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REVIEW
Deciphering internal and external factors inuencing intestinal junctional
complexes
Zachary Markovich
a,b,c
, Adriana Abreu
a
, Yi Sheng
a
, Sung Min Han
a
, and Rui Xiao
a,c,d,e,f
a
Department of Physiology and Aging, College of Medicine, University of Florida, Gainesville, FL, USA;
b
Graduate Program in Biomedical
Sciences, College of Medicine, University of Florida, Gainesville, FL, USA;
c
Center for Smell and Taste, University of Florida, Gainesville, FL, USA;
d
Institute on Aging, University of Florida, Gainesville, FL, USA;
e
Genetics Institute, University of Florida, Gainesville, FL, USA;
f
UF Health Cancer
Center, University of Florida, Gainesville, FL, USA
ABSTRACT
The intestinal barrier, an indispensable guardian of gastrointestinal health, mediates the intricate
exchange between internal and external environments. Anchored by evolutionarily conserved
junctional complexes, this barrier meticulously regulates paracellular permeability in essentially all
living organisms. Disruptions in intestinal junctional complexes, prevalent in inammatory bowel
diseases and irritable bowel syndrome, compromise barrier integrity and often lead to the
notorious “leaky gut” syndrome. Critical to the maintenance of the intestinal barrier is a nely
orchestrated network of intrinsic and extrinsic factors that modulate the expression, composition,
and functionality of junctional complexes. This review navigates through the composition of key
junctional complex components and the common methods used to assess intestinal permeability.
It also explores the critical intracellular signaling pathways that modulate these junctional compo-
nents. Lastly, we delve into the complex dynamics between the junctional complexes, microbial
communities, and environmental chemicals in shaping the intestinal barrier function.
Comprehending this intricate interplay holds paramount importance in unraveling the pathophy-
siology of gastrointestinal disorders. Furthermore, it lays the foundation for the development of
precise therapeutic interventions targeting barrier dysfunction.
ARTICLE HISTORY
Received 13 June 2024
Revised 29 July 2024
Accepted 1 August 2024
KEYWORDS
Gut barrier; junctional
complex; microbes; intrinsic
modulators; extrinsic
modulators; C. elegans;
Drosophila; mouse model;
Gastrointestinal diseases
1. Introduction
The digestive system is the first organ to develop
during animal evolution.
1
While many species can
thrive without specialized organs like the brain,
heart, kidney, or lung, none can endure without a
functioning gut. In fact, the digestive system
emerges as the linchpin of survival across multi-
cellular organisms.
1
In humans, the gastrointestinal
(GI) tract stands as the largest interface between
the organism and its environment, spanning an
estimated area of 20–30 square meters.
2
This vast
expanse of the adult human intestinal epithelium
forms a critical barrier, akin to the protective func-
tion of the skin albeit on a much larger scale.
2,3
Within this intricate ecosystem, an estimated 100
trillion to over 1,000 trillion microbes reside, many
in symbiosis with the host by supporting metabo-
lism and competing against pathogens. Despite this
symbiosis, numerous microbes also constantly pose
a threat of pathogenic invasion, which can severely
disrupt bodily balance. To maintain stability
amidst this constant assault, the intestine must
uphold a strong barrier, tightly regulating the
entry of foreign particles into the body.
4,5
Arguably the most important role of the intestinal
epithelium cells (IECs) is their highly-tuned selec-
tivity for the passage of molecules across the intest-
inal epithelium, a single-cell thick layer of tissue that
is selectively permeable to different molecules
depending on their size and charge.
6,7
The ‘Leak
Pathway’ describes a route in which larger, non-
charged molecules (>5 Å) can move across the bar-
rier (albeit in limited capacity), whereas the “Pore
Pathway” allows for smaller (~4.5 Å), charged ions
and water molecules to permeate.
8–10
While these
pathways differ in their functions, they are con-
trolled by a large group of evolutionarily conserved
protein structures with similar features that form
connections to anchor adjacent cells, namely, intest-
inal junctional complexes.
7
CONTACT Rui Xiao rxiao@ufl.edu Department of Physiology and Aging, College of Medicine, University of Florida, PO Box 100274, Gainesville, FL
32610, USA
GUT MICROBES
2024, VOL. 16, NO. 1, 2389320
https://doi.org/10.1080/19490976.2024.2389320
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted
Manuscript in a repository by the author(s) or with their consent.
Disruptions in the integrity of the intestinal bar-
rier frequently result in the abnormal leakage of gut
contents into the bloodstream, triggering increased
inflammation, autoimmune reactions, and malnu-
trition, among other effects.
11,12
This phenomenon
is implicated in a broad spectrum of diseases,
including inflammatory bowel diseases (IBDs),
irritable bowel syndrome (IBS), liver disorders,
and even neurodegenerative conditions.
13–18
The
increasing evidence linking intestinal permeability
with IBDs, alongside their significant economic
burden, has prompted researchers to propose tar-
geting the intestinal barrier as a strategy to manage
various intestinal and extraintestinal ailments.
Notably, studies indicate that increased intestinal
permeability often precedes the onset of IBDs and
other related conditions, suggesting a potential
causal role.
13,19
For instance, longitudinal studies
with first-degree relatives of Crohn’s Disease
patients show that these individuals often experi-
ence increased permeability years before
diagnosis.
19
Additionally, research suggests a posi-
tive correlation between the severity of IBD cases
and elevated permeability levels.
13
IBDs are prevalent in developed nations, affect-
ing over 0.3% of the population, with an estimated
3 million adults diagnosed in the United States
alone by 2015.
20,21
In 2004, the direct medical
costs for IBD patients in the United States sur-
passed $6 billion annually, while Europeans
incurred direct costs ranging from €4.6–5.6 billion
despite a lower incidence rate.
22,23
Given the sig-
nificant implications of intestinal permeability in
IBDs and related conditions, understanding how
junctional complexes regulate intestinal perme-
ability is of utmost importance. In this review,
we focus on the intestinal junctional complexes
by firstly examining their components across dif-
ferent species, and then exploring techniques for
assessing intestinal permeability. Subsequently,
we survey some of the known signaling pathways
and microbial elements involved in influencing
intestinal permeability and maintaining gut bar-
rier integrity. Lastly, we spotlight the intricate
relationship between the gut microbiome, parti-
cularly bacteria, in both fostering and protecting
against leaky gut conditions, highlighting recent
advances and posing questions for the future of
the field.
2. The structure and organization of the
intestinal barrier complexes
2.1. Mammalian intestinal barrier complexes
The mammalian intestinal barrier, traditionally con-
ceptualized as a tripartite junctional complex, com-
prises tight junctions, adherens junctions, and
desmosomes (Figure 1(a)).
6,24
In mammalian cells,
the most apical connection between intestinal cells is
the tight junction, a protein complex that forms a
firm seal between neighboring cells.
25
Also known as
zonulae occludens, tight junctions were first described
in the mid-1960s.
24
A characteristic of these junctions
is the fusion of adjacent cell membranes at multiple
points along the cells’ surface. At these points of
fusion, the intercellular space is eliminated, forming
a very tight seal between cells.
24,26
In addition, the
tight junction acts as a “fence” on the cell membrane
that separates the apical and basolateral sides and
controls the traffic of components across the cell
membrane.
7,27
Their formation is dependent on the
expression of several proteins that vary in size and
function. Foremost are the zonula occludens proteins
(ZO-1, ZO-2, and ZO-3), a family of scaffolding
proteins that anchor other transmembrane proteins
to the cytoskeleton. ZO-1, the primary member of
this family, is hypothesized to play a crucial role in
regulating tight junction structure. It functions as a
bridge linking transmembrane proteins like claudin
and occludin to the actin cytoskeleton.
28,29
Claudin proteins are integral membrane proteins
that span the cell membrane four times and extend
outward to engage in either homophilic or hetero-
philic interactions with other claudins.
30
The mam-
malian claudin family consists of approximately 27
different proteins with varying functions. While the
functions of many claudin family members remain
unknown, several (such as claudins 1, 3, 4, 5, 7, 11,
14, and 19) are known for their sealing properties,
which enhance the integrity of tight junction
barriers.
31–33
By contrast, other claudins are not
involved in tight junction sealing, rather forming
selective channels that allow for the passage of
cations (claudins 2, 10b, 12, and 15), anions (clau-
dins 10a and 17), and water molecules (claudin-2)
across tight junctions.
30,31,34
Nonetheless, all claudin
proteins play crucial roles in the formation of tight
junctions, and ongoing research aims to uncover the
functions of the remaining family members.
30,31,34
2Z. MARKOVICH ET AL.
Figure 1. Comparative analysis of junctional complexes across evolutionary models of intestinal barriers. (a), in the vertebrate
intestine, junctional complexes comprise tight junction, adherens junction, and desmosomes, with each complex containing multiple
GUT MICROBES 3
Occludin is a member of the tight junction-
associated MARVEL-domain protein (TAMP)
family of proteins. Despite differences in length
and the specific cytosolic domains of the proteins,
occludin and other TAMPs function similarly to
claudins in their membrane transversal and pairing
with other proteins in the tight junction.
