International Journal of

Molecular Sciences

Review
Probiotics, Prebiotics and Epithelial Tight Junctions:
A Promising Approach to Modulate Intestinal Barrier Function
Elizabeth C. Rose 1 , Jack Odle 2 , Anthony T. Blikslager 1 and Amanda L. Ziegler 1, *

1 Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University,
Raleigh, NC 27606, USA; [email protected] (E.C.R.); [email protected] (A.T.B.)
2 Laboratory of Developmental Nutrition, Department of Animal Science, College of Agriculture and
Life Sciences, North Carolina State University, Raleigh, NC 27607, USA; [email protected]
* Correspondence: [email protected]; Tel.: +1-(919)-315-8099

Abstract: Disruptions in the intestinal epithelial barrier can result in devastating consequences and
a multitude of disease syndromes, particularly among preterm neonates. The association between
barrier dysfunction and intestinal dysbiosis suggests that the intestinal barrier function is interactive
with specific gut commensals and pathogenic microbes. In vitro and in vivo studies demonstrate that
probiotic supplementation promotes significant upregulation and relocalization of interepithelial
tight junction proteins, which form the microscopic scaffolds of the intestinal barrier. Probiotics
facilitate some of these effects through the ligand-mediated stimulation of several toll-like receptors
that are expressed by the intestinal epithelium. In particular, bacterial-mediated stimulation of toll-
like receptor-2 modulates the expression and localization of specific protein constituents of intestinal
tight junctions. Given that ingested prebiotics are robust modulators of the intestinal microbiota,





 prebiotic supplementation has been similarly investigated as a potential, indirect mechanism of
barrier preservation. Emerging evidence suggests that prebiotics may additionally exert a direct
Citation: Rose, E.C.; Odle, J.;
Blikslager, A.T.; Ziegler, A.L.
effect on intestinal barrier function through mechanisms independent of the gut microbiota. In this
Probiotics, Prebiotics and Epithelial review, we summarize current views on the effects of pro- and prebiotics on the intestinal epithelial
Tight Junctions: A Promising barrier as well as on non-epithelial cell barrier constituents, such as the enteric glial cell network.
Approach to Modulate Intestinal Through continued investigation of these bioactive compounds, we can maximize their therapeutic
Barrier Function. Int. J. Mol. Sci. 2021, potential for preventing and treating gastrointestinal diseases associated with impaired intestinal
22, 6729. https://doi.org/10.3390/ barrier function and dysbiosis.
ijms22136729

Keywords: probiotics; prebiotics; bioactive compounds; intestinal barrier function; tight junctions;
Academic Editor: Miguel Gueimonde toll-like receptors; intestinal microbiota

Received: 9 June 2021

Accepted: 21 June 2021
Published: 23 June 2021
1. The Emergence of Pre- and Probiotic Therapy
Publisher’s Note: MDPI stays neutral
The first campaign for host-friendly bacteria occurred in the late 19th century when
with regard to jurisdictional claims in
Dr. Élie Metchinkoff, who later received the Nobel Prize in Physiology or Medicine, began
published maps and institutional affil- to prescribe a new treatment for old age: soured milk [1]. Metchinkoff’s rationale for the
iations. innovative medicine stemmed from his realization that rural, Bulgarian peasants were
outliving their rich, city-dwelling peers. Metchinkoff attributed this lifespan discrepancy to
the peasants’ consumption of soured milk, which likely resembled an early-day counterpart
to today’s ever-expanding yogurt aisle. Although host-bacterial mutualism is now a well-
Copyright: © 2021 by the authors.
established paradigm, the mechanisms through which commensal organisms support
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systemic health are largely unresolved. Given that the intestinal tract hosts the body’s
This article is an open access article
largest bacterial ecosystem, elucidation of this mutualistic relationship relies upon an
distributed under the terms and advanced appreciation of the microbiota’s effects on intestinal homeostasis.
conditions of the Creative Commons Intestinal homeostasis is largely dependent on careful maintenance of the intestinal
Attribution (CC BY) license (https:// barrier, a physical boundary which separates the host from noxious luminal microbes and
creativecommons.org/licenses/by/ compounds. Intestinal barrier function is regulated through complex interactions among
4.0/). several intrinsic and extrinsic components, including intestinal epithelial cells (IECs), a

Int. J. Mol. Sci. 2021, 22, 6729. https://doi.org/10.3390/ijms22136729 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 6729 2 of 18

