum [8]. gests that AMPs that have been doomed to be
Insects, like moths and flies, have a much less relatively less potent and consequently less
developed inflammatory response that consist of important have to all be re-evaluated.
hemocytes (fagocyting cells) and an AMP
humoral defense. Therefore, AMPs are a more
crucial segment of the inflammatory response in 8.4 AMPs Grouping
insects than in vertebrates [9]. In 1962 Stephens
and Marshall [10] discovered that heat stable Some reviews on AMPs focus on defensins and
relatively small (compared to lysozyme) AMPs cathelicidin [13, 18], some have more inclusive
were produced by wax moth larvae when they approach, and include metal ion chelators, pro-
were exposed to Pseudomonas aeruginosa. teinase inhibitors, peroxidases, and agglutinating
Interestingly, the protective effect of the AMPs peptides [19, 20]. This is mainly because defen-
could transfer to another moth by hemolymph sins and cathelicidin have been more extensively
transfusion, showing that these innate protective researched than any other AMPs. The grouping
peptides are soluble. here is inclusive and thusly a bore detailed
8 Antimicrobial Peptides: Roles in Periodontal Health and Disease 99
Table 8.1 Categorization of AMP based on their antimicrobial action

Group Antimicrobial action Members
Cationic peptides To destabilize cell membrane and Alpha- and beta-defensins, LL-37, azurocidin,
to neutralize LPS CCL-28, heparin-binding growth factor,
histatins, statherin, adrenomedullin, calcitonin
gene-related peptide, neuropeptide Y, substance
P, and vasoactive intestinal peptide
Lipid-binding peptides Also a cationic peptide, but the Bactericidal/permeability increasing protein,
lipid-binding action intensifies the parotid secretory protein and palate lung and
binding to cell membrane and LPS nasal epithelial clone family
Lysomzyme Damages surface exposed Lysozyme
peptidoglycans
Agglutinating and adhesive Agglutinate bacteria and prevents Mucin 7, β-2-microglobulin, fibronectin,
peptides and proteins them from attaching to surfaces surfactant protein A, proline-rich proteins,
prolactin-inducible protein
Ion chelators Disturb intracellular signaling Lactoferrin, S100 proteins, and ATP
pathways of bacteria, and thereby
restrict its growth and vitality
Protease inhibitors Inactivate proteases from bacteria Cystatins, trappin gene family members
secretory leukoprotease inhibitor protein and
elafin
Peroxidases Cell degradation and loss of action Lactoperoxidase and myeloperoxidase
through the peroxidase system
description of all AMPs would be too compre- CCL28 [24, 25], heparin-binding growth factor
hensive. For further, more detailed description, [26], histatin 1, 3, and 5 [27, 28], statherin [29],
please see the references. Table 8.1 summarizes and the neuropeptides adrenomedullin [30, 31],
the categorization of AMPs. calcitonin gene-related peptide [31], neuropeptide
Cationic peptides—Cationic peptides are Y [31], substance P [31], and vasoactive intestinal
small (< 10 kDa) positively charged molecules peptide [31].
that can perforate the cellular membrane of both Lipid-binding AMPs—This group includes
gram-positive and -negative bacteria and interact bactericidal/permeability increasing protein (BPI)
with the LPS/CD14-signaling cascade that initi- and its homologs; BPI-like proteins (including
ates production of pro-inflammatory cytokines. parotid secretory protein (PSP)) and PLUNC
The human cathelicidin LL-37, beta- and alpha- (palate lung and nasal epithelial clone family).
defensins dominate the group. LL-37 is expressed PLUNC can further be categorized into short
in neutrophils, macrophages, and epithelial cells. and long PLUNC, also known as BPI fold con-
LL-37 has a broad-spectrum antimicrobial effect taining family A and B, respectively. BPI is a
and many immunomodulatory effects [12, 18]. boomerang-shaped cationic molecule that exerts
LL-37 is also important in wound healing, often bactericidal activity by perforating the cell mem-
lacking in chronic ulcers [21]. brane (preferably gram-negative bacteria) and
In general, alpha-defensins are the defensins binds endotoxins, including LPS which it binds
of neutrophils (and also Paneth cells), and beta- with high affinity [32]. Neutrophils and epithelial
defensins the epithelial defensins. Both LL-37 cells, including salivary duct cells, express the
and alpha-defensins are an important part of BPI-proteins [33]. PLUNC-proteins are multifac-
the intracellular phagocytosis mechanism of eted and not only possess strong LPS-neutralizing
neutrophils, and released into the extracellular capacity, but also inhibit dendritic cell growth, act
space when the cells burst. Beta-defensins are as chemoattractant and opsonization agent [34].
released through LPS-mediated signaling [18, BPIs are actually upregulated in mucosa by
22]. Cationic AMPs also include: azurocidin [23], resolvins [35], which may offer a pharmacological
100 D. Jönsson
approach to treating periodontal disease [36]. comitans. Cystatin SA demonstrated an