30
The
pairing of these proteins along with other tight
junction proteins such as Junctional Adhesion
Molecules (JAMs, which span the membrane only
once) and tricellulin leads to the formation of a
barrier that prevents the free flow of molecules
across epithelial membranes.
30
More basal than tight junctions are the adherens
junctions, also known as zonulae adherens (Figure
1a). Unlike tight junctions, adherens junctions do
not fuse the membranes of adjacent cells and instead
leave a gap between the cells approximately 20 nm
in length.
35
Protruding into the gap and bridging the
cells are 9 nm thick, rod-shaped proteins known as
cadherins.
35
Cadherin proteins are anchored into
the membrane and form binding pairs with other
cadherins on neighboring cells.
35,36
Within the cell
they bind catenin proteins through which they are
associated with the actin cytoskeleton, ultimately
providing a support network and rigidity for the
cellular junction.
36
While their morphology differs
in different cell types, adherens junctions in polar-
ized epithelial cells, like those found in the intestine,
form a continuous ring with intracellular F-actin
(known as the adhesion belt) that wraps around
the entirety of the cell and contributes to the barrier
function of the tissue.
36
Closest to the basal surface of the cell and the last
of these three junctional complexes are desmo-
somes (Figure 1(a)), also recognized as Macula
Adherens.
37
In contrast to adherens junctions reli-
ance on actin microfilaments, desmosomes utilize
intermediate filaments.
38
Bundles of intermediate
filaments extend outwards from the nucleus to the
cell membrane where they anchor the desmosome
junctional proteins.
38
Nonetheless, desmosomes
are similar to adherens junctions in that
desmosomes also employ a family of cadherin pro-
teins to mediate their junctional capacity. Within
the intercellular space, two cadherin subtypes (des-
mogleins and desmocollins), recognize and bind to
each other, fostering a robust cell-to-cell junction
that withstands mechanical stress.
38,39
2.2. Invertebrate intestinal barrier complexes
Many of the intestinal junctional proteins found in
mammals are evolutionarily conserved across
diverse species, including lower organisms. For
instance, the model organism Drosophila melanoga-
ster (D. melanogaster) possesses analogues of both
adherens junctions and tight junctions, although
their organization differs from vertebrates. In D.
melanogaster, the adherens junction occupies the
most apical position among junctional complexes
(Figure 1(b)).
40
It is predominantly composed of
Drosophila epithelial (DE)-cadherin, which serves
as a counterpart to vertebrate cadherin proteins.
40
DE-cadherin circumscribes the cells near the apical
surface and interacts with the sole D. melanogaster
β-catenin homologue, known as Armadillo.
41
Similar to mammalian adherens junctions, the bind-
ing of DE-cadherin to Armadillo β-catenin facili-
tates interaction with α-catenin, which in turn
directly interacts with actin cytoskeleton.
41
In con-
trast to mammalian tight junctions, the analogous
structure in D. melanogaster is located subapical to
the adherens junction, known as the “septate junc-
tion” (Figure 1(b)).
42
Septate junctions serve a simi-
lar function to tight junctions by sealing cells
together to form a barrier across epithelia.
43
Similar to tight junctions, septate junctions contain
claudin proteins such as Megatrachea (Mega),
Sinuous (Sinu), and Kune-Kune that regulate the
structure and function of the junction.
42
They also
contain two important structural proteins named
Neurexin IV and Coracle.
43
While mammalian
tight junctions appear as fused regions between
adjacent cells, septate junctions have a ladder-like
intercellular structure and are not as tightly
key junctional proteins. (b), in the Drosophila intestine, adherens junctions and septate junctions form the protective junctional
complex among intestinal epithelial cells. Many evolutionarily conserved junctional proteins are expressed in the fly intestinal barrier.
(c), the C. elegans apical junction consists of the cadherin-catenin complex and DLG-1/AJM-1 complex, with many junctional proteins
sharing homology with higher species.
4Z. MARKOVICH ET AL.
associated.
42
Overall, while the organization of junc-
tional complexes in D. melanogaster differs from
that in mammals, many homologous proteins func-
tion similarly to create an protective cell barrier.
In Caenorhabditis elegans (C. elegans), the
intestine consists of just 20 cells that together
form a tubular structure within the organism.
44
Despite their apparent simplicity, these intestinal
cells are interconnected via an apical junctional
complex of remarkable sophistication. As a func-
tional homologue to the bipartite cell junctions in
the Drosophila intestine (adherens junctions and
septate junctions) and tripartite structures in ver-
tebrate intestines (tight junctions, adherens junc-
tions, and desmosomes), the C. elegans Apical
Junction (CeAJ) exhibits a more condensed struc-
ture, featuring two distinct domains: the cad-
herin-catenin complex (CCC) and the DLG-1/
AJM-1 complex (DAC) (Figure 1(c)).
44
The
CCC in C. elegans, akin to classical adherens
junctions in vertebrates, comprises proteins such
as HMP-1, HMR-1, and HMP-2 which are
homologues of human α-catenin, E-cadherin,
and β-catenin, respectively.
45
Notably, HMP-1
directly binds to F-actin and associates with
HMR-1 via HMP-2, mirroring the interactions
observed in mammalian cells.
45
Additionally, the
Juxtamembrane Domain-associated Catenin
(JAC-1), a homologue of human p120 catenin,
plays a crucial role in regulating cadherin func-
tion, as evidenced by exacerbating morphological
defects in HMP mutants when absent.
46
More
basal in the CeAJ is the DAC which is formed
by the two key proteins: AJM-1 and DLG-1.
AJM-1, an orthologue of human AJM1, is a
coiled-coil protein, while DLG-1 shares homology
with the Drosophila discs-large protein.
47
DLG-1
aids in recruiting AJM-1 to the DAC, with its
spatial localization mediated by another protein,
LET-413.
47
Mutations in LET-413 result in severe
adhesion and polarity defects, emphasizing its
role in proper protein positioning.
48
The DAC
also contains a homolog of mammalian claudin
named CLC-1.
44
Together, the CCC and DAC
establish a densely packed region between C.
elegans intestinal cells, mirroring the junctional
complexes observed in mammalian intestines and
featuring numerous homologous proteins.
2.3. Animal models for studying intestinal
permeability and related conditions
Exploring junctional complexes in genetic model
organisms such as Drosophila and C. elegans offers
several advantages and invaluable insights into the
conservation of critical proteins across species.
Despite variations in protein structure and organi-
zation, key proteins like cadherin, catenin, and
claudin are well conserved in intestinal junctions
across diverse organisms, highlighting their pivotal
role in establishing a robust barrier system essential
for intestinal protection, digestion, and immune
function.
49
In addition, model organisms often
feature rapid life cycles, large broods, and sophis-
ticated genetic toolkits, enabling the genetic study
of intestinal junctional complexes in vivo with
unparalleled efficiency. Moreover, the transparent
bodies of C. elegans and Drosophila larvae facilitate
direct visual assessment of gut leakage, enhancing
our ability to study intestinal barrier function.
50,51
Furthermore, Drosophila and C. elegans are ideal
for high-throughput genetic and chemical screens.
Researchers can quickly test the effects of many
genes or compounds on intestinal barrier function,
aiding in the discovery of potential therapeutic
targets and drugs.
While D. melanogaster and C. elegans offer sig-
nificant advantages for understanding basic and
evolutionary aspects of intestinal barriers, using
these invertebrate models to study human intest-
inal diseases presents several limitations. For exam-
ple, the macro-structure of the intestine and
signaling pathways can significantly differ among
mammals, nematodes, and insects.
52
Moreover, D.
melanogaster and C. elegans lack an adaptive
immune response, thus missing key cell types and
cellular processes integral to mammalian intestinal
epithelium.
52
Additionally, the composition of gut
microbes differs drastically between invertebrate
models and humans, with D. melanogaster and C.
elegans harboring much simpler and less diverse
microbial communities tailored to their evolution-
ary and physiological needs.
52
Thus, to better
mimic human intestinal diseases such as IBDs,
rodent models are often preferred to mitigate
these limitations.
53
Mice have a GI tract anatomically similar to
humans, with analogous elements of adaptive
GUT MICROBES 5
immune response.
52
The gut microbiome in mice is
also similar in composition to humans with the
same species diversity of bacteria phyla.
52
Genetically, mice are invaluable for studying spe-
cific genes due to their high homology with
humans and the ability to generate specific genetic
knockouts. Various methods induce IBD-like
symptoms in mice, such as trinitrobenzene sulfonic
acid (TNBS) treatment in the BALB/c background
to mimic Crohn’s disease-like symptoms, whereas
C57BL/6 mice are relatively unaffected.
52,53
Another common model is sodium dextran sulfate
(DSS)-induced colitis, which induces inflammation
and ulcerative colitis symptoms, with severity vary-
ing across different mouse strains.
52,53
The robust-
ness of murine systems in modeling IBDs has been
pivotal for advancing research in this field. Many
modern discoveries in IBD treatment and manage-
ment stem from foundational studies in mice. For a
comprehensive discussion on the advantages and
disadvantages of various model organisms and
their contributions to understanding IBDs and
related intestinal conditions, please refer to insight-
ful reviews by Jiminez et al. and Kiesler et al., which
provide extensive coverage of this topic.