superficial mucus layer, the enteric nervous system (ENS) and various populations of
immune cells. The maintenance of intact barrier function is largely dependent upon
paracellular tight junctions (TJs), which link adjacent IECs at their apical surface and
create a polarized monolayer with an apical and basolateral domain. Relocalization and
altered expression of TJ protein constituents can result in rapid deterioration of intestinal
barrier function, ultimately hindering normal absorptive and secretory activity as well as
dissolving the host’s physical barricade against noxious luminal stimuli.
Interestingly, studies utilizing a pig model for intestinal ischemia have demonstrated
that barrier recovery of the small intestines is significantly reduced in neonates as compared
to juveniles [2]. These findings suggest that infants may be particularly predisposed to
serious complications of barrier insult because of their inability to mount a robust reparative
response. Neonatal barrier function can be challenged by the birthing process alone,
which can prompt adverse, perinatal outcomes such as necrotizing enterocolitis (NEC),
a life-threatening condition characterized by inflammation and inappropriate bacterial
overgrowth throughout the small and large intestines [3–5]. Although most publications
demonstrate that probiotic administration for intestinal disease prevention or treatment is
overall safe and variably effective, the mechanisms of action remain poorly understood.
The rising popularity of probiotic supplementation has fueled interest in the precise
mechanisms through which IECs recognize probiotics, commensal organisms and other mi-
crobes. Although this concept was initially dichotomizing, there is now a general consensus
that IECs express certain pattern-recognition receptors (PRRs), which are encoded proteins
that recognize and bind specific microbial ligands. Although many sub-families of PRRs
have been described, a complete review of these subfamilies is beyond the scope of this
review. Instead, we will focus on the specific sub-family of toll-like receptors (TLRs) due to
emerging evidence for TLR’s interactions with the intestinal microbiota and epithelial cell
TJs. Given their direct interaction with intestinal TJs, TLRs are an exciting potential target
for probiotic therapy.
Furthermore, we introduce the budding realm of prebiotic supplementation and its
effects, both direct and indirect, on the intestinal barrier. Although specific investigations
into the effects of prebiotic supplementation on intestinal barrier function are relatively
recent compared to those with probiotics, the limited data that have emerged informs
exciting prospects for future endeavors. Given their speculated shared health benefits,
pre- and probiotics have become increasingly lumped together as bioactive compounds,
which are extra-nutritional food constituents that provide health benefits beyond a basic
nutritional value. This review aims to highlight how the intestinal TJs, and consequently
intestinal barrier function, can be directly and indirectly influenced by specific interactions
among gut commensals, TLRs, and select bioactive compounds, namely pre- and probiotics.

2. Extrinsic and Intrinsic Perinatal Variables Influence the Neonatal

Intestinal Microbiota
Concurrent with probiotics’ rising popularity, manuscripts reporting the genesis of
the intestinal microbiota have numerically increased by more than one hundred-fold
over the past few decades. Intestinal gut colonization is now speculated to commence
in utero, thus challenging the long-standing dogma that the microbiota is established at
birth [6–10]. Sequencing technologies have revealed that the neonatal intestine is rapidly
populated by microorganisms that, after just a couple of days, exponentially expand to
a number exceeding that of all host cells [11]. Initial colonization by aerobic bacteria
transforms the intestinal lumen into an oxygen-poor niche that is subsequently dominated
by anaerobes [12–14]. Many of these anaerobic bacteria are genetically adapted to obtain
energy from host-derived glycans including human milk oligosaccharide, a naturally
occurring prebiotic present in the diet of breastfed infants [15].
Initial microbial colonization of the neonatal intestine can significantly vary depend-
ing on an assortment of factors including delivery method, feeding source, antibiotic
administration, and gestational age [16–18]. Preterm infants, for instance, often experience
sterile formula feedings, widespread use of antibiotics and reduced exposure to maternal
Int. J. Mol. Sci. 2021, 22, 6729 3 of 18

microflora. Furthermore, formula fortification with pre- and probiotics has become a high
priority given that bovine milk, from which most commercial formulas are derived, is
comparatively low in prebiotic oligosaccharides utilized by gut commensals as a primary
energy source [19]. Therefore, although developed to protect against pathogen transmis-
sion and infection, these protocols diminish early-life exposure to commensal bacteria and
can impede the efficiency of gut colonization. Importantly, preterm infants with altered
intestinal flora due to antibiotic use are more likely to develop NEC [20]. Necrotizing
enterocolitis is among the most common gastrointestinal emergencies in preterm neonates
worldwide, occurring in approximately 7% of very low birth weight infants and resulting
in mortality rates of up to 30% [3,21,22]. The precise pathogenesis of NEC are unclear but
it is presumably multifactorial with several identified risk factors in addition to preterm
birth. This knowledge gap in disease pathogenesis, as well as NEC’s alarming occurrence
rate, have motivated researchers across the globe to determine whether probiotics may
ameliorate or reduce the incidence of NEC. In 2010, meta-analysis of eleven randomized,
controlled trials confirmed that preterm, very low birth weight infants who receive pro-
biotic supplementation have a significantly lower risk for developing NEC [23]. A more
recent trial demonstrated reduced NEC incidence and severity in infants given a probiotic
that contains 4 species of Bifidobacterium [24]. These trials provide promise for probiotics’
beneficial effects in neonates, particularly preterm infants.

