Innate Immunity: Viktor Honti, Éva Kurucz, Gyöngyi Cinege, Gábor Csordás, István Andó
Innate Immunity: Viktor Honti, Éva Kurucz, Gyöngyi Cinege, Gábor Csordás, István Andó
Innate Immunity: Viktor Honti, Éva Kurucz, Gyöngyi Cinege, Gábor Csordás, István Andó
1):1-15, 2015
Acta Biologica Szegediensis
http://www.sci.u-szeged.hu/ABS
Review
Innate immunity
Viktor Honti1, Éva Kurucz1, Gyöngyi Cinege, Gábor Csordás, István Andó*
Immunology Unit, Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
ABSTRACT In this review, we discuss how studying the Drosophila immune system contributes Key Words
to a better understanding of the basic principles of innate immunity. We describe the homolo- blood cell
gies between the insect and the vertebrate immune-regulatory mechanisms and convergent Drosophila
evolutionary traits of the Drosophila and the vertebrate immune system. immune defense
Acta Biol Szeged 59(Suppl.1):1-15 (2015) immune system
innate immunity
Metazoans have inborn structures and mechanisms for lo- All multicellular organisms are constantly exposed to mi-
comotion, sensing, reproduction, maintaining the homeo- crobes, which are generally in balance with the host organ-
stasis and defending against foreign invaders. The proper, ism. On the epithelial surfaces of the skin, in the respiratory
coordinated functioning of these systems is essential for the system and in the gut, they far outnumber cells of the body.
evolutionary success and survival of the species. The inborn Some organisms, such as Drosophila, even live in a soup of
defense system, which protects the organism from infection microorganisms, in fermenting material. In Drosophila, these
by microorganisms and parasites, includes mechanical, hu- microbes normally serve as a source of food or survive in
moral and cellular barriers. The innate immune system con- equilibrium with the organism by becoming part of the normal
sists of cells and proteins which are present in the organism gut flora (Broderick et al. 2014). Injury or any disturbance of
from birth and are ready to combat invaders. The basic task the gut flora permits microbes to pass through the epithelial
of this defense system is to recognize and eliminate anything surfaces, multiply in the body fluids and use the host’s com-
foreign, i.e. to discriminate self from non-self, thereby pro- ponents as a source of energy. This leads to pathogenicity
tecting the organism from invaders (Janeway Jr and Medzhi- and a systemic activation of the immune system (Ferrandon
tov 2002). In consequence of its versatility and the available et al. 2007; Fig. 1).
powerful genetic, genomic and immunological tools, the fruit In vertebrates the immune system is composed of two
fly Drosophila melanogaster has become a model organism arms, the innate immune system and the adaptive immune
in which to study and understand the basic mechanisms of system. The innate immune system, which is evolved in all
innate immunity and host-pathogen interactions (Sackton et metazoans, is already functional at birth, constantly present
al. 2007; Kounatidis and Ligoxygakis 2012). The results of and ready to act immediately upon detecting an invader. It
such studies have revealed that there are substantial similari- comes into action efficiently through preformed effectors,
ties between the Drosophila immune response and vertebrate encoded for by the germ line, with a restricted array of
innate immunity (Hoffmann et al. 1999), and that most of the specificity to common molecular patterns of invaders (Beutler
genes involved in the regulation of Drosophila immunity are 2004). The adaptive immunity present in vertebrates is nor-
similar to those involved in the innate immunity of vertebrates mally silent, however, when an invader is sensed, it adapts to
(Engström et al. 1993; Hoffmann et al. 1999). its presence and develops mechanisms to eliminate it. This
development after exposure to an invader requires time for the
protective action to become functional and needs the contribu-
Submitted Febr 15, 2015; Accepted June 1, 2015
tion of the innate system (Medzhitov et al. 1997). Its specific-
*Corresponding author. E-mail: [email protected] ity is engineered by somatic cell gene rearrangements, which
1
Contributed equally to this work generate an enormous array of receptors which distinguish
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Honti et al.
minor differences between closely-related structures of the Sensing and signaling in innate immunity
invaders (Cook and Tomlinson 1995; Krangel 2009).