52,53
3. Techniques for assessing intestinal
permeability
Due to the importance of intestinal barrier integrity,
numerous experimental procedures have been devel-
oped to measure intestinal permeability both in vitro
and in vivo. Here we highlight a selection of the most
widespread methods for assaying gut leakage.
3.1. Oral probe excretion assays
One of the more traditional methods for mea-
suring human intestine permeability is through
administration of saccharide-like or radiolabeled
probe molecules. These probes are chosen for
specific properties such as their resistance to
bacterial degradation, solubility in water, and
relative inertness.
54
Also key to these probes is
their inability to pass through the intestinal
barrier via transcellular pathways.
55
Following
ingestion, the probe molecules pass through
the GI system where they may leak across the
intestinal barrier. The leakage can then be
quantified via renal excretion assays and serve
as a marker for intestine permeability (Figure 2
(a)). Depending on their sizes and the severity
of leakage in gastric tissues, different ratios of
the probes can be detected in the urine of
subjects.
56
For example, the monosaccharide
probe mannitol is relatively small with a dia-
meter of 6.5 Å and believed to cross the epithe-
lial barrier through the pore pathway.
54
In
contrast, the disaccharide probe lactulose is a
larger molecule with the diameter of 9.5 Å and
thought to only cross via the leak pathway.
54
The lactulose:mannitol ratio (LMR) in urine
samples can therefore provide insight on the
activity of these two pathways. Studies have
reported a LMR in healthy individuals to be
0.014, while in IBD patients the LMR increases
10-fold to 0.093–0.133.
57
Intriguingly, healthy
individuals who are first-degree relatives of
IBD patients often exhibit elevated LMRs prior
to disease onset and have a markedly increased
predisposition to developing IBD later in life.
19
While the use of oral probes in detecting intest-
inal permeability is convenient, it is important to
note its complications. First, oral probes are indir-
ect measurement of intestinal permeability.
Second, recent research has challenged the accu-
racy of oral probes in measuring gut permeability.
For example, the sizes of two popular oral probes
mannitol and lactulose may not differ
significantly.
58
However, mannitol is excreted in
orders-of-magnitude higher concentrations than
lactulose: 31.2 ± 3.4% of administered
13
C-manni-
tol is excreted within 24 hours in healthy adults,
compared to just 0.32 ± 0.03% for lactulose.
59
Thus,
the evidence for similarly-sized probes with differ-
ing excretion rates questions the validity of the leak
versus pore distinction since both molecules may
be crossing via the same pathway. Taken together,
cautions need to be taken to interpret the result of
using oral probes to detect leaky gut.
3.2. Transepithelial electrical resistance assay and
Ussing chamber assay
The Transepithelial Electrical Resistance (TEER)
assay is an in vitro method for real time and direct
measurements of barrier integrity in cellular
monolayers.
60
TEER is frequently used to study
6Z. MARKOVICH ET AL.
intestinal tissue permeability in Caco-2 cells
derived from a human colorectal adenocarcinoma,
but is also useful for other, intact epithelial tissues
such as the pulmonary alveolar epithelial barrier,
urinary tract epithelial barrier, and intestinal
epithelial barrier.
61,62
Caco-2 cells grow naturally
into monolayers and have many similar functions
to in vivo small intestine villi epithelium.
63
For grown cellular monolayers, the TEER assay
involves culturing the tissue of interest as a single-
cell layer on a semi-permeable membrane, forming a
tight monolayer that mimics the epithelial barrier in
vivo. With electrodes placed in the apical and baso-
lateral compartments, a small electrical current is
applied across the cell monolayer, and the resistance
to this current is measured (Figure 2(b)).
64,65
TEER measurements exhibit considerable varia-
bility due to factors such as cell culture conditions,
experimental setup, and the specific laboratory
conducting the assay. Typically, data are presented
as unit area resistance, calculated by dividing resis-
tance values by the membrane area.
66
For instance,
TEER values in Caco-2 cells have been documented
to range from 49 to 137 Ω/cm
2
depending on their
degree of differentiation.
61
Additionally, studies
have reported TEER measurements as high as
nearly 400 Ω/cm
2
in healthy Caco-2 cell controls,
while exposure to lipopolysaccharide (LPS) can
lead to a decrease of up to 50% in TEER values.
66
For an extensive compilation of TEER values in
Caco-2 monolayers across various experimental
setups, refer to Srinivasan et al.
60
When studying whole-tissue samples like the
intestine, researchers often utilize the Ussing
chamber assay. This method preserves the hetero-
geneity and morphology of intact intestinal tissue
by mounting it in a specialized chamber that allows
separate solutions on both apical and basolateral
sides.
62
Unlike the TEER assay, which measures the
resistance of cellular monolayers, the Ussing cham-
ber assay evaluates the electrical properties such as
voltage and the movement of ions across the
epithelial barrier under controlled conditions.
Combining the Ussing chamber with radio-labeled
probes like
14
C and
3
H allows for quantitative
assessment of barrier permeability.
67
This
Figure 2. Popular methods for assessing intestinal permeability. (a), the oral probe excretion assay is a widely employed indirect
method where probe molecules traverse the gastrointestinal system and potentially cross the intestinal barrier, subsequently
quantified through renal excretion assays to gauge intestinal permeability. (b), the transepithelial electrical resistance (TEER) assay
measures the electrical resistance across a cell monolayer, commonly used to assess the integrity and barrier function of epithelial or
endothelial cell layers. (c), the gut permeability staining assay involves the visualization of fluorescently labeled molecules or tracers
that permeate through the gut epithelium, thus providing insights into intestinal barrier integrity and permeability. This method is
commonly used in model organisms with transparent anatomy.
GUT MICROBES 7
approach provides valuable insights into ion trans-
port, secretion, and absorption across epithelial
tissues ex vivo.
3.3. Staining assays
In addition to the methods outlined above, the
Smurf assay and Fluorescein isothiocyanate
(FITC)-Dextran staining assay are commonly used
to visualize leaky gut. The Smurf assay involves
feeding a non-absorbable blue dye to animals and
monitoring the leakage of the dye from the intestine
into the body (Figure 2(c)).
51
The most frequently
used dye is FD&C Blue No. 1 (disodium 2-[[4-
[ethyl-[(3-sulfonatophenyl)-methyl]amino]phenyl]-
[4-[ethyl-[(3-sulfonatophenyl)methyl]-azaniumyli-
dene]cyclohexa-2,5-dien-1-ylidene]methyl]benze-
nesulfonate) which is more commonly known as
Brilliant Blue FCF. This dye has been widely used
in commercially processed foods as a coloring agent
since it was first approved by the FDA in 1993.
68
The Smurf assay, pioneered in D. melanogaster
and later adapted for C. elegans, allows for the
identification of ‘blue Smurfs’ – organisms exhi-
biting increased intestinal leakage, indicated by a
blue-stained appearance.
51
Following dye inges-
tion and an incubation period, animals can be
sorted based on the color of their bodies, either
normally colored or stained blue. Animals with
blue bodies are a result of increased leakage from
the intestine. This leakiness leads to the charac-
teristic blue stained appearance by which the
assay gets its name, i.e. blue Smurfs.
51,69
Notably, the Smurf phenotype is typically
assessed as a binary outcome, reflecting either
increased permeability or an intact barrier. The
proportion of Smurf animals within a population
serves as a measure of overall leakiness and has
been linked to lifespan in various organisms.
69
Importantly, studies have demonstrated a corre-
lation between the proportion of ‘blue Smurfs’
and lifespan. In Drosophila, the proportion of
wildtype flies exhibiting the Smurf phenotype
increases with age.
51
Conversely, the eat-2
mutant of C. elegans, known for its extended
lifespan due to dietary restriction, displays a
markedly lower Smurf proportion compared to
wildtype counterparts.
70
These findings align
with observations in humans, indicating that
age plays a pivotal role in determining intestinal
permeability.
71
Another method for evaluating intestinal perme-
ability is the FITC-Dextran staining assay.
72
Operating on a principles akin to the Smurf assay,
this method may be performed by orally administer-
ing fluorescently labeled FITC-Dextran to animals or
by staining in vitro cellular monolayers and three-
dimensional animal and human intestinal organoids.
-
73
Subsequent monitoring allows detection of leakage
from the tissue of interest into the surrounding tissue
(Figure 2(c)), serving as an indicator of permeability.
Enhanced fluorescence observed in across the barrier
tissue corresponds with heightened levels of intestinal
leakage and compromised integrity of junction pro-
tein complexes.
66
It is important to note that the
commonly used FITC-4 kDa dextran probe for asses-
sing intestinal permeability cannot differentiate
between leak and unrestricted pathways, as 4 kDa
dextran can cross both. Additionally, 4 kDa dextran
is too large to pass through the pore pathway, thereby
providing no information about paracellular flux
through this route. To quantitatively measure barrier
permeability across pore, leak, and unrestricted path-
ways, and to accurately assess flux changes due to
defects in cell junctions or overall epithelial damage,
a three-probe fluorescent system has been developed.