3. IECs Recognize Intestinal Microbes through Toll-Like Receptors

Toll-like receptors contribute to IEC homeostasis by governing appropriate host re-
sponses to the intestinal microbes. Downstream effects of nearly all TLRs are mediated
by Myd88, which is a cytosolic adaptor protein that converts extracellular signals to in-
tracellular activity including NF-κB activation and cytokine production. A few seminal
publications on TLR expression by IECs will be summarized below. For a more detailed
discussion, readers are referred to relevant reviews [25,26].
To further investigate the significance of TLR stimulation in barrier function, it is
initially important to identify when and where IECs express specific receptors. Particular
attention has been paid to TLR2 and TLR4, which recognize pathogen-associated molecule
patterns (PAMPs) expressed by gram-positive and gram-negative bacteria, respectively.
In mice, TLR expression significantly varies throughout the length of the intestinal tract.
Colonic IECs exhibit robust expression of TLR2 and TLR4 while small intestinal IECs
express very low levels of each receptor [27]. Spatial compartmentalization of these two
receptors is perplexing given that bacterial constituents can be isolated along the entire
gastrointestinal tract. Speculatively, it is possible that the small intestinal barrier may be
particularly sensitive to injury following robust TLR-signaling. Therefore, decreased TLR
expression may mitigate small intestinal injury through reduced bacterial-TLR signaling.
Limited TLR expression may also be explained by the fact that, in health, the small intestine
contains a sparce microflora compared to the colon. Therefore, small intestinal cells may
restrict TLR expression so that they can dedicate energy to more essential functions, namely
nutrient absorption.
Where expression is detectable, TLR2 and TLR4 are constitutively localized to the
epithelium’s apical surface [28–30]. Localization to the apical surface of IECs promotes
TLR stimulation by the contents of the intestinal lumen, including commensal bacteria as
well as injurious pathogens. Interestingly, TLR expression in undifferentiated, or immature,
intestinal epithelial cell lines is limited to the cytoplasmic compartment [30]. Therefore,
IECs’ ability to sample the intestinal lumen may evolve with age, possibly paralleling shifts
in the microbiota that naturally occur throughout life.
Caution must be taken when proceeding with this singular interpretation, however,
as undifferentiated intestinal cell lines are defined by a lack of polarity. It therefore re-
mains plausible that undifferentiated, unpolarized IECs retain TLR domains within their
cytoplasmic compartments until distinct apical and basolateral surfaces are defined. This
speculation is furthered by a recent demonstration that the largest amount of lipopolysac-
Int. J. Mol. Sci. 2021, 22, 6729 4 of 18

charides (LPS) is taken up from IECs along the villus tip rather than within the crypts [31].
Therefore, it is equally plausible that IECs alter TLR expression as they migrate up the
crypts to the tip of the intestinal villi.
Differentiation between the beneficial, or at least benign, microbiota and harmful
pathogens is further complicated by the fact that TLR-specific ligands are expressed by
both commensal and pathogenic organisms. To mitigate this challenge, IECs rely upon a
fundamental principle of bacterial pathogenicity: harmful bacteria are more likely to breach
the epithelial barrier. Many pathogenic bacteria rely upon a timely production of certain
virulence factors to breach the mucosal surface and invade the subjacent parenchyma.
Salmonella spp., for example, encode a type III secretion system that allows for direct
‘injection’ of Salmonella invasive proteins (SIPs), which stimulate enterocyte apoptosis, con-
sequently creating a physical defect in the epithelial barrier. Therefore, cellular responses
may be customized according to the physical location of the TLR on the cell surface. As
an example, toll-like receptor 9 (TLR9) is activated by unmethylated CpG domains in
bacterial DNA. Downstream effects of TLR9 stimulation are determined by the origin of
the receptor’s ligand. TLR9 activation along the apical surface of IECs inhibits NF-κB
activation. Conversely, activation from the basolateral surfaces promotes canonical acti-
vation and nuclear translocation of NF-κB [32]. Therefore, pathogenic organisms which
translocate across the IEC barrier and access basolateral TLR9 are more likely to exert a
proinflammatory effect which is intended to clear the pathogens but has the potential to
become deleterious causing local tissue destruction and further barrier injury.
Similarly, cellular responses can be customized by restricting the expression of certain
TLRs to specific IEC domains. For instance, toll-like receptor 5 (TLR5), which recog-
nizes bacterial flagellin, is expressed along only the basolateral surface of colonic IECs
in vitro [33]. Interestingly, TLR5 expression by murine small intestinal epithelial cells
gradually decreases throughout the neonatal period until expression is largely restricted to
Paneth cells in adult mice [27]. These findings reinforce the speculation that TLR expression
may evolve with age. Robust TLR5 expression in neonates may act to provide further
protection from pathogenic bacteria while the immature intestinal tract and immune system
continue to develop. Alternatively, one may speculate that decreased TLR5 expression by
neonatal Paneth cells may predispose the neonate to prolonged and more severe intesti-
nal injury. Paneth cells support the intestinal stem cell niche and are arguably necessary
for stem cell proliferation and differentiation [34]. Therefore, decreased TLR5-mediated
stimulation by Paneth cells may result in hindered epithelial proliferation and recovery
of the neonatal intestines following injury. By promoting a healthy commensal microbial
community with pre- and probiotic interventions, these pathogens can be reduced by
competitive inhibition, quite possibly preventing their harmful TLR-mediated effects on
the intestinal barrier.

4. Intestinal Tight Junctions Are Regulated by Bacterial Stimulation of

Toll-Like Receptors
Intestinal TJs regulate epithelial permeability through the dynamic nature of their
three-dimensional, multiprotein conformation. Essential constituents of all TJs include
integral membrane proteins, such as occludin and various claudins, as well as cytoplas-
mic plaque proteins, such as zonular occludins-1 (ZO-1) and zonular occludins-2 (ZO-2).
Previous reviews have detailed these constituent’s differing effects on intestinal perme-
ability [35–37]. Provided that IECs express specific TLRs along their apical and basolateral
domains, researchers have reasonably speculated that microbial ligands regulate TJ pro-
teins through their interactions with TLRs. Consequently, there is a steady increase in
the number of studies that investigate alterations in specific TJ proteins following sup-
plementation with probiotics. For example, activation of MyD88, the cytosolic adaptor
protein for most TLRs, is required for appropriate claudin-3 expression in neonatal mice.
MyD88−/− neonatal mice demonstrate impaired intestinal barrier function with increased
permeability to large polysaccharides. Therefore, TLR-mediated induction of claudin-3
protein expression may be necessary for appropriate TJ formation and maintenance [38].
Int. J. Mol. Sci. 2021, 22, 6729 5 of 18