The generated specificities are fixed in memory cells
and, upon repeated exposure, they ensure a fast, specific When barriers are damaged, microorganisms or parasites
response. The efficient immune response develops through can enter the tissues. The non-self recognition of the innate
the cooperation of the two arms of the immune response and immune system is based on a limited number of germline-
any malfunction in the innate immune system has severe encoded pattern recognition receptors (PRRs), which detect
consequences, and in many cases lethality, which underlines evolutionarily conserved structures on pathogens, termed
the importance of the innate immune system. Although the pathogen-associated molecular patterns (PAMPs), which
cellular and the humoral elements have different functions, are not found in the host (Janeway Jr and Medzhitov 2002).
they interact with each other in the course of an efficient im- PRRs are also involved in sensing endogenous ‘danger’
mune response (Fig. 2). signals by recognizing danger-associated molecular patterns
(DAMPs). PRRs are expressed by professional immunocytes
that engulf and destroy pathogens or act as humoral factors in
Epithelial surfaces provide physical barriers the extracellular compartment. Ligand engagement of PRRs
induces receptor oligomerization, which subsequently trig-
gers intracellular signaling pathways, and induces effector
The mechanical barriers are the epithelium of the skin, the mechanisms, the activation of gene expression and the synthe-
respiratory system and the digestive tract. The tight junctions sis of proinflammatory cytokines, chemokines, antimicrobial
between epithelial cells block the entry of the microorganisms peptides and cell adhesion molecules. These responses also
into the body, but these cells also have anatomical and bio- initiate the development of adaptive immunity (Barral and
logical constituents, such as cilia to sweep away and mucus to Brenner 2007).
trap microorganisms and prevent their entry into the tissues. The cellular components of innate immunity express the
The mucus also contains peptides with a broad spectrum of Toll-like pattern recognition receptors (TLRs) at their cellular
antimicrobial activity, e.g., the defensins kill Gram-positive or endosomal membranes (Medzhitov et al. 1997). TLRs are
and Gram-negative bacteria, fungi and parasites. The respi- glycoproteins characterized by an extracellular or ligand-
ratory tract and the digestive system accommodate a natural binding domain containing leucine-rich repeat (LRR) motifs
(commensal, symbiotic) flora of fungi and bacteria which are and a cytoplasmic signaling Toll/interleukin-1 (IL-1) receptor
required for normal development and metabolism. This flora homology (TIR) domain. The TLRs derived their name from
forms an important part of the first-line defense as it prevents their Drosophila homolog Toll receptor, which is involved
invasion by other, pathogenic microorganisms by competing in embryogenesis and the antimicrobial response in the fruit
for sources of energy. Invasion by pathogenic bacteria elicits fly (Lemaitre et al. 2004). In humans, 10 TLRs have been
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Innate immunity
identified that recognize a variety of PAMPs from bacteria, eliminate the invaders and facilitate the healing of the lesion;
fungi, parasites and viruses, including lipid-based bacterial these processes involve the complement system in vertebrates
cell wall components such as lipopolysaccharide (LPS) and (Dunkelberger and Song 2010), the melanization reaction in
lipopeptides, microbial protein components such as flagellin, insects and the blood clotting system in all metazoans with
and nucleic acids such as single-stranded or double-stranded a circulatory system (Cerenius et al. 2010; Theopold et al.
RNA and CpG DNA (Akira et al. 2003). 2014).
Components of internalized or intracellular pathogens In insects, melanin and proteins produced after activation
and their derivatives are detected by cytosolic PRRs. The of proteolitic cascades are important to prevent loss of body
nucleotide binding oligomerization domain (NOD) receptors fluids through lesions, or, for directly killing certain patho-
NOD1 and NOD2 receptors sense bacterial molecules derived gens by their toxic properties (Cerenius and Söderhäll 2004).
from the synthesis and degradation of peptidoglycan. The Melanin is the final, toxic product of a proteolytic cascade.