Decreased PLUNC expression in nasal polyps antimicrobial activity on A. actinomycetemcomi-
has been associated with multibacterial coloniza- tans in vivo, which was reversed by cystatin SA
tion in chronic rhinosinusitis [37]. antibodies. Interestingly, the antimicrobial effect
Lysozyme—The first AMP to be categorized was independent of protease inhibitory function.
was lysozyme [5]. It is a small protein (145 kDa) Cystatin 9 also exerts an immunomodulatory
present in body fluid, including saliva, but also in function, increasing the efficiency of macrophage
neutrophils. Lysozyme mainly exerts its activity phagocytosis and promoting an upregulation of
against cell membrane integrity of gram-positive macrophage proteins involved in anti-
cells by damaging surface exposed peptidogly- inflammation and anti-apoptosis while restrain-
cans [38]. ing pro-inflammatory associated proteins [58].
Agglutinating and adhesive peptides and pro- Peroxidases [63] in the oral cavity are lacto-
teins—Agglutinating bacteria inactivates them peroxidase [64] and myeloperoxidase [63] which
and prevents them from attaching to surfaces. form the peroxidase system of saliva. The reac-
This group consist of peptides, like mucin 7 [39– tion products are active against several bacteria
41] and β-2-microglobulin [42], but also big mol- associated both with dental caries and periodon-
ecules that are technically proteins rather than tal disease [63].
peptides, like fibronectin [43–46], surfactant pro- Importantly, the oral AMPs are not separate
tein A [47], proline-rich proteins [48], and entities, but complement each other. Choi et al.
prolactin-inducible proteins [49]. allowed P. gingivalis LPS to interact with pooled
Ion chelators interact with and disturb vital saliva and then investigated what AMPs were
intracellular signaling pathways of the bacteria, attached to the LPS molecule. They found inter-
and thereby restrict its growth and vitality. action with alpha-amylase, cystatin, prolactin-
Examples of AMP chelators are lactoferrin [50], inducible protein, lysozyme C, immunoglobulin
S100 proteins [51, 52], and, according to a newly components, serum albumin, lipocalin-1, and sub-
published paper, also ATP [53]. Lactoferrin maxillary gland androgen regulated protein 3B
single-nucleotide polymorphisms (SNPs) are [65]. This indicates that the saliva, and probably to
associated with aggressive periodontitis [54] and a similar extent GCF, is to be considered as a pot of
have broad antibacterial, antiviral, and antifungal AMPs that share the ability to interact with bacteria
properties [55]. Lactoferrin knockout mice are and to neutralize their endotoxins. Indeed, there is a
more susceptible to A. actinomycetemcomitans- synergistic relationship between AMPs [66].
induced periodontitis [56]. Lactoferrin reportedly
inhibit P. gingivalis proteases by its ion chelator
mechanism [57]. This also inhibited the biofilm 8.5 AMPs in the Periodontium
formation capacity of P. gingivalis.
Protease inhibitors inactivate proteases from In gingiva and oral mucosa, the inflammatory
bacteria, but also through other mechanisms [58, responses face unique challenges confronting an
59]. The group includes cystatins [58–61], the immense quantity of bacteria. The mucosal
trappin gene family members secretory leukopro- response can first be broken down into the innate
tease inhibitor protein and elafin [62]. Cystatins and adaptive immune response, and the innate
reportedly exert antibacterial effect, specifically inflammatory response can be further categorized
on P. gingivalis [61] and A. actinomycetemcomi- into the acellular and cellular response. The acel-
tans [59]. Ganeshnarayan et al. [59] investigated lular response comprises AMPs, which is the
the affinity of salivary peptides to A. actinomy- very first line of defense. The AMPs are mainly
cetemcomitans in subjects with high salivary found in saliva and pellicle as well as the epithe-
anti-A. actinomycetemcomitans activity and lium. If or when the microbes cross the epithe-
found that lactoferrin, immunoglobulin A, kalli- lium through the epithelial barrier, they are
krein, and cystatin SA bind to A. actinomycetem- exposed to the phagocytic cells, like stationed
macrophages and Langerhans cells. In the gingi- different histones [75], but there is also neutrophil