Furthermore, maturation of barrier function and claudin-3 protein expression in mouse

pups is induced by enteric feeding of Lactobacillus rhamnosus GG, which is an intestinal
commensal species and a commonly used probiotic [38]. Taken together, these findings
suggests that expression of crucial barrier-forming TJ proteins, such as claudin-3, may be
induced via TLR signaling associated with select probiotics.
Specific TLRs play distinct roles in the regulation of intestinal TJs. Following treatment
with a TLR-2 ligand, for instance, Caco-2 monolayers demonstrate robust apical redistribu-
tion of ZO-1 as well as increased intracellular protein kinase C (PKC) activity [39]. This may
suggest that TLR2 stimulation promotes structural modifications in TJs by promoting PKC
activity. Therefore, deficient levels of host-protective TLR2 ligands may compromise barrier
function. For instance, antimicrobial administration could compromise barrier function
by decreasing luminal numbers of gram-positive gut commensals such as Lactobacillus
spp. and Bifidobacterium spp., both of which are important sources of TLR2-stimulating
ligands [40].
Notably, TLR activation does not always result in increased barrier function. Stim-
ulation of TLR4 by LPS causes decreased barrier function in mouse models and human
intestinal epithelial cell lines [41]. This detrimental effect is attributable to TLR4/MyD88
upregulation of myosin light chain kinase (MLCK). Myosin light chain kinase induces the
opening of the TJs through contraction of actin-myosin filaments, followed by internaliza-
tion of TJ proteins [41–45]. Therefore, intestinal homeostasis and barrier function requires
a delicate balance between commensal- and pathogen-induced activation of TLRs.
To maintain this delicate balance and further investigate the effects of deliberate
TLR stimulation, researchers must complement their research studies with a foundational
understanding of TLR pathophysiology. Each TLR is defined by a unique combination of
stimulating ligands, co-receptors, adaptor proteins and effector pathways. Given TLR-20 s
potential as a therapeutic target for intestinal barrier function, its unique protein dynamics
merit careful consideration. Interestingly, TLR-2 is the only TLR described thus far to
form functional heterodimers with two different types of TLR as well as a large number
of non-TLR molecules [46–50]. This unique ability to interact with multiple co-receptors
and adaptor proteins, such as Declin-1 and CD14, facilitates TLR-20 s ability to bind a broad
repertoire of structurally diverse ligands [51–53]. A thorough understanding of TLR-20 s
various co-receptors, adaptor proteins and ligands can inform probiotic formulations so
as to effectively and specifically stimulate TLR-2 and promote it’s beneficial effects on
intestinal TJs.

5. Probiotics Promote Barrier Function through Regulation of Tight Junction Proteins

Lactobacillus and Bifidobacterium species are prominent genera within the human gut
microbiota and have become the most commonly-used probiotics in human medicine,
including obstetrics [54,55]. The proposed mechanism of these commensals was initially
based upon in vitro experiments that utilize intestinal epithelial cell lines. Preincubation
of Caco-2 cells with specific strains of Lactobacillus plantarum and Lactobacillus rhamnosus
attenuate pathogen-mediated disruptions in the intestinal epithelial barrier, including a
highly reductionist bacterial LPS in vitro model of NEC, which entailed TJ disruption
through LPS derived from Escherichia coli [56–58]. Although NEC is often associated
with bacterial overgrowth and dysbiosis, the exact pathogenesis remains unknown and
is generally accepted to be multifactorial. Therefore, an LPS model is likely an over-
simplification of NEC and until the pathogenesis of NEC is further elucidated, conclusions
gleaned from such simplified models should be made with caution. However, this probiotic-
mediated barrier protection in a model of bacterial intestinal insult is certainly worth noting.
Certain Lactobacillus species abate barrier disruption through upregulation of TJ pro-
teins. L. acidophilus and L. plantarum increase occludin protein expression within in vivo
and in vitro models, respectively [59,60]. In addition to increasing occludin protein expres-
sion, L. plantarum induces apical relocalization of ZO-1 and occludin through stimulation of
TLR2 [58,61]. However, Caco-2 cell coincubation with L. plantarum simultaneously results
Int. J. Mol. Sci. 2021, 22, 6729 6 of 18

in increased transcription of genes involved in tight junction disassembly and occludin