-
74
This assay utilizes three separate probes: creatinine
(6 Å diameter), FITC-4 kDa dextran (28 Å diameter),
and rhodamine-70 kDa dextran (120 Å diameter),
each probing different leakage routes. Creatinine can
permeate all three pathways, while 4 kDa dextran is
restricted to leak and unrestricted pathways, and 70
kDa dextran is limited to the unrestricted pathway.
74
Importantly, this technique can be applied either in
vivo via oral gavage or ex vivo using the Ussing cham-
ber, thereby combining the advantages of both
approaches.
4. Intrinsic factors involved in regulating
intestinal barrier complexes
As previously described, alterations in intestinal per-
meability often precede the onset of many intestinal
and extraintestinal disorders. Thus, unraveling the
factors that contribute to changes in intestine perme-
ability has become an important facet in combating
these diseases. In the subsequent sections, we focus on
8Z. MARKOVICH ET AL.
modulators that impact the integrity of the intestinal
barrier complexes, highlighting endogenous (Figure
3) and exogenous (Figure 4) contributors to intestinal
leakage. Herein, we present a compilation of signaling
pathways and effectors widely implicated in GI dis-
eases, most of which have been predominantly stu-
died using mouse models of intestinal barriers. While
this list is not exhaustive, it highlights some of the
most recognized and influential pathways known to
regulate the intestinal epithelial barrier.
4.1. Myosin light chain kinase (MLCK) pathway
Among the myriad signaling pathways implicated in
the regulation of gut barrier complexes, the myosin
light chain kinase (MLCK) stands out as a primary
player. Encoded by the MYLK1 gene, intestinal
MLCK exists in two splice variants: MLCK1 and
MLCK2.
75
Both variants are calmodulin-activated
serine/threonine kinases long known to orchestrate
tight junction organization in intestinal epithelial
cells, a process intricately linked to Na
+
-glucose
cotransport.
75
Mechanistically, the demand for Na
+
transport across the barrier triggers MLCK activation,
leading to the phosphorylation of myosin II regulatory
light chain (MLC) within the perijunctional actomyo-
sin ring (PAMR). This phosphorylation triggers a
contractile force that increases physical tension on
tight junctions, potentially causing structural defor-
mations and thereby elevating paracellular
permeability.
76
Enhanced expression and activity of
MLCK are frequently observed in cases of IBDs in
both rodent models and human patients, making it an
attractive therapeutic target.
77,78
Several recent studies
Figure 3. A summary scheme of the intrinsic cellular signaling pathways modulating intestinal permeability.
GUT MICROBES 9
Figure 4. Gut microbes and common chemicals regulate intestinal junctional complexes in a complex manner. (a), many beneficial
microbes (e.g., L. plantarum, A. muciniphila, B. longum) promote the gut barrier integrity and reduce cytokine release and
10 Z. MARKOVICH ET AL.
have explored the potential of MLCK
inhibition in controlling IBD pathology and reducing
intestinal permeability.
79,80
In a recent study, researchers demonstrated that
recruitment of MLCK1 to the PAMR, followed by
MLC phosphorylation, heightened permeability, as
evidenced by a reduction in TEER.
80
Through
screening a library of 140,000 compounds for
MLCK1 inhibitory activity, they identified a novel
compound termed “divertin”, which impedes the
recruitment of MLCK1 to the PAMR. Addition of
divertin to TNF-treated cells restored TEER levels
to those of healthy, non-TNF-treated cells and
reversed MLC phosphorylation.
80
These findings
were corroborated in vivo, as divertin protected
mice administered TNF to induce barrier dysfunc-
tion. In the absence of MLCK1, MLC phosphoryla-
tion is inhibited, preventing occludin endocytosis
and thereby preserving tight junctions integrity.
80
4.2. AMP-activated protein kinase (AMPK) pathway
AMPK signaling has also been implicated as a
key pathway for regulating inflammation and
tight junction expression within the intestine.
In a DSS-induced colitis model, genetic deletion
of AMPK exacerbated the severity of IBD symp-
toms, potentially attributed to heightened release
of proinflammatory cytokines and increased
macrophage activity.
81
Conversely, activation of
AMPK directly influences tight junctions assem-
bly. For instance, treatment with AICAR, a non-
specific AMPK activator, accelerates the
recruitment of ZO-1 protein to the membrane,
reduces permeability to FITC-dextran, enhances
TEER, and upregulates markers indicative of
intestinal differentiation.
82
Notably, the tran-
scription factor CDX2, crucial for cell differen-
tiation, is upregulated following AMPK
activation. Inversely, inhibition of AMPK abol-
ished the protective permeability effects seen
and CRISPR/Cas9 deletion of CDX2 abolished
differentiation.
82
Overall, AMPK serves as a cri-
tical modulator of intestinal permeability and
tight junction assembly, likely mediated through
mechanisms involving cellular differentiation
and recruitment of tight junction proteins to
the membrane.
83
4.3. cAMP-responsive element-binding protein H
(CREBH) pathway
The cAMP signaling pathway has been exten-
sively explored for its impact on intestinal per-
meability modulation. Central to this pathway is
the cAMP-responsive element-binding protein H
(CREBH), acting as a pivotal transcription
factor.
84
Given reports of defective cAMP signal-
ing in pediatric colitis and the presence of
CREBH in intestinal epithelial cells, it was postu-
lated that they play a pivotal role in regulating
intestinal permeability.
85
In a mouse model of
DSS-induced colitis, both CREBH mRNA and
protein expression were diminished.
85
Notably,
both DSS-treated wild type mice and CREBH
knockout mice exhibited decreased expression of
tight junction proteins critical for maintaining
intestinal barrier integrity, including claudin-1,
claudin-3, claudin-5, claudin-8, and ZO-1.
85
Intriguingly, there was also a substantial increase
in the expression of claudin-2, a known mediator
of leaky gut and promoter of IBD progression.
85
This regulation of tight junction proteins via
CREBH may be related to IGF signaling, which
is known to stimulate epithelial cell proliferation
following injuries to the intestine.
86
Further
investigations unveiled a downregulation of
IGF1R in both DSS-treated wild type mice and
CREBH knockout mice, with expression levels
being restorable through forced expression of
CREBH.
85
Together, these findings suggest that
CREBH plays a crucial role in stimulating IGF1R
expression, ultimately leading to an increase in
the expression of essential tight junction proteins.
This mechanism contributes to the enhancement
of gut barrier health and the reduction of perme-
ability, offering potential therapeutic avenues for
addressing intestinal disorders.
inflammation. (b), detrimental microbes (e.g., C. albicans, E. faecalis, S. aureus) undermine gut barrier integrity by diminishing
junctional protein expression and organization. (c), a summary of microbes and chemicals that are known to affect gut barrier
permeability through diverse mechanisms.
GUT MICROBES 11
4.4. TLR4 pathway
The Toll-like receptor 4 (TLR4) signaling pathway
stands as a cornerstone of innate immunity, and it
is a pivotal signaling pathway governing gut
permeability through its activation of downstream
proinflammatory signaling events. Specifically,
TLR4 serves as a pivotal receptor known for its
recognition of bacterial remnants, notably LPS.
87
Within the intestine, LPS engagement with epithe-
lial TLR4 receptors initiates a signaling cascade
culminating in the activation of nuclear factor–κB
(NF-κB) transcription factors and other proinflam-
matory processes.
88,89
Elevated NF-κBp65 levels
and intestinal inflammation have been associated
with decreased expression of ZO-1 and occludin.
90
While basal TLR4 expression in intestinal tissue
is low, it plays a crucial role in safeguarding against
intestinal and bacterial injuries.
88,91
Notably, stu-
dies have consistently observed elevated TLR4
expression in the intestinal cells of individuals
with IBD, with overexpression in murine models
correlating with increased susceptibility to chemi-
cally induced colitis.
87,92
Moreover, experiments
with TLR4-overexpressing mice have demon-
strated compromised intestinal barrier function,
leading to heightened permeability and leakage of
FITC-dextran into the serum.
93
4.5. Wnt/β-catenin pathway
The canonical Wnt signaling pathway, also referred
to as the Wnt/β-catenin pathway, plays a pivotal
role in cell proliferation by stabilizing and translo-
cating β-catenin into the nucleus.
94
In the intestine,
this pathway is indispensable for the maintenance
and regeneration of intestinal stem cells and tissue
integrity.
95
Dysregulation of Wnt signaling is
implicated in various intestinal diseases, including
necrotizing enterocolitis and IBDs, which are char-
acterized by intestinal injury, inflammation, and
compromised gut barrier function.
95,96
Activation of Wnt signaling leads to the nuclear
translocation of β-catenin, which then activates
many target genes through β-catenin-T-cell fac-
tor/lymphoid enhancer-binding factor (TCF/LEF)
transcription factors. Notably, these target genes
include proteins crucial for tight junction assembly
in the intestine, such as ZO-1 and occludin.