val sulcus there is also a high presence of the elastase [75], S100 A8 and A9 [75], azurocidin
phagocytic neutrophils. In a prolonged exposure [75], cathepsin [75], lactotransferrin [75], calpro-
of microbes to the antigen-presenting cells, tectin [75], cathelicidin LL-37 [76], and several
T-cells will be recruited and activated, and others [75]. For more detailed description of
B-cells will differentiate into plasma cells. NETs, please see Cooper et al. [74].
The oral epithelium is highly specific, and the Due to the high presence of neutrophils in the
AMP expression is different between the differ- periodontal lesion, the AMP levels become so high
ent mucosal sites, and in response to different that they do not only lyse bacteria, but also resi-
infections. When comparing the expression of dent cells, like periodontal fibroblasts and osteo-
defensins in healthy oral tissue samples from gin- blasts [7, 77]. This may be due to the need of more
giva, tongue, buccal mucosa, labial mucosa, sub- space to allow the periodontal lesion to expand.
mandibular glands, small labial glands, and When the inflammatory response is sup-
dental pulp, the expression is higher in gingiva pressed, due to, e.g., severe immunosuppressive
and in the submandibular gland [67]. In addition, therapy, bacteria may reach the alveolar bone,
the tissue response of AMP-expression in peri- which potentially can cause osteonecrosis [78]. It
odontitis causes a higher expression of defensins could be that the threat of deadly osteonecrosis
than candidosis-infections [67]. (prior to Fleming’s discovery of penicillin [8]) is
The gingival sulcus has a challenging mis- the evolutionary incentive of the strong peri-
sion—to maintain the epithelial barrier around the odontal inflammatory response that unfortunately
tooth, which penetrates the mucosa. To hinder the causes tissue breakdown and tooth loss.
down-growth of bacteria and to sustain the junc-
tional epithelial barrier there is a high presence of
AMPs in the sulcus, due to the high inflammatory 8.6 Double Edge Swards
activity, even at clinically healthy sites [68–70].
Particularly, the high density of neutrophils in the Many of the AMPs display a large array of func-
periodontium causes a high concentration of tions; LL-37 can affect apoptosis (both pro- and
AMPs, as several important AMPs, such as LL-37 anti-) [7, 77, 79–81] and chemoattractant [82],
and alpha-defensins, are abundant in neutrophils fibronectin can agglutinate bacteria [43–45], but
[69, 71]. The high bacterial load in sulcus in itself is obviously also a key component in extracellu-
also induces AMP- expression through toll-like lar matrix. The functional duality of AMPs does
receptor (TLR) and nucleotide oligomerization in some instances cause a paradox. This can be
domain (NOD) signaling, causing a feedback exemplified by LL-37, which is important in
loop [72]. In periodontal disease, the epithelial wound healing [21] and has well-known antimi-
barrier in sulcus is lost, and the only remaining crobial features, but in psoriatic lesions LL-37 is
barrier between the bacteria and the alveolar bone found to reach concentrations exceeding that of
is a strong inflammatory response together with healthy skin [83, 84]. Because of the high con-
neutrophil extracellular traps (NETs). centrations in psoriatic lesions and the capacity
When neutrophils in the periodontium undergo to activate the innate immune response when
cell death, they release a whole range of AMPs released extracellularly, they are often termed
that end up in gingival crevicular fluid of the peri- alarmins (for an excellent review on alarmins
odontal pocket. Importantly, neutrophils also [85]). As visualized in Fig. 8.1, the concentration
form NETs [73]. NETs are DNA strings from of LL-37 found in healthy GCF is primarily anti-
neutrophils that compose the extracellular matrix inflammatory, and the concentrations found in
of the innate immune response of the periodontal GCF in periodontitis reaches the concentrations
pocket that capture bacteria, degrade their viru- proapoptotic in periodontal ligament cells [7].
lence factors, and kill them [74]. The NETs con- The duality aspect of AMPs is important to
tain large quantities of AMPs, mostly (70%) are bear in mind, as focusing on the antimicrobial
102 D. Jönsson
Pro-apoptotic situation, a disruption of homeostasis causes an