degradation [60]. Therefore, increased occludin expression and apical localization may
actually be a protective response provoked by initial bacterial-mediated degradation, not
maintenance, of the TJs.
While Bifidobacterium supplementation has been repeatedly shown to decrease the in-
cidence and severity of NEC, the potential mechanism by which this gut commensal exerts
its beneficial effects has only recently been described [62–64]. Upon the onset of NEC, inter-
nalization of claudin 4 and occludin disrupts the intestinal TJs [64]. Oral supplementation
with B. infantis prevents this disturbance by preserving claudin 4 and occludin localization
along the vicinity of the TJs. Therefore, Bifidobacterium supplementation preserves intesti-
nal barrier function, and consequently prevents severe NEC, though the maintenance of
TJ conformation.
Bifidobacterium and Lactobacillus spp. are the primary constituents of a commercial
probiotic formulation called VSL#3. For nearly 20 years, VSL#3 has dominated the dietary
supplements market by claiming to mitigate clinical symptoms of certain gastrointestinal
disorders. The formulation’s beneficial effects are hypothesized to result from bacterial-
mediated preservation of TJ protein expression and localization as well as inhibition of
intestinal epithelial cell apoptosis. Increased colonic permeability and epithelial cell apop-
tosis resulting from DSS-induced colitis in mice were prevented by VSL#3 supplementation,
which additionally increased expression of ZO-2, occludin and several claudins [65]. How-
ever, the VSL#3 dose required to produce these beneficial effects in this mouse colitis model
is twice that recommended for inflammatory bowel disease treatment in humans. These
findings warrant a reevaluation of current probiotic dose recommendations for VSL#3
as well as other commercial probiotics. Probiotics advertised as dietary supplements do
not require approval by the United States Food and Drug Association before marketing.
Therefore, many probiotic manufacturers have not had the incentive to research physiologic
differences among various dosages. As the probiotics market continues to expand, the
supplementation efficacy and dosing demands more intensive study.
For some probiotics, realization of their beneficial effects relies upon prior disruption of
TJ homeostasis. For instance, E. coli Nissle 1917 (EcN) is a gram-negative probiotic that has
been studied since the discovery of its potential benefit during World War I. Alfred Nissle
isolated the strain in 1917 from the feces of a German soldier after noting that the soldier
did not develop diarrhea despite a regional Shigellosis outbreak [66]. Nissle speculated
that gastrointestinal EcN populations prevented the soldier from falling ill. Nearly one
century following this initial isolation, studies have expanded upon Nissle’s findings by
noting that incubation of T84 human intestinal epithelial cells with EcN alone results in
no significant changes to intestinal epithelial barrier function [67]. However, following
T84 co-incubation with enteropathogenic E. coli (EPEC) and associated barrier disruptions,
EcN supplementation restores barrier permeability to nearly the same value as seen in
control cells. The beneficial properties of EcN might therefore be realized post-infection
and may not require preventative supplementation, which is clinically challenging and
often not feasible. Additionally, in concurrence with decreased barrier permeability, EcN
supplementation results in increased ZO-2 expression and robust ZO-2 relocalization to the
TJs [67]. Although these early findings do not prove absolute causation, they suggest that
EcN’s beneficial effects on intestinal barrier function may be modulated by the bacteria’s
regulation of ZO-2 expression and localization.
E. coli Nissle 1917 supplementation can enhance IEC expression of ZO-1 under healthy
and inflammatory conditions in vivo. However, EcN administration following DSS-
induced colitis does not significantly decrease colonic permeability [68]. Therefore, the
practical significance of increased ZO-1 expression in vivo remains equivocal. Interestingly,
ZO-2 expression is not altered upon in vivo administration of EcN, which is unexpected
given that it has been shown to increase in vitro [67]. One potential explanation for this
discrepancy in ZO-2 expression is that the aforementioned in vitro studies utilized a human
intestinal epithelial cell line while the in vivo studies used a mouse model. The patho-
Int. J. Mol. Sci. 2021, 22, 6729 7 of 18

physiology of mouse intestinal TJs may differ from that of human intestinal TJs. Although
animal models play an invaluable role in the advancement of human medicine, species
variation can limit data extrapolation from these models. Future studies evaluating EcN
effects on TJ protein expression across numerous species, including humans, under normal
conditions as well as models of intestinal injury would provide interesting insight into how
mechanisms of intestinal repair may vary across species.
The skin and gut barrier share many morphological and pathophysiological similar-
ities. Therefore, some researchers have utilized normal human epidermal keratinocytes
(NHEK) to infer potential mechanisms by which probiotics modulate intestinal TJs. Ker-
atinocyte cell cultures demonstrate increased barrier function and TJ protein expression
following treatment with bacterial lysates from certain strains of Lactobacillus and Bifidobac-
terium spp. [69]. These phenotypes are abolished with neutralization of TLR2, suggesting
that these bacteria augment TJ closure through TLR2 stimulation. Unfortunately, key
physiologic differences between keratinocytes and enterocytes, notably the robust secretory
and absorptive capacity of the latter, prohibit a blind application of these findings to the
intestinal barrier. Instead, it would be interesting to repeat the referenced experiment using
intestinal cell lines or in vivo models. A summary of selected probiotics’ target TLRs and
proposed beneficial effects on intestinal tight junctions is available in Table 1.

Table 1. Select probiotics target TLRs and proposed beneficial effects on intestinal tight junctions.

Probiotic Target TLR(s) Tight Junction Effects Reference(s)

Lactobacillus rhamnosus GG TLR2, TLR9 Increased claudin-3 protein expression [38,70]
Lactobacillus acidophilus TLR2 Increased occludin protein expression [59]
Increased occludin protein expression and apical
Lactobacillus plantarum TLR2 [58,60,61]
redistributionIncreased ZO-1 apical redistribution
Preserves apical distribution of claudin-4
Bifidobacterium infantis TLR2 [64]
and occludin
Increased ZO-2 mRNA and protein expression and
E. coli Nissle 1917 TLR4 apical redistributionIncreased ZO-1 mRNA and [67,68]
protein expression
Lactobacillus rhamnosus TLR2 Preserves apical distribution of ZO-1 and occludin [71]