90
Studies in mice have shown that disruption of
Wnt/β-catenin signaling results in reduced
mRNA expression of ZO-1 and occludin, accom-
panied by increased intestinal permeability, as indi-
cated by FITC-dextran staining. Conversely,
restoration of Wnt signaling reverses these effects,
highlighting the importance of Wnt signaling in
maintaining intestinal barrier function.
90
Furthermore, Wnt signaling has been found to
inhibit NF-κB activity through direct interaction
of β-catenin with NF-κB, thereby mitigating
inflammation and tissue damage.
97,98
Together,
these results implicate Wnt/β-catenin signaling as
another important pathway affecting intestinal
inflammation and junctional organization.
4.6. Notch pathway
NOTCH receptors also play a crucial role in reg-
ulating the integrity of the intestinal barrier. These
membrane-bound proteins undergo endocytosis
and nuclear translocation upon activation by their
binding partners.
99
Once cleaved in the nucleus,
NOTCH acts as a transcription factor, orchestrat-
ing the expression of numerous genes involved in
development, tissue repair, and cell differentiation.
-
99
In the GI tract, NOTCH activity is specifically
linked to the regulation of tight junction proteins.
Interestingly, in mice lacking lamina propria lym-
phocytes that induce intestinal epithelial differen-
tiation, the absence of cleaved Notch-1 protein
correlated with increased intestinal permeability.
This was evidenced by reduced TEER measure-
ments and increased FITC-dextran staining.
100
Further evidence from Notch-1 knockdown experi-
ments in Caco-2 cell lines underscores NOTCH’s
direct impact on barrier integrity, with increased
intestinal permeability observed compared to con-
trol lines.
100
These studies suggest that cleaved
NOTCH proteins transcriptionally regulate the
expression of proteins crucial for the architecture
of intestinal junction complexes.
100
While the relationship between NOTCH activity
and specific junction proteins may vary, research
indicates a direct correlation between NOTCH
activity and the expression of proteins such as
12 Z. MARKOVICH ET AL.
occludin and claudin-1, while an inverse relation-
ship exists with proteins like claudin-5.
100,101
Overall, the NOTCH pathway emerges as another
critical signaling pathway intricately linked to the
expression and maintenance of junction complex
proteins in the intestinal epithelial barrier.
4.7. Autophagy
Routine cellular processes such as autophagy, along
with other stress-response factors, play a pivotal
role in regulating tight junction architecture and,
consequently, are essential for maintaining barrier
integrity. Autophagy is a highly conserved mechan-
ism of cellular recycling that eliminates damaged
and aged proteins.
102
This homeostatic process is
crucial for responding to cellular stress and is par-
ticularly active in the proliferative component of
colonic crypts.
103
Under stress conditions, the
autophagic machinery engulfs cytoplasmic compo-
nents, which are subsequently degraded upon
fusion with lysosomes.
103
Regarding its impact on permeability, autophagy
induction protects the intestinal barrier by influen-
cing the expression of tight junction proteins. For
instance, claudin-2, a pore-forming claudin protein,
undergoes lysosomal degradation during starvation-
induced autophagy.
102
Conversely, TNF-mediated
inhibition of autophagy leads to increased claudin-
2 expression and heightened permeability.
103
Beyond claudin-2, autophagic activity also impedes
the endocytosis of occludin protein, thereby preser-
ving tight junction structure.
104
5. Microbes involved in regulating intestinal
barrier complexes
The gut microbiome constitutes a complex ecosys-
tem of microorganisms inhabiting the human intest-
inal epithelium. This ecosystem undergoes dynamic
changes throughout life, with newborns initially
possessing a sterile intestinal tract. However, by
adulthood, the gut microbiome can comprise up to
10
14
cells, greatly outnumbering their own host cells
nearly tenfold.
105
Among these microorganisms,
bacteria predominate, though the microbiome also
encompasses fungi, viruses, and other organisms,
with estimates suggesting the presence of 300 to
1000 bacterial species.
106,107
While many microbes
are pathogenic and cause damage to the epithelium,
countless others have been investigated for their role
as probiotics, improving the health and integrity of
the intestine. The gut microecosystem maintains a
delicate balance between beneficial and pathogenic
microbes, and disruptions to this balance can lead to
dysbiosis and the overgrowth of opportunistic
organisms. In healthy individuals, beneficial
microbes effectively outcompete specific pathogenic
strains, thereby limiting their ability to spread and
cause infections.
108
In contrast, patients suffering
from IBDs and other related conditions often exhibit
a disturbed microbiome that exacerbates inflamma-
tion and compromises barrier function.
109
Although
these observations are now recognized as hallmarks
of IBD pathology, whether they are causes and/or
effects of the disease is still debated.
109
Understanding how microbiome alterations influ-
ence IBDs and contribute to intestinal barrier func-
tion is essential for developing effective treatment
strategies for these diseases. Due to the open ques-
tions that remain in this field of research, numerous
studies over the past decade have highlighted the
profound impact of bacterial colonization on gut
permeability (Figure 4).
5.1. Benecial microbes for intestinal barrier
complexes
Of particular interest is the utilization of probiotics
to mitigate gut inflammation and subsequent per-
meability. Probiotics often exert their beneficial
effects on permeability by modulating proteins
within cell-adhesion junctions. For example,
Akkermansia muciniphila (A. muciniphila) and its
associated compounds have demonstrated efficacy
in reducing intestinal inflammation and perme-
ability in both in vitro and in vivo models through
diverse molecular pathways (Figure 4(a)).
High-fat diets (HFD) are recognized for their
propensity to increase permeability in experimen-
tal colitis models and exacerbate symptoms of IBD
in humans.
110
HFDs reduce the expression of tight
junction components such as ZO-1 and occludin,
compromising gut barrier integrity and elevating
overall permeability.
111
Interestingly, HFD-fed
mice administered A. muciniphila-derived
GUT MICROBES 13
extracellular vesicles (AmEVs) exhibited a marked
reduction in permeability compared to those on the
HFD alone.
112
Notably, AmEV-treated mice dis-
played elevated expression of tight junction pro-
teins like occludin and claudin, countering the
detrimental effects of the HFD.
112
This suggests
that barrier reinforcement is associated with
enhanced cellular connectivity within the
intestine.
113
Mechanistically, the upregulation of
tight junction proteins induced by AmEVs appears
to involve multiple pathways including the afore-
mentioned phosphorylation of AMPK and cAMP
signaling via CREBH (Figure 4(c)).
85,112
Consequently, it is unsurprising that treatment of
Caco-2 cells with AmEVs resulted in elevated
AMPK phosphorylation, accompanied by a corre-
lated reduction in intestinal permeability. By con-
trast, pharmacological inhibition of AMPK in
AmEV-treated cells attenuated the protective
effects observed in previous experiments.
112
In
addition to AMPK, A. muciniphila may also be
exerting its effect through the activation of
CREBH. Infection of Caco-2 cells with A. mucini-
phila resulted in the upregulation of CREBH
expression, leading to the mitigation of gut
leakage.
85
This effect may be attributed to the pre-
sence of Amuc_1100, an abundant outer mem-
brane protein of A. muciniphila known to
regulate CREBH expression.
85
Furthermore,
Amuc_1100 was shown to upregulate the expres-
sion of toll-like receptor 2 (TLR2), a key player in
intestinal wound healing.
85,114
Taken together,
these findings suggest that A. muciniphila and its
protein components confer a protective effect on
intestinal barrier integrity, potentially through
modulation of multiple signaling pathways.
The supplementation of human diets with gram-
positive, lactic-acid producing bacteria (LAB) has
long been recognized for its beneficial effects in
combating various human diseases.
115
Among
these bacteria, multiple species of the
Bifidobacterium genus, commonly found in the
human gut microbiome, have shown promise in
regulating proinflammatory cytokine expression
and promoting epithelial barrier function. Studies
have revealed that Bifidobacterium longum ssp.
longum can downregulate the expression of proin-
flammatory markers TNF-α and IFN-γ, along with
the anti-inflammatory cytokine IL-10, indicating a
reduced inflammatory state and decreased demand
for anti-inflammatory responses (Figure 4(c)).
116
This bacterial strain has also demonstrated the abil-
ity to decrease permeability as assessed by FITC-
dextran measurements in a DSS-induced colitis
mouse model.
116
Likewise, Bifidobacterium bifidum
(B. bifidum) has demonstrated beneficial effects on
barrier integrity. In experiments using Caco-2 cell
monolayers treated with TNF-α to induce a decrease
in TEER, concurrent administration of B. bifidum
effectively restores TEER levels and enhances occlu-
din expression.
117
Lactobacillus, another genus of LAB prevalent in
the human intestine, has been shown to modulate
intestinal permeability. In a randomized controlled
trial involving healthy human subjects, administra-
tion of Lactobacillus plantarum (L. plantarum)
directly to the duodenum resulted in alterations in
the expression pattern of the tight junction protein
ZO-1.
118
Interestingly, the presence of L. plantarum
appears to force apical localization of ZO-1 and
increase tight junction presence on cell surface
membranes.
118,119
While this increase in tight junc-
tion presence alone did not enhance barrier integrity
in a Caco-2 monolayer, L. plantarum colonization
significantly attenuated the increase in permeability
induced by phorbol 12,13-dibutyrate (PDBu), a deri-
vative of TPA known to cause dislocation of tight
junction proteins.