overgrowth of a specific microbe, as in the case of
Anti-inflammatory
Helicobacter pylori in peptic ulcers [88]. In
another situation, the commensal microbiota can
0 1 2 µM LL-37 be disturbed and become pathogenic from changes
H CP in the inflammatory response or milieu and there-
fore display a nonspecific microflora, as in
Fig. 8.1 The antimicrobial peptide LL-37 is anti- Crohn’s disease [89] and periodontal disease in
inflammatory in concentrations associated with periodon- which the composition of the microbiota can dif-
tal health (H) but proapoptotic in concentrations associated
fer even between sites in the same mouth [90].
with chronic periodontal disease (C P). The width of the
boxes represents the upper and lower quartile of LL-37 in Unfortunately, we know less about the changes in
GCF in health and chronic periodontitis [68] AMP-profile during these diseases than we know
about the changes of the microbiota.
capacity may attract ideas on increasing the levels As illustrated in Fig. 8.2, AMPs can change
of these peptides pharmacologically to intervene the microbiota, through neutralizing endotoxins
infections, such as periodontitis. On the contrary, and disintegrating cell membrane integrity.
focusing on the pro-inflammatory and proapop- Reversely, the oral microbiota can alter the AMP-
totic effects may generate thoughts on suppress- profile by LPS-TLR/CD14 cell signaling. For
ing them, which would probably cause severe obvious reasons studies proving causal changes
microbiological dysbiosis. One way to brake this in the microbiota in response to alterations of
catch 22 may be to utilize agents that increase specific AMPs is primarily performed in mice
AMP-levels to healthy physiological concentra- models and human in vitro benchtop models.
tions. Vitamin D deficiency causes LL-37 levels There are however case presentations of diseases
to drop, reportedly increasing susceptibility to and single mutations in humans that also add to
infections in the lung; however vitamin D supple- the knowledge of the importance of AMP-
mentation can increase the LL-37 levels revering microbiota homeostasis.
the risk of lung infection [86]. One example of mouse model are the trans-
Considering that the antimicrobial capacity of genic mice models. To show the impact and mag-
AMPs have been known for about 90 years and nitude of effect from human alpha-defensin 5
that the concept of alarmins was introduced about (HD-5), Salzman et al. [91] developed a trans-
a decade ago [87], our knowledge of AMPs in the genic mouse model expressing HD-5. Mice lack
oral cavity mainly focuses on the antimicrobial defensin 5 and the transgenic mouse expression
capacity. of HD-5 in their Paneth cell epithelium.
Interestingly the transgenic mice were resistant
to Salmonella typhimurium, compared to
8.7 The Homeostasis Between wild-
type mice, indicating the importance of
AMPs and the Microbiota HD-5 in defense against Salmonella infections.
In another example Wehkamp et al. [92] report a
From the day we are born, until that final day lower expression of HD 5 and 6 in ilium Paneth
there is a constant homeostasis between the cells and intestinal mucosal extract from subjects
inflammatory defense and the commensal micro- with Crohn’s disease compared to healthy con-
biota. The moment we die microbiota in the oral trols. Using a similar model of transgenic mice as
cavity, in the gut, on the skin, and elsewhere no Salzman et al. [91], they found a difference in the
longer has AMPs and cell mediated inflammatory luminal microbiota when comparing HD-5-
response as counterpart, and consequently our positive and -negative mice.
bodies start to molder. During our lifetime, dis- There are mouse models that show importance
ruption of this complex homeostasis causes dis- of the adaptive immune system in turning a com-
eases, including periodontal disease. In one mensal microbiota into a pathogenic. In the models
Fig. 8.2 The reciprocal Neutralizing endotoxins and disintegrating cell membrane integrity
relationship between the
AMP-profile and the
oral microbiota
AMP-profile Oral
microbiota
Changing AMP-profile through LPS -TLR/CD14 cell signaling
the microbiota is disturbed by changes in the pores [94–96]. At a low concentration the pep-
innate immune response, including AMPs, which tides are bound parallel to the lipid bilayer, but
causes a dysbiotic/pathogenic microbiota, that as the concentration of peptides increase, the
can then be transferred to wild-type mice in peptides begin to orient in a perpendicular ori-
which dysbiosis is sustained. One example is a entation and form “barrel-stave” shaped pores in
study by Garrett et al., where transgenic T-bet the lipid membrane [95]. This research is based
(transcription factor) knockout mice developed on lipid bilayer models, and although it is a pos-
ulcerative colitis, causing a dysbiotic microbiota. sible model, it may be simplistic since different
When the dysbiotic microbiota was transferred to membrane proteins constitute about 50% of the
genetically intact wild-type mice, they too devel- microbial membranes [96]. Zwitterionic phos-
oped ulcerative colitis. Similarly, mice that lack pholipids and cholesterol are prominent constitu-
the bacterial flagella sensitive toll-like receptor 5 ents of eukaryotic cell membranes, and they will
(TLR5) express metabolic syndrome features and strongly reduce the interaction of cationic AMPs
insulin resistance and also a dysbiotic gut micro- and the cell membrane by changing the mem-
biota. Transferring this gut microbiota to wild- brane net charge to less anionic [96]. Therefore,
type mice caused similar metabolic syndrome there needs to be higher cationic AMP levels to
and insulin resistance in wild-type mice [93]. form pores in cell membranes of eukaryotic cells
What these studies show is that even though it than in bacteria.
may be tempting to “prove” microbiological cau- The first step of the AMP-lysis of bacterial
sality by transferring dysbiotic microbiota from a membrane is attraction of AMPs to the bacteria.
diseased animal to a healthy that then gets the The obvious mechanism for AMP attraction is
same disease/condition, the inflammatory through electrostatic bonding between the cat-
response may very well still be of key importance ionic AMPs and the anionic LPS and teichoic acid
in creating this dysbiotic microbiota. on gram-negative bacteria and gram-positive bac-
teria, respectively. For the AMPs to reach the lipid
bilayer, the AMPs have to penetrate the LPS and
8.8 MP Cell Membrane
A peptidoglycans. This may be through a process
Interaction termed “self-promoted uptake,” in which AMPs
first bind to LPS, causing destabilization, which
Because the cell wall lipid bilayer is negatively then allows excess to the lipid bilayer [97].
charged, it attracts cationic peptides, including Microbiome specific lipid receptors may also be
both cationic peptides, like LL-37 and defen- involved in the AMP antimicrobicidal action [98].
sins, but also the lipid-binding AMPs and lyso- To disrupt the integrity of eukaryotic cell
zyme. Cationic AMPs reduce the cell membrane membranes much higher levels are required than
integrity through membrane perturbation. At a for prokaryotic cell, that high levels are reached
certain peptide/lipid ratio on the cell surface the in some pathologic conditions, such as in the
peptides orient in a perpendicular manner and gingival sulcus during periodontal disease and
insert into the bilayer, forming transmembrane in psoriatic lesions [7, 68, 83, 84]. The effect
104 D. Jönsson
of cationic AMPs on eukaryotic cell vitality is 8.9 AMPs and Endotoxins