NOD-like receptors are characterized by a tripartite-domain When active forms of the proteins are present in the body fluid
organization with a conserved NOD and leucine-rich repeats of insects, melanin is deposited on the eggs of parasites and
(LRRs) (Kanneganti et al. 2007). The RIG-I-like receptors, tumours (Ashida and Brey 1995). Pathogens are insulated by
which are involved in the recognition of viruses, constitute a blood clotting too. Besides preventing blood loss, the main
family of three cytoplasmic RNA helicases that are critical for purpose of the coagulation is to localize the infectious agents
host antiviral responses through the detection of exogenous on the site of injury. The blood clot is formed by the contribu-
dsDNA, leading to the induction of interferons and/or the tion of several proteins, some of them functioning in cleavage
processing of pro-inflammatory cytokines (Yoneyama et al. of precursor proteins (Karlsson et al. 2004).
2004). C-type lectin receptors (CLRs), including the dectin-1 In vertebrates, lactoferrin and transferrin limit the growth
and mannose binding lectin (MBL), bind to carbohydrates of bacteria by sequestering free iron, and thereby remove
in a calcium-dependent manner through their carbohydrate- this essential substrate (required for bacterial growth) (Ac-
recognition domains. CLRs are involved in fungal recogni- tor et al. 2009). The interferons limit virus replication, the
tion and in the modulation of the innate immune response enzyme lysozyme breaks down bacterial cell walls, and the
(Geijtenbeek and Gringhuis 2009). Cytosolic dsDNA sensor interleukins induce inflammatory proteins, and some have
molecules bind dsDNA to prevent cytokine induction. antimicrobial activity too. There are three major families of
The sensing of microbes in the extracellular compartment antimicrobial peptides (AMPs) in vertebrates: the defensins
is prompted by humoral factors such as lysozyme, lactoferrin, (Lehrer and Ganz 2002), the cathelicidins and the histatins
complement proteins and antimicrobial peptides. Lysozyme (Ganz and Lehrer 1998). The defensins are an ancient class
destroys the cell wall of Gram-negative and Gram-positive of antimicrobial peptides that are produced by insects, plants
bacteria by enzymatic mechanisms, while lactoferrin forms and vertebrates. There are several families of defensins: over
biofilms on certain bacteria. Complement proteins are prote- 13 defensins are known in plants, over 15 in Drosophila,
olytic enzymes activated in the presence of microbes (Dunkel- and at least 20 in humans, their genes forming a single gene
berger and Song 2010). The classical pathway of complement cluster on chromosome 8. These peptides are characteristic of
action is activated by antibodies combined with microbes to a disulfide-bound stabilized amphipathic region and are able
which the host has previously been exposed. The alternative to disrupt bacterial and fungal cell membranes and also en-
pathway is triggered directly through the contact of certain velopes of some viruses. It is supposed that they are incorpo-
complement components C3, factor B, factor D and properdin rated into the cell membrane and form pores, which make the
to microbial surfaces. The lectin pathway is initiated by the cell membranes leaky. Cathelicidins have been identified in
interactions of microbial polysaccharides with the soluble humans and mice (Kościuczuk et al. 2012). They are produced
mannose-binding lectins. The activation of the complement constitutively by neutrophil granulocytes, macrophages and
cascades generates complement component C3b, which epithelial cells in the gastrointestinal system and in the lung
binds to microbes and opsonizes them for phagocytosis. The after infection. They are activated by proteolytic cleavage.
generation of the inflammatory and chemotactic C5a and the The histatins are secreted into the saliva and kill pathogenic
activation of C5 initiates the membrane attack complex to lyse fungi such as Candida albicans in the oral cavity (Yan and
Gram-negative bacteria, and also to inactivate viruses. Bennick 1995).
In insects, large families of antimicrobial peptides have
been identified (Lemaitre et al. 1997; Bulet et al. 1999).
Humoral defense mechanisms They are produced by the fat body (the equivalent of the
vertebrate liver) and by blood cells, called hemocytes and are
secreted into the body fluid, the hemolymph. Their produc-
Injuries and the cell wall components of microbes activate tion is induced in response to infection by microbes or some
humoral responses like proteolytic cascades, which kill and parasites. On the basis of their sensitive target, they can be
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Honti et al.