6. Specific Bacterial Components May Contribute to Probiotics’ Beneficial Effects

Given that different probiotic formulations exhibit similar effects on the intestinal
epithelial barrier, we can plausibly speculate that probiotics’ mechanisms of action are
regulated by shared bacterial components rather than specific bacterial species. In other
words, probiotics’ effects on intestinal TJs and barrier function may be mediated by distinct
structural and chemical bacterial constituents rather than the bacterium as a whole.
Soluble proteins isolated from Lactobacillus rhamnosus ameliorate intestinal epithelial
barrier disruption in vitro by preventing injury-induced redistribution of occludin and
ZO-1 [71]. Furthermore, pretreatment with the isolated proteins prevents increased barrier
permeability to ions and large sugar molecules. The introduction of a PKC- or MAP-kinase
inhibitor, however, attenuates these phenotypes. Therefore, soluble proteins secreted by
select probiotic bacteria exert their protective effects through a PKC- and MAP-kinase-
dependent mechanism.
Indole is an intercellular signaling molecule produced by many gram-positive and -
negative organisms. Repeated indole administration results in increased protein expression
of vital TJ constituents as well as resistance to DSS-induced mucosal damage and colitis [72].
Therefore, the protective effects of some probiotics may be reliant upon indole production
and signaling.
Peptidoglycans are the primary constituent of most bacterial cell walls. Co-incubation
of NHEKs with bacterial peptidoglycans results in increased barrier function [69]. Again,
Int. J. Mol. Sci. 2021, 22, 6729 8 of 18

relying upon the morphologic similarities between the dermal and intestinal epithelium,
these results suggest that bacterial cell walls contribute to probiotics’ promotion of intestinal
homeostasis and barrier function. However, NHEK co-incubation with bacterial peptido-
glycans does not alter TJ protein expression levels. Therefore, peptidoglycans appear to
promote barrier function through mechanisms that do not influence TJ protein expression
levels. These findings force us to acknowledge that protein activity is not determined
by expression levels alone. Peptidoglycan and other bacterial ligands may influence TJ
proteins through structural modifications, such as phosphorylation or even ubiquitination.
Investigations into probiotics’ mechanisms of action must therefore expand beyond simple
protein expression and localization to include post-translational modifications.
Investigations into the role of peptidoglycans in intestinal homeostasis is further
complicated by the ligand’s nearly ubiquitous nature: peptidoglycan is present in the cell
wall of both commensal and pathogenic bacteria. Therefore, there must be some physical
or chemical property of specific peptidoglycans that differentiate these two populations of
bacteria. In fruit flies, immune cells rely on peptidoglycan recognition proteins (PGRP),
which are a type of PRR similar to TLRs, to differentiate bacterial peptidoglycans [73].
More specifically, PGRPs stimulate customized cellular responses based upon the amino
acid that resides in specific positions of the peptidoglycan peptide [74,75]. The binding of
some peptidoglycans, and consequently some bacteria, illicit a potent immune response
while other bacteria and their associated peptidoglycan will dampen the immune response.
Although these findings are not directly translatable to the mammalian intestinal
microbiota, they illuminate that TLRs are only one of several types of PRRs. Other PRRs,
including PGRP, have been implicated in the homeostasis of the gut barrier as well as the
maintenance of gut commensal populations in mice [73,76]. Therefore, PRRs other than
TLRs are promising therapeutic targets for repairing intestinal epithelial TJs and promoting
intestinal barrier function.

7. Prebiotics Promote Barrier Function through Microbial-Dependent and

-Independent Mechanisms
Despite probiotics’ promising effects on intestinal barrier homeostasis and repair,
potential drawbacks and limitations of probiotic therapy cannot be ignored. Over the
past few decades, reviews have highlighted potential dangers of probiotic therapy in
perinatal infants, immunocompromised individuals and patients with compromised in-
testinal barrier function [77–79]. The hindered immune system and barrier function in
these demographics exponentially increase the risk for microbes, even those considered
to be commensal, to breach the mucosal barrier and establish multisystemic infections.
For instance, a recent report described three unrelated cases of Bifidobacterium longum
bacteremia in very low birth weight infants following administration of a commercially
available probiotic preparation [80].
This and similar case series emphasize that very few studies have investigated appro-
priate doses for probiotic therapy. To date, essentially every study has utilized not only a
different probiotic preparation but also a different dosage regime. Investigating the proper
doses for safe and effective probiotic supplementation is further complicated by the fact
that probiotics must endure the hostile journey from the oral cavity to the intestines as well
as survive within the intestinal lumen long enough to illicit their beneficial properties.
Limitations of probiotic therapy have organically fueled an emergence of prebiotic
supplementation and research. Prebiotics directly influence the composition of the gut
microbiota and stimulate growth of beneficiary commensals such as Bifidobacterium spp.
and Lactobacillus spp. [81–85]. The term “prebiotics” was introduced to the scientific
community in a 1995 publication that defined the bioactive substances as “nondigestible
food ingredients that beneficially affect the host by selectively stimulating the growth
and/or activity of one or a limited number of bacterial species already resident in the
colon.” [86] Since this publication, prebiotics’ proposed benefits have expanded beyond
colonic health to include the treatment and prevention of select gastrointestinal, skin,
genitourinary and skeletal diseases in both humans and animals [81,83,87–95]. For this
Int. J. Mol. Sci. 2021, 22, 6729 9 of 18