cell specific and may be cytotoxic in some cells
types, proapoptotic in others, and anti-apoptotic The most important pathway for triggering dys-
yet another [79–81, 99–105]. Part of the explana- biosis through an inflammatory response is when
tion of this cell specificity may be endogenous LPS, a gram-negative bacteria cell wall segment,
peptides and proteins that can reverse the action binds to LPS-binding protein (LBP) at macro-
of cationic peptides, including mucin [106, 107] phage surfaces. When CD14 and toll-like recep-
and the membrane protein p33 [105, 106]. tor 4 (TLR4) recognize the LPS/LBP complex, a
Lysozyme exerts its anti-microbiological signaling cascade is initiated through MyD88
activity preferentially against gram-positive and TRIF that activate the transcription factors
cells, because of its peptidoglycan-degrading like NF-κB and AP-1, which subsequently acti-
property. Lysozyme hydrolyzes the bond between vates the transcriptional activity of genes of pro-
N-acetyl glucosamine and N-muramic acid lead- inflammatory cytokines [110–113]. The LPS/
ing to degradation of peptidoglycan in the gram- CD14 signaling cascade is not macrophage or
positive cell wall, and thereby access to the lipid even leukocyte exclusive, but can occur in most
bilayer. This catalytic process on the peptidogly- cells, including periodontal fibroblasts [114]. As
can layer described is termed muramidase activ- iAMPs have the ability to interrupt the initiation
ity; however, this was recently challenged by of this signaling cascade through several differ-
Nash et al. [108]. They reported antibacterial ent mechanisms—(a) neutralizing LPS by bind-
activity also from muramidase deficient recombi- ing to the molecule, (b) disrupting aggregates of
nant lysosome in vitro, and in muramidase defi- LPS, (c) binding of AMPs to CD14, and (d) by
cient mice in vivo. Nevertheless, lysozyme is an scavenging LPS (Fig. 8.3).
important AMP against gram-positive cells. LPS is an anionic molecule and is build up by
To underline the importance of the whole AMP- a hydrophilic O-antigen, a polysaccharide core
profile, a study investigating the effect of BPI and with a negative charge and a glycophospholipid,
alpha-defensins on Escherichia coli cell growth lipid A. Lipid A is the active segment expressing
found a synergistic effect between the AMPs [109]. the endotoxic activity. The interaction between
Other groups have confirmed synergy between LPS and AMPs depends on the net charge of the
AMPs [66], which indicates an importance of the AMP and hydrophobicity [115]. Electrostatic
heterogeneity of the AMP-profile. binding prefers binding to the polysaccharide
a b c
LPS LBP LBP LPS
LPS
LPS
LPS
CD14
TLR4
d LPS LBP e
Pro-inflammatory transcription LPS