118
Previous research has demon-
strated that administration of Pam3-Cys-SK4
(PCSK), an artificial Toll-like receptor 2 (TLR2)
ligand, restored tight junction integrity in DSS-
induced colitis mice.
119
Given evidence suggesting
that TLR2 agonism increases translocation of ZO-1
to tight junctions and confers protection, researchers
investigated whether the effects of L. plantarum are
mediated by TLR2 signaling. By assessing TEER and
employing phorbol treatment, they found that acti-
vating TLR2 with PCSK replicated the effects
observed with L. plantarum treatment alone.
118
These findings imply that changes in ZO-1 localiza-
tion induced by L. plantarum may indeed be
mediated by TLR2 signaling (Figure 4(c)).
Lactobacillus paracasei (L. paracasei) and
Lactobacillus acidophilus (L. acidophilus), two other
species within the Lactobacillus genus, have also
demonstrated protective effects against epithelial
barrier damage.
120
Salmonella typhimurium (S.
typhimurium), a pathogenic bacterial strain known
14 Z. MARKOVICH ET AL.
for inducing intestinal epithelial destruction and
irregular remodeling of tight junction proteins,
poses a significant threat to gut health.
121
However,
a recent study revealed that a metabolite derived
from L. paracasei effectively counteracts the dama-
ging effects of S. typhimurium.
120
Specifically, L.
paracasei CNCM I-5220-derived postbiotic (LP-
PBF) was found to prevent the disorganization of
ZO-1 in tight junctions and increase TEER values in
cell monolayers; remarkably, these protective effects
were achieved without adversely affecting the com-
mensal gut microbiota.
120
Unlike antibiotics, which
target bacterial growth, LP-PBF appears to neutra-
lize S. typhimurium and restrict its ability to invade
the intestinal epithelium, possibly by impeding the
formation of S. typhimurium biofilms.
120
Unlike L.
paracasei, L. acidophilus exerts its protective func-
tion by directly modulating the expression of tight
junction proteins. As early as 2005, studies demon-
strated that treating rats with a probiotic cocktail
containing L. acidophilus increased the expression
of occludin.
122
More recent research has shown that
mice fed high-fat diets and subsequently adminis-
tered fecal transplants of L. acidophilus experienced
reduced inflammation and microbiome dysbiosis.
Similar to the effect observed with A. muciniphila,
these mice also showed improved permeability out-
comes, indicated by decreased FITC-dextran stain-
ing and increased occludin expression.
123
Improved
barrier integrity is attributed to decreased activation
of the TLR4 and NF-κB signaling pathways, as well
as reduced expression of downstream proinflamma-
tory cytokines.
123
Numerous studies on L. plan-
tarum, L. paracasei, and L. acidophilus collectively
underscore the critical role of the Lactobacillus
genus in maintaining gut barrier integrity.
5.2. Detrimental microbes for intestinal barrier
complexes
While approximately 93% of known bacterial species
are deemed nonpathogenic, the gut harbors a diverse
range of microbes, some of which can cause diseases.
-
124
Among these, Staphylococcus aureus (S. aureus), a
gram-negative bacterium commonly found in the
human nasal mucosa, poses a significant health risk.
-
125
While S. aureus infections are prevalent on the
skin, they become especially dangerous when they
penetrate deeper tissues, leading to conditions like
sepsis and organ failure, particularly when the strains
are antibiotic-resistant (methicillin-resistant S. aur-
eus, or MRSA) (Figure 4(b)).
124,125
MRSA-induced sepsis can also have profoundly
negative effects on gut barrier function, possibly
due to increased expression of cytochrome
P4501A1 (CYP1A1) (Figure 4(c)). In patients
with sepsis, increased levels of CYP1A1 have been
documented and CYP1A1 inhibitors exhibit anti-
inflammatory, antitumor, and other protective
immune functions.
126,127
Similarly, studies invol-
ving mouse models of MRSA infection have shown
that mice lacking CYP1A1 have a much higher
survival rate and better preservation of gut barrier
proteins (e.g., ZO-1, occludin, and E-cadherin) fol-
lowing MRSA infection compared to wildtype
mice.
127
Further investigations revealed the invol-
vement of cadaverine, a byproduct of lysine meta-
bolism produced by gut microflora, in MRSA-
induced gut permeability.
127
Cadaverine levels
rise significantly during MRSA infection, with
CYP1A1 potentially playing a crucial role in its
synthesis.
127
Interestingly, pretreatment with cada-
verine abolishes the protective effects against
MRSA infection in Cyp1a1 knockout mice, indicat-
ing the importance of CYP1A1 in this context.
127
Among the various commensal gut microbiota,
Enterococcus faecalis (E. faecalis) stands out as one
of the most prolific producers of cadaverine. In the
cecal contents of both Cyp1a1
+/+
and Cyp1a1
–/–
mice, cadaverine levels were increased following
oral gavage with E. faecalis.
127
Co-infection of
Cyp1a1
+/+
mice with E. faecalis and MRSA leads
to notable outcomes, including a decrease in ZO-1
expression, heightened intestinal permeability, and
reduced survivability compared to untreated
Cyp1a1
–/–
mice. Interestingly, Cyp1a1 knockouts
maintain a protective phenotype against these
effects, indicating the crucial role of CYP1A1 in
gut homeostasis.
127
Mechanistically, the increased
activities in MLCK and NF-κB pathways are likely
main drivers behind the increase in intestine per-
meability, as cadaverine can cause downstream
activation of both MLCK and NF-κB via agonism
of histamine receptor (HRH) family member
HRH4 (Figure 4(c)).
127
Notably, HRH4 expression
is also upregulated in the epithelium of Cyp1a1
+/+
mice with MRSA infection.
127
These findings
GUT MICROBES 15
collectively underscore the contribution of both
MRSA and E. faecalis—despite their natural pre-
sence in the human gut microbiome – to the
pathology of leaky gut. Their induction of cadaver-
ine levels, leading to NF-κB pathway activation,
highlights the intricate interplay between gut
microbiota and intestinal barrier integrity.
Interestingly, despite Cyp1a1
-/-
mice being pro-
tected against MRSA infection, Cyp1a1 expression
does not consistently indicate permeability status.
The aryl-hydrocarbon receptor (AhR) is a ligand-
activated transcription factor that detects xenobio-
tic compounds and regulates genes involved in
xenobiotic metabolism, including Cyp1a1.
128
Activation of AhR by the microbial metabolite
Urolithin A (UroA) mitigated symptoms of DSS-
induced colitis in mice, but this effect was absent in
Cyp1a1-/- animals.
129
Furthermore, wildtype mice
treated with UroA showed reduced FITC-dextran
staining and significant increases in ZO-1, occlu-
din, and claudin-4, whereas Cyp1a1-/- mice did not
exhibit improvement.
129
This suggests that while
AhR activation by certain immune pathways can
protect barrier integrity, prolonged activation (as
seen in MRSA infection) may impair the intestinal
epithelium barrier.
130
AhR is activated by a variety of tryptophan-
based ligands such as indole, indole-3-acetic acid,
and indole-3-aldehyde, all of which are derived
from indigenous microbiome members.
131
The
role of AhR as a regulator of intestinal barrier
function is underscored by its responsiveness to
diverse microbial-derived ligands. Ongoing
research into the generation of AhR ligands by
the microbiota will further clarify the influence of
this receptor on intestinal permeability.
131
5.3. Context-dependent bacterial modulators of
the intestinal barrier complexes
Not all bacteria species follow the binary classifi-
cation of either promoting or suppressing leaky
gut pathology. For example, some evidence sug-
gests that gut colonization with Escherichia coli (E.
coli) may reduce permeability while other studies
report an increase in permeability, dependent on
the specific strain of E. coli.
132,133
E. coli Nissle
1917 (EcN), for instance, has been observed to
increase ZO-1 expression in healthy and DSS-
induced colitis mouse models (Figure 4(c)).
132
While the precise mechanism by which EcN con-
trols ZO-1 expression remains unclear, it is
known that ZO-1 plays a crucial role in the for-
mation of tight junctions, thereby maintaining
intestinal barrier integrity.
134
Conversely, other strains of E. coli have been
reported to weaken barrier integrity through dif-
ferent mechanisms. For instance, E. coli of the B2
phylotype commonly found in human intestines
may produce α-hemolysin (HlyA), a toxin known
to induce intestinal leakage, which is often present
in higher levels in individuals with ulcerative
colitis.
133
Moreover, infection of T84 cell mono-
layers with E. coli strain C25 reduced TEER mea-
surements and activated NF-κB signaling.
135
Collectively, these examples underscore the diverse
effects that gut bacteria can exert on permeability
and junction remodeling, even within the same
species, highlighting the complexity of interactions
between microbes and host intestinal barriers.
5.4. Viral modulators of the intestinal barrier
complexes
While bacteria dominate the human gut microbiota,
comprising roughly 90%, the remaining 10%
encompasses a diverse array of viruses, fungi,
archaea, and protozoa species.