reason, the definition of prebiotics was updated in 2017 to encompass substrates that are
“utilized by host microorganisms conferring a health benefit.” [87]
Given probiotics’ beneficial effects on the intestinal barrier, it is not surprising that pre-
biotic supplementation similarly promotes intestinal barrier function and repair [84,96–100].
In vitro and in vivo studies are largely centered around select formulations of numerous
oligosaccharides and polysaccharides, both of which have gradually come to dominate the
forefront of prebiotic research [38,83,90,97,99,101,102]. Many publications highlight that
not all formulations can be treated equally because the degree of beneficial effects is unique
to each tested prebiotic as well as each prebiotic–probiotic combination. These findings
introduce the complexity of synbiotics, which rely upon synergism between prebiotics and
probiotics to amalgamate the two bioactive substances into a single supplement.
Mimicking probiotics’ proposed mechanisms of action, many prebiotics promote in-
testinal barrier function [85] through modulation of intestinal TJs. In vitro supplementation
with inulin fermentation products results in the significant upregulation of TJ genes includ-
ing occludin, claudin-3 and ZO-1 [102]. Supplementation with fructooligosaccharides and
butyrate, which is a bacterial metabolite produced during prebiotic fermentation, results
in the redistribution of select proteins, including ZO-1 and occludin, to the vicinity of
the TJs [103,104]. In vivo and in vivo studies demonstrated that galactooligosaccharide
pretreatment results in upregulation of ZO-1, occludin and claudin-1 gene expression
in LPS-challenged mice [105,106]. A key feature of this particular study is prebiotic ad-
ministration prior to intestinal injury. As previously mentioned, prebiotic and probiotic
pretreatment is often unrealistic in human medicine given that many intestinal injuries
are unpredictable. In the case of preterm infants with NEC, pretreatment with bioactive
compounds is simply impossible. Therefore, future endeavors must demonstrate beneficial
effects of pre- and probiotic supplementation following, not just prior, to intestinal injury
in order to support impactful advancements in clinical medicine.
Prebiotics’ direct effects on the gut microbiota is a reasonable explanation for these
observed changes in TJ protein expression and distribution. Given that prebiotics stimulate
the growth of select probiotic species, it is unsurprising that prebiotic supplementation
mirrors the beneficial effects produced by the same probiotics that those carbohydrates
fuel. Furthermore, prebiotic supplementation results in robust activation of AMP-activated
protein kinase (AMPK) in conjunction with the described changes to the epithelial bar-
rier [98,103,104,106]. Prebiotic-mediated activation of AMPK may therefore play a signif-
icant role in prebiotics’, and potentially probiotics’, effects on intestinal tight junctions.
Contrarily, data from two of the aforementioned studies suggest that some of prebiotics’
beneficial effects may occur independently of the surrounding microbiota [96,104]. Al-
though these microbial-independent effects are less robust than those produced with
concurrent probiotic supplementation, the remarkable possibility of prebiotic-mediated
effects despite concurrent dysbiosis, is undoubtedly worth pursing further. A summary of
selected prebiotics’ target TLRs and proposed beneficial effects on intestinal tight junctions
is available in Table 2.

Table 2. Select prebiotics target TLRs and proposed beneficial effects on intestinal tight junctions.

Prebiotic Tight Junction Effects Reference

Inulin Increased occludin, claudin-3 and ZO-1 RNA expression [102]
Fructo-oligosaccharide ZO-1 and occludin apical redistribution [104]
Galactooligosaccharide Increased ZO-1, occludin and claudin-1 protein expression [105,106]

8. Future Directions: Embracing the Enteric Glial Cell Network, Large Animal Models,
and Promising Clinical Interventions
As previously mentioned, the epithelial mucosa and its associated tight junctions
are only one piece to the intricate puzzle that constitutes the intestinal barrier. Intestinal
barrier function is additionally modulated by the ENS, which is a complex network of
Int. J. Mol. Sci. 2021, 22, 6729 10 of 18

densely intertwined neurons and glial cells that extends along the entire length of the
gastrointestinal tract. Enteric neurons regulate intestinal barrier function through a va-
riety a mechanisms including the promotion of epithelial cell turnover and increased
expression of TJ proteins [107–109]. Given that enteric neurons express select TLRs, their
regulatory effects are hypothesized to be mediated, at least in part, by their interactions
with the intestinal microbiota [110–112]. However, an exciting frontier of intestinal barrier
research, a comprehensive review of enteric neuron’s regulation of the intestinal barrier
function and TLR expression is beyond the scope of this review and readers are referred to
relevant publications.
While the neuro-epithelial unit has received extensive evaluation in intestinal barrier
research, enteric glial cells have been historically characterized by their supportive role in
neuronal maintenance and activity. However, emerging research demonstrates that the
enteric glial cell (EGC) network acts as more than a mere support system to the enteric
neurons and actually plays a direct role in intestinal barrier function. The EGC network
is consequently gaining increased interest in the gastrointestinal community due to its
implication in intestinal barrier homeostasis and repair, including regulation of TJs and cell
motility [107,113–117].
Interestingly, microbial fermentation products, specifically short-chain fatty acids
(SCFA), have been shown to modulate EGC network development [118]. The presence of
the intestinal microbiota additionally contributes to appropriate, perinatal development of
EGCs [119,120]. The microbiota’s effects on the EGC network development are postulated
to be regulated, at least in part, by microbes’ direct interactions with intestinal neurons
and glia. Enteric neurons and glia within the myenteric and submucosal plexi of adult
rodents express TLRs 2, 3, 4, 7 and 9 [111,112]. Given that enteric neurons and glia have
no known direct contact with the intestinal lumen, TLR-activation by intestinal microbes
is presumably limited to the subepithelial compartment. Therefore, TLR-mediated stimu-
lation of the EGC must occur consequent to barrier disruption by pathogenic organisms
or, in physiologic conditions, translocation of gut commensals across the mucosal barrier.
Furthermore, TLR protein expression by the EGC network fluctuates depending on the
specific microbial organisms that are present. For instance, TLR2 protein expression is
upregulated when EGCs are co-cultured with a specific species of Lactobacillus [121]. Given
EGC expression of certain TLRs and the networks’ regulatory effects on barrier function,
we can reasonably speculate that pro- and prebiotics’ preservation of intestinal barrier
function may be mediated, or at least enhanced, through their interactions with the EGC
network. Figure 1 illustrates our current understanding of TLR expression of IECs and the
EGC network.
In order to unravel this expanding web of intestinal barrier regulation, future endeav-
ors must further investigate these interactions among the intestinal microbiome, EGC and
nutrition. As a natural source of SCFAs, oligosaccharides are a rational stepping stone for
these future endeavors. Oligosaccharide supplementation has already shown to increase
intestinal epithelial transepithelial resistance, a marker of intestinal barrier function, as well
as ileal lactobacilli concentrations in formula-fed pigs [122,123]. Insights into prebiotics’
fermentation profiles and synergistic activity with specific probiotics may provoke even
more interesting results, eventually informing future symbiotic supplementation formulas
and protocols for mitigating gastrointestinal diseases.
Int. J. Mol. Sci. 2021, 22, 6729 11 of 18