CD14
TLR4
Fig. 8.3 Panel (a) illustrates LPS/LBD/CD14/TLR4 ing of LPS and LBP, (panel c) AMPs disintegrating LPS
transcription, which is an important pathway for pro- aggregation, (panel d) AMPs attaching to CD14 and
inflammatory transcription. This pathway of pro- thereby disrupting the subsequent cell signaling. Lastly,
inflammatory transcription can be disrupted by AMPs (panel e) AMPs can attach to cell membrane and attract
through (panel b) AMPs binding LPS disabling the bond- scavenged LPS
segment of LPS and hydrophobic interaction Patients with aggressive periodontitis and dys-
dominates binding to lipid A [116], however functional lactoferrin may benefit from supple-
small cationic peptides are able to penetrate the mentation with recombinant lactoferrin. Perhaps
LPS layers and bind to lipid A [117]. Binding of a salivary test checking the AMP-profile of
AMPs to lipid A segment of LPS neutralizes the saliva from subjects with periodontal disease
endotoxic properties and prevents the LPS/LBP could be a first step along that line. The next
complex to form and downstream transcription step, producing the drug may be more challeng-
of pro-inflammatory cytokines to take place ing due to the functional duality of many AMPs.
[118–120]. BPIs have a high affinity to lipid A With continued research in this field, we may
due to hydrophobicity and are cationic. Due to however be able to pinpoint the separate func-
the high affinity of BPI to LPS, LPS favors BPI- tional entities of the different peptides.
mediated endotoxin neutralization to binding to The intricate homeostasis between AMPs
LBP, resulting in an attenuation of the pro- and the oral microbiome works perfectly well
inflammatory cellular response at neutrophil-rich in most people and may very well be the
inflammatory sites, such as the periodontal lesion answer to the question the general dentists
[121–123]. The cationic peptides LL-37 and sev- asks themselves after seeing an older patient
eral defensins also bind and neutralize LPS [18]. with less impressive oral hygiene and no peri-
LPS aggregation is an important event, as odontal disease. Therefore, it may be a poor
monomer endotoxins exert less endotoxic activ- strategy to use AMPs on a larger population in
ity [124]. LL-37 as well as other AMPs can disin- toothpastes, and rather have tailor made sup-
tegrate LPS aggregation and thereby exert an plementation. As mentioned in this chapter
anti-inflammatory effect [118, 125]. changing the AMP-profile can alter the micro-
AMPs also have the ability to bind directly to biome and thereby disrupt this homeostasis.
CD14 without activating the receptor and by
doing so blocking LPS [126, 127].
Finally, incorporation of cationic AMPs in References
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Periodontal Pathogenesis:
Conclusions and Future Directions
9
Georgios N. Belibasakis and Nagihan Bostanci
This textbook on periodontal pathogenesis Microbes may just be transient travelers in the
aspired to provide an overview of our current oral cavity, or find favorable conditions for their
understanding of biological processes underlying growth and survival. The stable composition of
the disease, and discuss their clinical relevance the oral microbiome is a result of the dynamic,
and applicability. We now realize that periodon- yet balanced, interaction of the microbes with
tal disease affects 10% population in its severe each other, with their host, and with their local
form, and that it is primarily a disease of aging. environment in general. Microbiome stability is
Despite advances in prevention measures over indeed a requirement for oral health because it
the past decades, the prevalence of the severe favors the survival and persistence of health-asso-
form of disease has not decreased. This high- ciated species. These species not only prevent the
lights periodontitis as an existing health problem, overgrowth of endogenous opportunistic patho-
unlikely to be completely resolved with our pres- gens or the invasion of exogenous pathogens, but
ent therapeutic approaches. Continuous enrich- they also prime the immune system to be at readi-
ment of our biological knowledge of the disease ness level. Changes in microbial composition are
and continuous efforts in bridging this knowl- perceived by the immune system as deviations of
edge to the clinical application will hopefully the normal, which then strives to correct them via
lead to more holistic approaches for diagnosis, inflammatory responses. Changes in microbial
evaluation of patient susceptibility, or selection composition can be induced by variations of the
of efficient treatment options. This conclusive oral milieu. Hence, unfavorable conditions for the
chapter summarizes updates and take-home mes- survival of commensals may lead to overgrowth
sages from previous chapters, to consider of opportunistic disease-associated species, a con-
further. cept that has led to the ecological plaque hypoth-
The microorganisms that reside in the oral cav- esis on how oral microbiota can cause periodontal
ity, or the “oral microbiome” are highly diverse. disease [1]. Although several of those species are
The number of the different known types is likely usually referred to as “periodontal pathogens,”
to increase, as the technologies to discover those they are suited for the definition “opportunis-
have become more sensitive and sophisticated. tic pathogens”; due to that they are frequently
also detected in health, albeit at low numbers.
This leads us to postulate that no single bacterial
G.N. Belibasakis (*) • N. Bostanci
Department of Dental Medicine, Karolinska Institute,
species alone is responsible for the disease, but
Stockholm, Sweden groups of them, under certain conditions, may act
e-mail: [email protected] together to cause disease.