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Honti et al.
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Honti et al.
involved in the hemocyte homing in the sessile compartment chromatin regions (heterochromatin) and less compacted
(Makhijani et al. 2011). In the circulation, many factors are regions (euchromatin) can be observed in the cell nuclei;
known to affect blood cell proliferation and differentiation, euchromatic chromatin regions are the subject of intensive
but the regulatory networks have been characterized only transcription, while heterochromatic genes are generally
tentatively (Zettervall et al. 2004). repressed. A distinction between active and inactive chro-
matin domains can therefore be formally made. The role of
epigenetic regulation is to set up and maintain the active and
Transcriptional and epigenetic regulation inactive states of the higher-order chromatin structure. The
of hematopoiesis structure of chromatin enables or inhibits the accessibility of
the chromatin for the transcription factors and the transcrip-
tion machinery, and thus maintains gene expression patterns
Similarly to other developmental processes, hematopoiesis in cell lineages throughout cell divisions. Various molecular
is mainly regulated by the formation and maintenance of signals, ranging from histone modifications to regulatory
gene expression patterns, which define the morphology and RNAs, are involved in the regulation of the higher chromatin
function of each blood cell type. Genes that are indispens- structure (Grimaud et al. 2006).
able for a certain function are activated, whereas other genes Most epigenetic factors involved in gene regulation have
are inactivated; as an example, in the B cells, B cell receptor been identified in Drosophila as the regulators of homeotic
genes are active, while the phagocytic receptor genes are in- genes. These factors are formally grouped into two categories:
active (Parra 2009). Gene expression patterns are formed by the Polycomb group of proteins involved in gene silencing,
transcription factors that specifically bind cis-regulatory target and the antagonistic trithorax group of proteins, which are
sequences on the DNA, thereby promoting the transcription activators of homeotic gene expression (Kennison 1995;
of target genes (Latchman 1997). To ensure the proper gene Pirrotta 1998). Their DNA targets have been identified, and
expression patterns, transcription factors are expressed cell- named PRE (Polycomb Response Element) (Simon et al.
type specifically. 1993) and TRE (Trithorax Response Element), respectively
The transcriptional regulation of blood cell differentiation (Rozovskaia et al. 1999). The members of the Polycomb
is based on evolutionarily well conserved regulatory factors and the trithorax group are conserved in mouse and human.
(Williams 2007). In mammals, 3 GATA factors (GATA-1, -2 These proteins have been shown to regulate not only homeotic
and -3) control various aspects of hematopoiesis and lineage genes, but many others, which are involved in development,
commitment (Fossett and Schulz 2001). Interestingly, one of tumorigenesis, ageing, maintenance of the stem cell state and
the 5 Drosophila GATA factors, Serpent, is expressed by all blood cell differentiation (Grimaud et al. 2006).
hemocyte types, and its role is essential for hematopoiesis Polycomb proteins form multimeric repressor complexes,
(Rehorn et al. 1996). Another Drosophila GATA homolog, which are responsible for maintaining the inactive chromatin
pannier, regulates the differentiation of both hemocytes and state. Three of these complexes have so far been identified,
the dorsal vessel, the Drosophila heart tube (Minakhina et and biochemically characterized. These are PRC1 (Poly-
al. 2011). Two other Drosophila proteins, which are also comb Repressive Complex 1), containing the Posterior Sex
homologs of mammalian hematopoietic factors, U-shaped Combs, Polyhomeotic and Polycomb proteins (Kingston et
(the Drosophila homolog of the mammalian FOG (Friend al. 1996; Shao et al. 1999; Saurin et al. 2001), PRC2 (Poly-
Of GATA) and lozenge (a RUN-X homolog) play key roles comb Repressive Complex 2) containing the Enhancer of
in the differentiation of plasmatocytes and crystal cells, Zeste and Extra Sex Combs proteins (Ng et al. 2000; Tie et
respectively (Waltzer et al. 2002, 2003; Bataillé et al. 2005; al. 2001; O´Connell et al. 2001) and phoRC, containing Pho
Muratoglu et al. 2006). The plasmatocyte fate also requires and dSfmbt (Klymenko et al. 2006). These complexes work
the expression of two other transcription factors, Gcm and agonistically both in Drosophila and in vertebrates, and their
Gcm2 in Drosophila, but the homologs of these proteins are sequential action is precisely regulated (Spivakov and Fisher
not known to play a role in hematopoiesis in mammals (Al- 2007). The antagonistic trithorax proteins possess much more
fonso and Jones 2002). Little is known about the target genes diverse functions. Some trithorax group genes encode tran-
of these transcription factors, although a few genes have been scription factors (e.g. kohtalo; Treisman 2001), while others
found, which contain GATA binding sites in their regulatory encode components of nucleosome remodeling complexes
regions (Tokusumi et al. 2009). (e.g. brahma; Tamkun et al. 1992).