Figure 1. Toll-like receptor expression of intestinal epithelial cells (IECs) and the enteric glial cell
(EGC) network, illustrating positive effects on tight junctions and intestinal barrier function.

These promising studies on oligosaccharide supplementation introduce another excit-

ing venture in gastrointestinal research: porcine models of human disease. A recent review
by Ziegler et al. highlights fundamental physiologic, anatomic and nutritional similarities
between pigs and humans [124]. Notably, pigs and humans share similar gut microbial
profiles while the murine microbiota is significantly divergent [125–127]. Taking these
interspecies comparisons into account, pigs can undoubtedly act as powerful models for
upcoming investigations into intestinal barrier function and its relationship with the micro-
biome, nutritional supplements and the EGC [128,129]. In vivo studies in suckling piglets
have already demonstrated that early-life supplementation with galactooligosaccharides
results in significantly increased ZO-1 and claudin-1 protein expression [106]. These studies
did not directly evaluate intestinal barrier function, however, therefore the significance of
these results with respect to upholding barrier function requires further investigation.
With the introduction of germ-free and gnotobiotic pigs, researchers can further
embrace the porcine model by investigating how the gestational maternal microbiota
affects neonatal health. Chapter 2 of this review highlights emerging evidence that the
neonatal intestinal microbiota begins to develop in utero. Therefore, the composition of the
maternal microbiota throughout gestation may directly influence that of the developing
neonate and consequently influence the pathophysiology of the neonate’s intestinal barrier.
Given that the intestinal microbiota of pigs and humans is remarkably similar, porcine
models may be utilized to investigate whether the maternal intestinal microbiota can be
optimized through pre- and probiotic supplementation throughout gestation. Furthermore,
such models can investigate whether these supplementation protocols are significantly
correlated to the composition of the neonatal intestinal microbiota as well as neonatal
intestinal disease outcome.
Int. J. Mol. Sci. 2021, 22, 6729 12 of 18

As many researchers have focused on the effects of pre- and probiotics on intestinal
barrier function, others have investigated pharmaceuticals that directly target intestinal
TJs. For instance, Larazotide acetate is a small, TJ-directed drug that is currently in clinical
trials for treatment of celiac disease, the pathophysiology of which includes disruptions to
the intestinal TJs [130–133]. Several in vivo and in vitro experiments have demonstrated
that Larazotide administration prevents relocalization of crucial TJ-proteins, ultimately
resulting in quantifiably enhanced epithelial barrier integrity [134–136]. Taken together,
these studies introduce the exciting prospect of compounding pre- and probiotic’s benefi-
cial effects with Larazotide or similar TJ-protein-directed pharmaceuticals. Future studies
should investigate the therapeutic potential of timed treatment protocols in which initial
administration of TJ-protein-directed pharmaceuticals enhances TJ integrity to prevent con-
tinued bacterial stimulation and subsequent pre- and probiotic supplementation carefully
restores a balanced intestinal microbiota.

9. Conclusions
Probiotics’ beneficial effects on intestinal barrier function are at least partially medi-
ated through organismal stimulation of TLRs, particularly TLR2, and sequential changes
to TJ protein expression and localization. Prebiotic supplementation produces similar
beneficial effects on barrier function, although their mechanisms of action have yet to be
extensively evaluated. These findings implicate probiotic and prebiotic supplementation
as a reasonable preventative and treatment modality for disease syndromes defined by
increased intestinal permeability. Some of these disease syndromes, including NEC in
preterm infants, have already shown a positive response to probiotic therapy. Future
endeavors must expand upon these findings to unravel the intestinal barrier’s dynamic
interactions with probiotics, prebiotics and enteric glia. Pigs can act as a powerful trans-
lational model to enhance these studies and provide innovative techniques for solving
unresolved quandaries. Dietary supplementation with bioactive compounds remains an
exciting frontier and presents promising possibilities for therapeutic and preventative
modalities in gastrointestinal medicine.

Author Contributions: Conceptualization, E.C.R.; writing- original draft preparation, E.C.R.; writing-
review and editing, E.C.R., A.T.B., J.O. and A.L.Z. All authors have read and agreed to the published
version of the manuscript.
Funding: Elizabeth Rose was supported by a COHA Translational Fellowship (U01 TR002953).
Jack Odle, Anthony Blikslager and Amanda Ziegler were supported by grants from U.S. National
Institute of Child Health and Human Development (R01 HD095876), USDA National Institute for
Food and Agriculture (AFRI-006609). Amanda Ziegler was also supported by U.S. National Institutes
of Health Office of Research Infrastructure Programs (K01 OD028207).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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