136
Beyond bacteria,
certain viral species emerge as significant exogenous
regulators of intestinal permeability. Viruses and
viral particles wield considerable influence over gut
permeability through interactions with resident bac-
terial populations. Viral infections can precipitate
shifts in microbiome composition, thereby impact-
ing intestinal integrity. Notably, individuals infected
with Human Immunodeficiency Virus (HIV) often
experience marked alterations in gut microbiota,
characterized by a decline in beneficial bacteria
such as Bifidobacterium and an increase in harmful
bacteria like Pseudomonas.
137
This dysbiosis, linked
to HIV infection, may directly contribute to leaky
gut pathology. Moreover, research spanning decades
has illuminated potential pathways through which
HIV infection exacerbates intestinal permeability.
Studies from the late 1990s revealed that HIV-posi-
tive patients exhibit a 1.5 to 3.1-fold greater lactu-
lose-to-mannitol ratio in their urine, indicative of
heightened intestinal leakage.
138
HIV infection
16 Z. MARKOVICH ET AL.
prompts host immune cells to release proinflamma-
tory cytokines, potentially increasing permeability.
Notably, two HIV proteins, envelope protein ‘gp120’
and transactivator protein ‘Tat’, directly modulate
tight junction protein architecture. For instance, one
study reported decreased expression of ZO-1, occlu-
din, and claudin proteins in HIV-1-infected cells,
with similar effects observed following treatment
with isolated gp120.
139
The host response to viral
insult may involve production of TNF-α, IL-6, and
IL-8 cytokines and activation of NF-κB signaling
which may contribute to the increased permeability.
-
139
Similarly, Tat protein induces expression of IL-18
and activity of MLCK, culminating in reduced clau-
din-2/occludin expression and compromised barrier
integrity.
140
Collectively, these findings underscore
the complex interplay among viral infections, gut
microbiota dynamics, and the pathophysiology of
leaky gut syndrome in HIV-infected individuals.
138
Influenza, a virus affecting various avian and
mammalian species, primarily targets the upper
respiratory tract but also induces secondary effects
in the lower GI tract. Experimental models of intra-
nasal influenza infection in mice demonstrate sig-
nificant consequences such as shortened colons,
diarrhea, and upregulation of proinflammatory
genes in the intestines.
141,142
Remarkably, direct
infection of the gastric mucosa (intragastric) does
not replicate the effects of intranasal injection,
resulting in minimal pathological injury to the
mice and complete clearance of the infection
within 72 hours.
142
These observations support
the hypothesis that viral infection in the upper
respiratory tract may adversely affect the distal
intestinal mucosa. Evaluation of intestinal perme-
ability reveals increased FITC-flux across the
intestinal lumen into the surrounding plasma
upon intranasal influenza infection.
141
It is
hypothesized that this elevation in inflammation
markers and subsequent leaky gut pathophysiology
could stem from a reduction in available short-
chain fatty acids (SCFAs) – critical molecules for
intestinal homeostasis.
141
SCFAs, including acetate
and butyrate, are produced by gut bacteria through
the fermentation of dietary fiber, conferring pro-
tection against gut injury.
143
Influenza infections,
akin to other viral infections discussed, alter the gut
microbiome, potentially favoring harmful
microbes over SCFA-producing bacteria. This
microbiome shift, coupled with decreased SCFA
levels, correlates with the observed intestinal
damage in influenza-infected mice and can be miti-
gated by direct administration of SCFAs.
141
Additionally, it’s noteworthy that the H5N1 sub-
type of avian influenza virus has been found to
directly downregulate E-cadherin, occludin, clau-
din-1, and ZO-1 in the alveolar tissue of infected
mice via activation of TAK1-Itch, an upstream
activator of NF-κB signaling.
144
Although this
data originates from a peripheral tissue rather
than the intestine, similar phenomena may occur
in the intestine, contributing to the observed
increase in intestinal permeability.
144
More recent studies have documented
instances of microbiota dysbiosis in patients
infected with SARS-CoV-2 (COVID-19).
145
Similarly, mice carrying COVID-19 exhibited
reduced microbial diversity and significant
alterations in intestinal epithelial composition.
-
145
Notably, severely ill mice showed diminished
Paneth cell numbers, along with abnormalities
in granule placement and morphology, coupled
with reduced gene expression of antimicrobial
factors such as lysozyme and defensins.
145
These changes to the Paneth cells are reminis-
cent of changes seen in human cases of IBDs. In
addition to mouse studies, researchers analyzed
stool samples from human COVID-19 patients
to monitor Faecalibacterium bacterium presence.
Faecalibacterium species, commonly found in
the human gut, play an immunosupportive role
and inhibit NF-κB activation and IL-8
production.
146
Reduced Faecalibacterium diver-
sity in COVID-19 patients correlated negatively
with nosocomial bloodstream infection (nBSI),
indicating potential bacterial translocation from
the intestine to the bloodstream.
145
Furthermore, COVID-19 infection’s impact on
alveolar tissue has been extensively studied,
revealing the release of various cytokines that
ultimately downregulate tight junction protein
expression.
147
Although these findings originate
from a different tissue, they suggest a plausible
link between COVID-19 infection and disrup-
tion of gut barrier integrity via similar mechan-
isms. While research on COVID-19‘s effects on
intestinal health and diversity remains in its
early stages, current evidence indicates that
GUT MICROBES 17
severe viral infection can compromise intestinal
barrier function, potentially exacerbated by anti-
biotic use.
145
5.5. Fungal modulators of intestinal barrier
complexes
Candida albicans (C. albicans), a commonly found
fungal species in the healthy human gut microbiome,
usually maintains a commensal relationship without
causing harm under normal circumstances. However,
when exposed to stress or changes in gut bacterial
composition, C. albicans may proliferate unchecked
by outcompeting commensal microbes, increasing
the risk of infection.
148
In a mouse model of DSS-
induced colitis, supplementation with C. albicans has
been found to worsen colitis severity and heighten
intestinal barrier permeability.
149
Moreover, co-infec-
tion with C. albicans and Klebsiella pneumoniae has
been shown to cause extensive damage to the gut
barrier.
150
This damage is attributed to increased
expression of proinflammatory cytokines and simul-
taneous reduction in occludin.
150
The primary means through which C. albicans
inflicts damage on the intestine are via the production
of Candidalysin, a cytolytic peptide toxin secreted
upon epithelial infection.
151,152
Candidalysin accu-
mulation induces intestinal inflammation by trigger-
ing IL-1β release and activating the NLRP3
inflammasome, culminating in direct tissue damage
and pyroptosis-mediated cell death.
151
The NLRP3
(NACHT, LRR, and PYD domains-containing pro-
tein 3) inflammasome pathway is a significant proin-
flammatory pathway that responds to stress signals
arising from injury or microbial invasion.
151
While
there are conflicting reports on the precise effects of
NLRP3 inflammasome signaling on exacerbating
IBDs, colitis, and intestinal permeability, NLRP3
inflammasome activation is considered a critical
step in the damage inflicted by C. albicans infection.
-
151,153,154
Previous studies have demonstrated that
specific inhibitors targeting NLRP3 and IL-1β lead
to increased expression of tight junction proteins.
155
Consequently, NLRP3 activation through candidaly-
sin secretion may have the contrary effect of reducing
tight junction protein expression, thereby exacerbat-
ing gut damage and increasing permeability.
151
6. Common chemicals involved in regulating
intestinal barrier complexes
Nonsteroidal anti-inflammatory drugs (NSAIDs)
are among the most commonly used medications
due to their effective anti-inflammatory, analgesic,
and antipyretic properties.
156
However, chronic
use of NSAIDs such as aspirin, especially in high
doses, has been associated with increased gut per-
meability and alterations in the gut microbiome.
-
157,158
Studies have shown that discontinuing
NSAID use can restore gut permeability and
microbiome composition to normal levels.
159
The
primary cause of NSAID-induced permeability
increases is thought to be their mechanism of
action as cyclooxygenase (COX) enzyme inhibi-
tors, particularly within the gastrointestinal tract.
More recent research has demonstrated that
NSAIDs can upregulate interleukin IL-17A
mRNA, and antibody neutralization of IL-17A
can mitigate barrier damage.
160
Additionally,
NSAIDs have been shown to uncouple mitochon-
drial oxidative phosphorylation and generate reac-
tive oxygen species (ROS).
156
Interestingly, the
intestinal damage caused by NSAID use can be
either exacerbated or mitigated by co-administra-
tion with other compounds. For instance, when
NSAID-administered mice were also given gliadin,
a component of wheat gluten implicated in the
progression of celiac disease, intestinal permeabil-
ity more than doubled compared to mice given
NSAIDs alone, and quadrupled compared to
untreated mice.
161
Conversely, NSAID-induced
barrier damage was ameliorated in rodents given
simultaneous doses of revaprazan, a potassium-
competitive acid blocker (PCAB). Revaprazan pre-
vented increases in intestinal permeability by
enhancing the expression of tight junction proteins
such as occludin, claudin, and ZO-1, likely due to
the inactivation of Rho-GTPase, MLC, and ERK
signaling pathways.