DOI 10.1007/978-3-319-53737-5_9
112 G.N. Belibasakis and N. Bostanci
Bacteria are prone to grow on tooth surfaces, tissue. The catalytically active MMP-8, which
in the form of organized biofilm communities. degrades interstitial collagens, is a predominant
This preferential polymicrobial lifestyle provides MMP in periodontitis-affected gingiva [4]. A large
to the individual species robustness and further body of scientific literature evidence demonstrates
survival possibilities. The microbial composition that the levels of aMMP-8 in gingival crevicular
of biofilms is very complex and can vary not only fluid and saliva correlate with the occurrence of
between individuals, but also between sites of the periodontal disease, and that periodontal treatment
same individual. Bacterial survival and growth is commensurate with the reduction of its levels in
depends on their adaptability to the microenvi- these fluids [5]. Hence, aMMP-8 can constitute a
ronmental conditions at a given niche of the oral biological indicator for excessive collagen tissue
cavity, such as availability of suitable nutrients, breakdown and thus a diagnostic biomarker for
oxygen, and other physicochemical parameters. progressive periodontal disease. Accordingly, a
Importantly, biofilms can form both under aero- limited number of molecules of the immune
bic and anaerobic conditions. Subgingival bio- response can tip the balance between bone forma-
films are formed by bacteria that can colonize the tion and resorption towards of the latter [6]. In par-
periodontal pocket and grow under its anaerobic ticular, the RANKL-OPG axis is a key in this
conditions, during the progression of periodontal process, as RANKL stimulates osteoclast forma-
disease. No one of the individual species populat- tion and bone resorption, whereas OPG inhibits
ing a subgingival biofilm can single-handedly this action [7]. An increase in the RANKL/OPG
cause the disease. Instead, groups of them can ratio in gingival crevicular fluid indicates the
synergize and establish a dysbiotic relationship occurrence of periodontal disease [8], but treat-
with the host, eliciting a chronic inflammatory ment of the disease by conventional means does
response [2, 3]. Opportunistic pathogens in the not affect this, thus indicating that pharmacologi-
subgingival biofilm, such as Porphyromonas gin- cal modulation of this set of molecules is a path to
givalis and Aggregatibacter actinomycetemcomi- explore in future treatment modalities [9, 10].
tans, produce a number of virulence factors that While it is clear that the dysbiotic interaction
can contribute to this dysbiotic relationship with between the host tissues and the microbes of the
the host and elicit a disadvantageous host biofilm community is accountable for inflamma-
response. tory periodontal tissue destruction, it is also clear
In periodontal health, a low grade of harmless that genetic susceptibility somehow holds a fun-
immune response is elicited by the periodontal tis- damental role in this process. Genetic factors of
sues. The purpose of this is to control microbial the host, or gene variants in different populations,
colonization and to “guard” the tissues against can influence microbial colonization and compo-
invasion and multiplication of oral microorgan- sition of the biofilms, and can reduce the bio-
isms, at the same time avoiding to inflict collateral logical threshold for triggering an inflammatory
damage to the tissues. Hence, the control of the response. This genetically driven modulation of
immune response without its complete abolition is the interaction between the host and its coloniz-
a key to maintenance of periodontal health. Yet, an ing microbes may underline the susceptibility
uncontrolled immune response will cause a to the disease processes by accelerating dysbio-
chronic inflammatory tissue destruction, which sis [11]. In other terms, some individuals may
will clinically manifest as periodontitis. Subsets of be genetically more predisposed than others for
cells of the immune system (initially neutrophils developing periodontal disease, or may exhibit
and later on lymphocytes) and inflammatory higher progression rate of the disease. The anti-
signaling molecules are orchestrating this destruc- microbial peptides present in saliva and oral cav-
tive process. The net result is the production and ity in general may also play a role in the genetic
release of proteolytic enzymes, such as matrix susceptibility to periodontal disease. They estab-
metalloproteinases (MMPs), within the gingival lish an intricate homeostatic relationship with the
9 Periodontal Pathogenesis: Conclusions and Future Directions 113
oral microbiome, and changes in one’s antimicro- intervention. Understanding genetic predisposi-
bial peptide profile may also lead to dysbiosis. tion to periodontal disease, and how this may
affect the microbial colonization patterns could
facilitate prevention or clinical management, par-
9.1 Future Directions ticularly in susceptible populations. In addition,
in Diagnosis and Treatment proactive modulation of signaling pathways and
of Periodontal Disease molecules that are involved in periodontal connec-
tive tissue and bone breakdown, such as aMMP-8
In terms of microbial etiology of periodontal and RANKL/OPG, are warranted. Such interven-
disease, it is becoming evident that shifts in the tions would help arrest periodontal disease on the
composition of the subgingival microbiota are molecular level, and could thus be included in the
more crucial than the mere presence of a single growing list of “adjunctive” periodontal treatment
or a handful of pathogens for the progression of modalities used in handling patients unresponsive
periodontal disease. Reduction or elimination of to traditional therapy. Hence, as molecular modu-
certain “marker” species such as A. actinomy- latory therapies are becoming more readily avail-
cetemcomitans and P. gingivalis could serve as able and applicable, they are expected to lead to
indicative measures of the efficiency of the peri- more predictive treatment outcomes.
odontal treatment, yet monitoring the stability of Steering towards a better understanding of
the pocket microbiome on a full scale, and in a biological mechanisms underlying periodontal
fast and cost-efficient manner, during the mainte- disease is the preamble for implementing this
nance would be ideal. In monitoring the disease, knowledge in particular clinical situations, and a
there is accumulated evidence over the years to drive for personalized dental medicine [13].
suggest that measuring aMMP-8 in oral biofluids Therefore, it is of great significance that the clini-
holds strong promise for the prediction, diagno- cal audience receives an updated understanding
sis and progression of the disease, and is already of the intricate biological mechanisms of peri-
applied in chair-side/point-of-care applications. odontal pathogenesis, in order to be in readiness
This could be used alone, or in combination with level for the next generation of diagnostic and
other host inflammatory or microbial biomarkers. treatment options. Reciprocally, it is important
Development of point-of-care devices for microbi- that biological researchers remain receptive to
ological and immunological detection that would feedback from their clinical counterparts in seek-
assist clinicians in the diagnostic, monitoring, and ing what would be a meaningful and feasible
selection of treatment aspects are underway [12]. application for clinical reality and patient
Biofilms are difficult microbiological targets to handling.
hit, and the “one size fits all” chemo-mechanical
disruption approach that we follow today cannot be
perfectly achieved on a full mouth scale. Thinking References
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Nagihan Bostanci