To exert their action, transcription factors must bind to The epigenetic maintenance can be regarded as a battle
their target sequences, which requires accessible DNA. The between activation and inactivation. This battle begins with
accessibility of a locus is dependent on the density of the signals that mark chromatin domains as prone to activation or
higher-order chromatin structure. In the interphase, dense silencing. DNA itself can be marked, since methylation of the
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Innate immunity
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Honti et al.
Epigenetic regulation of blood cell recently shown to be the key regulators of the hemocyte fate
differentiation in Drosophila in Drosophila. One of them is the nucleosome remodeling
factor (NURF) complex, which plays a role in the silencing
of JAK/STAT activated genes when the ligand is absent. In
Several signal transduction pathways have been identified the case of activation, NURF dissociates from the chromatin
as regulators of the hemocyte fate in Drosophila. They are of the regulated genes, and hemocyte differentiation can take
associated either with the maintenance of the progenitor place (Kwon et al. 2008).
hemocyte pool in the lymph gland or with the differentiation These data imply that epigenetic factors play a major role
of effector blood cells, and especially lamellocytes during in the regulation of lamellocyte differentiation. Lamellocytes
the immune response to parasites (Fossett 2013). The tran- are normally absent from the circulation, but they differentiate
scriptional targets of some of these pathways, e.g. the JAK/ rapidly in the event of immune induction or certain tumorous
STAT pathway have also been identified (Bina et al. 2010). A conditions. It was recently recognized that not only do lamel-
large-scale genetic screen led to the identification of several locytes differentiate from precursors of the lymph gland and
genes, including transcription and epigenetic factors, cell the sessile tissue, but circulating plasmatocytes, which were
cycle regulators, signal transduction pathway components previously believed to be terminally differentiated blood cells,
and their regulatory networks that are required for the proper can also convert into lamellocytes upon immune induction
organization and function of the hematopoietic niche of the (Honti et al. 2010). This transition is most probably regulated
lymph gland (Tokusumi et al. 2012). However, our knowledge by an interaction of several epigenetic factors and may serve
on the regulatory connections between epigenetic factors as a key to the understanding of the molecular events that lead
and signal transduction pathway targets is still fragmentary, to the lamellocyte fate.
despite the fact that most of the Polycomb and trithorax group U-shaped is believed to be the transcription factor that
genes in Drosophila have been identified. suppresses lamellocyte differentiation (Sorrentino et al.
Although the Polycomb and trithorax group genes have an 2007), and it may therefore be a candidate master gene of
indispensable function in the regulation of homeotic genes, the conversion event, together with Serpent, which is an
surprisingly, few of the classical Polycomb and trithorax activator of the transition (Kroeger et al. 2012). The epige-
group genes have been investigated and proven to be involved netic regulatory events of the transition are mostly unknown.
in the epigenetic regulation of hemocyte differentiation in Recent data indicated that one of the main factors responsible
Drosophila. However, extensive screens have never been car- for the plasticity of macrophages is Charlatan (Stofanko
ried out to identify the Polycomb and trithorax group genes on et al. 2010). Since Charlatan is a member of the CoREST
the basis of the regulation of hemocyte differentiation. complex, which is an epigenetic repressor, the silencing of
One member of the Polycomb group that has been shown plasmatocyte-specific genes may be essential for lamellocyte
to have a function in hematopoiesis is multiple sex combs differentiation.