162
Proton pump inhibitors (PPIs) effectively treat
gastro-esophageal reflux disease (GERD), but their
long-term use has been associated with gut barrier
damage and worsening symptoms of IBDs.
163
A
2023 study by Nighot et al. demonstrated that
prolonged PPI use decreased TEER in cell culture
by activating MLCK and exacerbated colitis in
mouse models.
164
This finding, along with other
18 Z. MARKOVICH ET AL.
concerns about chronic PPI use, has spurred inter-
est in identifying alternative therapies such as
PCABs. For example, the novel PCAB tegoprazan
has shown promise in improving barrier function
by addressing microbiome dysbiosis, promoting
the growth of beneficial bacteria, and increasing
the expression of occludin and ZO-1.
165
Further
development of PCABs for conditions like GERD
holds the potential to offer alternative therapeutic
strategies that are less detrimental to gut barrier
integrity.
Selective serotonin reuptake inhibitors (SSRIs) are
widely prescribed for various mental health conditions,
particularly depression and anxiety disorders. Some
research suggests that SSRIs may contribute to gastro-
intestinal symptoms such as diarrhea, constipation, and
abdominal discomfort, which may indicate potential
effects on gut barrier function.
166
While the direct
causal link between SSRIs and leaky gut syndrome is
not definitively established, SSRIs are known to mod-
ulate the intestinal barrier through several mechanisms.
By altering serotonin levels, SSRIs may affect the tight
junctions between intestinal epithelial cells, potentially
influencing intestinal permeability. Moreover, seroto-
nin plays a role in shaping the gut microbiota composi-
tion, and changes in serotonin levels induced by SSRIs
can disrupt this balance, thereby impacting intestinal
barrier function.
167
Additionally, serotonin receptors
present on immune cells within the gut can be influ-
enced by SSRIs, potentially altering immune responses
and inflammation. These immune-mediated changes
have the potential to affect the integrity of the intestinal
barrier and its permeability.
In contrast to the harmful effects associated with
chronic use of NSAIDs, PPIs, and SSRIs, vitamin E
has been shown to confer protection toward the intest-
inal barrier. In a murine model of DSS-induced colitis,
dietary supplementation with vitamin E prevented the
depletion of the tight junction protein occludin, indi-
cating enhanced barrier integrity.
168
Additionally,
owing to its recognized anti-inflammatory and antiox-
idant properties, vitamin E was hypothesized to miti-
gate the effects induced by TNF-α and IFN-γ
treatment. In experiments using Caco-2 cell mono-
layers, α- and γ-tocopherol (natural derivatives of vita-
min E) were found to preserve TEER levels and restore
ZO-1 protein expression following cytokine exposure.
-
168
While the precise mechanism underlying vitamin
E’s protective action is not fully elucidated, studies
indicating reduced expression of TLR-4 and NF-κB
with vitamin E supplementation suggest its potential
involvement in the established signaling pathways dis-
cussed previously in this review.
168,169
Other natural compounds derived from sources
beyond our microbiome, such as certain plant species,
also play a role in modulating gut permeability.
Schisandra chinensis, commonly known as the five-
flavor fruit plant, has a rich history in herbal medicine,
particularly for treating respiratory conditions.
170
One
of its derivatives, Schisandrin C, has demonstrated the
ability to decrease FITC-dextran staining and enhance
electrical resistance across Caco-2 cell layers exposed
to IL-1β.
170
Through its anti-inflammatory effects,
Schisandrin C reduces the phosphorylation and
nuclear translocation of NF-κB, while also reducing
the expression of MLCK and p-MLC. These collective
actions culminate in elevated levels of ZO-1 and
occludin expression.
170,171
Moreover, in an in vivo
model using C. elegans infected with barrier-dama-
ging bacteria, Schisandrin C demonstrated a reduc-
tion in FITC-dextran staining.
170
Another noteworthy plant-derived compound is
resveratrol, a natural polyphenol abundant in
grapes, seeds, and berries.
172
Renowned for its
reported anti-cancer, anti-inflammatory, antioxi-
dant, and neuroprotective effects, resveratrol mod-
ulates various signaling pathways involved in
inflammation and tight junction protein
expression.
172
Despite some conflicting findings,
resveratrol treatment in LPS-aggravated Caco-2
cells generally reduces inflammation by limiting
NF-κB and TLR4 signaling.
173,174
Additionally,
resveratrol directly enhances tight junction protein
expression, thereby reducing intestinal permeabil-
ity. Treatment with resveratrol in cells with LPS-
induced inflammation led to increased expression
of ZO-1, occludin, and claudin-1.
175
This increased
expression is attributed to decreased inflammatory
cytokine expression and attenuation of Notch-1
signaling, known inducers of barrier damage and
modulators of tight junction proteins.
175
Similarly, Aloe vera L. plant pulp, known for its
antioxidative and anti-inflammatory effects, con-
tains various polysaccharides and phytochemicals,
with polysaccharides believed to be the primary
therapeutic agent.
176,177
Recent research demon-
strates that processed gel from Aloe vera pulp
replicates the beneficial effects of other naturally-
GUT MICROBES 19
derived polysaccharides on tight junction
formation.
176
Administration of Aloe vera gel
reduced leakage in vivo and increased TEER and
translation of tight junction proteins (ZO-1, occlu-
din, claudin-1) in vitro.
176
It is suggested that Aloe
vera gel regulates tight junction protein expression
by enhancing phosphorylation of ERK1/2 and acti-
vating MAPK/ERK signaling, another pathway
influencing tight junction assembly.
176
7. Concluding remarks
The integrity of the intestinal barrier, essential for
maintaining gastrointestinal health, is intricately
regulated by both intrinsic and extrinsic factors.
Intrinsic factors, such as the composition of junc-
tional complex proteins and intracellular signaling
pathways acting on these proteins, play a funda-
mental role in maintaining the structural and func-
tional integrity of the intestinal barrier. These
factors ensure precise regulation of paracellular
permeability, thereby preserving the selective per-
meability essential for nutrient absorption and
defense against pathogens. Extrinsic factors,
including diet, microbial communities, and envir-
onmental chemicals, significantly influence the
function of intestinal junctional complexes. For
instance, the gut microbiome profoundly impacts
the modulation of junctional proteins, with bene-
ficial microbes promoting barrier integrity and
pathogenic microbes contributing to barrier dys-
function. Environmental factors, such as dietary
components and xenobiotics, further interact with
junctional complexes, either strengthening or com-
promising barrier function.
Disruptions in the intestinal barrier are impli-
cated in various gastrointestinal and extraintest-
inal disorders, making it crucial to understand
the complex interplay between these intrinsic
and extrinsic modulators to elucidate the patho-
physiology of these disorders. While the compo-
sition of intestinal junctional complexes is
relatively well-studied, many open questions
remain. For example:
●What biomarkers can be identified to reliably
assess the integrity and function of intestinal
junctional complexes in clinical settings?
●How can noninvasive techniques be developed
or improved to monitor intestinal permeabil-
ity and junctional complex function?
●What are the precise molecular mechanisms by
which intrinsic factors, such as genetic muta-
tions, epigenetic modifications, and intracellular
signaling pathways, regulate the expression and
function of junctional proteins?
●How do post-translational modifications (e.g.,
phosphorylation, ubiquitination) of junctional
proteins influence their stability and function?
●How do specific microbial species and their
metabolites influence the composition and
function of intestinal junctional complexes?
●What are the mechanisms through which
pathogenic microbes disrupt junctional com-
plexes, and how can these pathways be tar-
geted to prevent or treat barrier dysfunction?
●How does chronic exposure to environmental
toxins and pollutants contribute to long-term
changes in barrier function?
●How do intrinsic factors (e.g., genetic predis-
positions) modulate the response of the intest-
inal barrier to extrinsic factors (e.g., diet,
microbiota, environmental chemicals)?
●What are the disease-specific alterations in
junctional complexes that occur in conditions
such as IBD, IBS, and other gastrointestinal
disorders?
●How do systemic diseases, such as metabolic
syndrome and autoimmune diseases, affect the
regulation and function of intestinal junctional
complexes?
●How can dietary interventions or probiotics/
prebiotics be optimized to support and
enhance the function of junctional
complexes?
Addressing these critical questions will advance
our understanding of the regulation and function
of intestinal junctional complexes and their roles in
health and disease. This knowledge will also inform
the development of novel therapeutic strategies to
maintain or restore intestinal barrier integrity.
Disclosure statement
No potential conflict of interest was reported by the author(s).
20 Z. MARKOVICH ET AL.
Funding
This work was supported by grants from the National Institute
on Aging [R01AG063766 and P30AG028740 to R.X.] and
National Institute on Deafness and Other Communication
Disorders [T32DC015994 to Z.M.]. All model figures in this
article were generated with BioRender.com.
ORCID
Rui Xiao http://orcid.org/0000-0001-5541-6685
Author contributions
Z.M. and R.X. wrote the manuscript; Z.M., A.A. and R.X.
prepared the model figures; Y.S. and S.M.H. reviewed and
edited the manuscript. All authors discussed and approved
the final version of the manuscript.
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