ISBN 978-3-319-53735-1 ISBN 978-3-319-53737-5 (eBook)

Library of Congress Control Number: 2017953835

© Springer International Publishing AG 2018

Printed on acid-free paper

This Springer imprint is published by Springer Nature

1 Periodontal Pathogenesis: Definitions and Historical

© Springer International Publishing AG 2018 1

Initital lesion Early lesion Established lesion Advanced lesion

© Springer International Publishing AG 2018 9

survival of Fusobacterium nucleatum, an obli- • Antibiotic resistance in dense biofilms. Biofilm

Table 2.1 Evolving concepts in the bacterial etiology of periodontal diseases

gram-negative, anaerobic bacterial composition, host-compatible organisms [38]. These findings

Chronic periodontitis: The bacterial pro- gressing periodontitis. Nevertheless, available

actions and successions during plaque development.

25. Van Hoogmoed CG, Geertsema-Doornbusch GI,

3.1  efinition of Subgingival

© Springer International Publishing AG 2018 21

3.2 Oral Biofilms:

3.2.3 Subgingival Biofilm RNA-based phylogenetic methods, which origi-

6. Jefferson KK. What drives bacteria to produce a bio-

4.1 Overview P. gingivalis is frequently detected in periodontal

© Springer International Publishing AG 2018 31

Fig. 4.3 Pathogenesis Environmental and acquired risk

Genetic risk factors

Tannerella forsythia, Treponema denticola, individuals with chronic gingivitis or periodonti-

p­ eriodontopathogens such as P. gingivalis, T. for- induce an inflammatory reaction in the gingival

cetemcomitans in individuals with an IL-6 poly-

of A. actinomycetemcomitans upregulates expres- while Type-II fimbriae are associated with

arginine within a peptide (peptidylarginine) [134]. The outcome of TLR2 activation in

Clinical Relevance Summary Points gingivalis to become reestablished in

tein from Aggregatibacter actinomycetemcomitans. 90. Brage M, Holmlund A, Johansson A. Humoral

5.1 Introduction fibroblasts, cementoblasts, and epithelial cells,

T. Sorsa (*) S. Nwhator

Division of Periodontology, Department of Dental N. Rathnayake

© Springer International Publishing AG 2018 51

collagenase or collagenase-2) is the predomi- after scaling/root planning (SRP), cleaning/depu-

GCF MMP-8 level Periodontal site level MMP-8 diagnostics

Active disease Monitoring during MMP-8 levels High risk of

MMP-8 above the

5.3  aliva and Mouthrinse

5.4 Chairside Monitoring phase of periodontitis, i.e., active periodotal degen-

> 25 ng/ml < 25 ng/ml

as surrogated markers of experimented or ongo-

6.1 Introduction determine the best course of action and to pro-

© Springer International Publishing AG 2018 59

damaged or infected tissues. Thus, most inflam- Conceptually, inflammation is categorized in

macrophages in an IFN-γ-independent pathway. Simultaneous analysis of multiple cytokines

elements in the determination of a host–microbe cytokines, endogenous pro-­resolving lipid medi-

6.3.1 Acquired Risk Factors

6.3.2 Innate Risk Determinants associated with an increased risk of periodontitis

with special emphasis on mechanisms of colla-

7.1 Introduction 7.2 Infectogenomics

© Springer International Publishing AG 2018 87

Host genetic variants Host genetic variants

Shift in the composition of the subgingival

Fig. 7.3 Schematic representation of periodontal infectogenomics

Periodontal genetic research still has a long way

38. Papapanou P, Neiderud AM, Sandros J, Dahlen

1 Periodontal Pathogenesis: Definitions and Historical

3.1 efinition of Subgingival

3.2 Oral Biofilms:

3.2.3 Subgingival Biofilm RNA-based phylogenetic methods, which origi-

4.1 Overview P. gingivalis is frequently detected in periodontal

p eriodontopathogens such as P. gingivalis, T. for- induce an inflammatory reaction in the gingival

5.1 Introduction fibroblasts, cementoblasts, and epithelial cells,

5.3 aliva and Mouthrinse

5.4 Chairside Monitoring phase of periodontitis, i.e., active periodotal degen-

6.1 Introduction determine the best course of action and to pro-

elements in the determination of a host–microbe cytokines, endogenous pro-resolving lipid medi-

6.3.1 Acquired Risk Factors

6.3.2 Innate Risk Determinants associated with an increased risk of periodontitis

7.1 Introduction 7.2 Infectogenomics

8.1 Antimicrobial Peptides The endogenous cationic AMPs, like cathelicidin

of cationic AMPs on eukaryotic cell vitality is 8.9 AMPs and Endotoxins