(mxc), which has been described as a regulator of proliferation Several genes, such as Peroxidasin, eater and nimC1,
and plasmatocyte differentiation (Remillieux-Leschelle et al. become silenced during lamellocyte differentiation (Honti et
2002). In mxc mutant larvae, the number of plasmatocytes is al. 2010; Kroeger et al. 2012). Eater and NimC1 are phago-
higher, due to overproliferation, and lamellocytes appear in cytosis receptors (Kocks et al. 2005; Kurucz et al. 2007a),
the circulation. A few of the Polycomb group genes tested and their expression is therefore not necessary for the lamel-
{(Sex comb on midleg (Scm), Polycomb-like (Pcl), Polycomb locyte function. This finding parallels the observation that
(Pc), Posterior sex combs (Psc) and extra sex combs (esc)} macrophage-specific csf1 is repressed in B cells (Tagoh et al.
did not display a synergistic effect with mxc in the regulation 2004), since plasmatocytes also lose their phagocytic capacity
of the hemocyte fate. Later, two other Polycomb group genes, while converting into lamellocytes (Honti et al. 2010).
polyhomeotic proximal (ph-p) and Enhancer of Polycomb There is evidence that signal transduction pathways are
(E(Pc)) were shown to affect lamellocyte differentiation as directly linked to the epigenetic regulation of differentiation
potential targets of the ROS-activated JNK signaling path- processes: the epigenetic regulator Split ends (Spen) serves as
way in the lymph gland (Owusu-Ansah and Banerjee 2009; an inducible switch. In uninfected larvae, spen is involved in
Theopold 2009). the activation of genes responsible for maintenance of “stem
Similarly as observed for the Polycomb group genes, cell-ness”. In cases of bacterial or fungal infection, the down-
only a few trithorax group genes are known to regulate the regulation of Notch activity results in the repression of spen,
differentiation of hemocytes. The genes domino and brahma which leads to hemocyte proliferation and differentiation (Jin
have been shown to be indispensable for normal hemocyte et al. 2009). Spen may therefore be considered as one of the
differentiation (Braun et al. 1998; Remillieux-Leschelle et main epigenetic regulators of hemocyte differentiation.
al. 2002). However, several other factors previously identi- The reason why most of the well-known Polycomb and
fied as chromatin modifiers and epigenetic regulators were trithorax group genes do not seem to be involved in hemocyte
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Innate immunity
differentiation may be that certain differences exist between between distant taxa is a proof that compartmentalization of
homeotic and blood cell fate determination. Homeotic genes blood cells furnishes a great evolutionary advantage.
are regulated epigenetically throughout the whole course
of ontogenesis; the chromatin state of the regulated genes
becomes fixed early in the embryo, and the actual state is Acknowledgements
maintained throughout development (Mihály et al. 2006).
However, in the case of hemocytes, the epigenetic regulatory
mechanism must act rapidly to enable the lamellocyte cell fate This work was supported by the TÁMOP-4.1.1.C-13/1/
upon immune induction. This ability to switch between differ- KONV-2014-0001 program entitled „Practice-oriented,
ent epigenetic states can be attributed to the poised chromatin student-friendly modernization of the biomedical education
state, which may require different epigenetic factors. for strengthening the international competitiveness of the
rural Hungarian universities”. The work was funded by grants
from the Hungarian Science Foundation (OTKA grant NK
Conservation and convergent evolution of 101730), and TÁMOP 4.2.2.A-11/1KONV-2012-0035 (IA).
blood cell differentiation in Drosophila and This research was supported by the European Union and the
vertebrates State of Hungary, co-financed by the European Social Fund
in the framework of TÁMOP 4.2.4.A/2-11-1-2012-0001
‘National Excellence Program’ (VH).
Drosophila obviously offers a versatile system in which to
study the regulatory events via which blood cell fates are
achieved. The similarities in hematopoiesis and blood cell References
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