Zonulin, A Regulator of Epithelial and Endothelial Barrier Function

TISSUE BARRIERS
2016, VOL. 4, NO. 4, e1251384 (19 pages)

http://dx.doi.org/10.1080/21688370.2016.1251384
REVIEW
Zonulin, a regulator of epithelial and endothelial barrier functions, and its

involvement in chronic inflammatory diseases
Craig Sturgeona,b and Alessio Fasanoa,c
a
Center for Celiac Research and Treatment, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital and Division of
Pediatric Gastroenterology and Nutrition, Boston, MA, USA; bGraduate Program in Life Sciences, University of Maryland School of Medicine,
Baltimore, MD, USA; cEuropean Biomedical Research Institute of Salerno (EBRIS), Salerno, Italy
ABSTRACT ARTICLE HISTORY

Beside digesting nutrients and absorbing solutes and electrolytes, the intestinal epithelium with its Received 20 September 2016
barrier function is in charge of a tightly controlled antigen trafficking from the intestinal lumen to Revised 14 October 2016
the submucosa. This trafficking dictates the delicate balance between tolerance and immune Accepted 14 October 2016
response causing inflammation. Loss of barrier function secondary to upregulation of zonulin, the KEYWORDS
only known physiological modulator of intercellular tight junctions, leads to uncontrolled influx of autoimmune disease; barrier
dietary and microbial antigens. Additional insights on zonulin mechanism of action and the recent function; intestinal
appreciation of the role that altered intestinal permeability can play in the development and permeability; tight junctions;
progression of chronic inflammatory disorders has increased interest of both basic scientists and zonulin
clinicians on the potential role of zonulin in the pathogenesis of these diseases.
This review focuses on the recent research implicating zonulin as a master regulator of intestinal
permeability linked to the development of several chronic inflammatory disorders.
Introduction environmental factors affecting clinical outcome has

led to the formulation of the microbiota hypothesis,
Increased intestinal permeability has been recently
which postulates the key lifestyle changes, including
proposed to be an integral element, along with
modality of birth, overuse of antibiotics and, most
genetic makeup and environmental triggers, in the
importantly, dietary differences in industrialized
pathogenesis of chronic inflammatory diseases (CID),
countries causes changes in the microbiome composi-
including allergic, autoimmune, and metabolic dis-
tion and ultimately fuel the onset of CID (Fig 1).6,7 It
eases.1-3 The incidence of these conditions in indus-
is now clear there is a symbiotic relationship between
trialized countries has been on a steady rise since the
the microbiome and the host. As early as 2001, it
1950s,4 leading to the formulation of the hygiene
was described that commensal bacteria have an effect
hypothesis.4,5 The rate and timeline of these epidem-
on intestinal permeability.8
ics imply that genetic factors are necessary but not
Evidence has shown the impaired gut barrier func-
sufficient in determining which individuals will
tion is a key pathogenic component rather than the
develop these diseases, pointing to a key role of envi-
epiphenomenon of several CID.9-11 In this review we
ronmental factors as driving forces to cause CID in
will discuss the role of zonulin, the only physiologic
genetically predisposed individuals. Increased hygiene
modulator of intercellular on tight junctions discov-
in developing countries was not paralleled by similar
ered so far, in the development of several CID.12
epidemics of CID, questioning the validity of the
hygiene hypothesis, while pointing out to a more
Intestinal physiology and tight junctions
complex dynamic of host-environment interaction
centered on the possible epigenetic role of the micro- The human intestine is lined up by a single layer of
bial ecosystem with which we co-exist since birth. epithelial cells that represents the largest interface
The appreciation of the microbiome composition as between the environment and the host. The structural
a key “transductor” of pre-, peri-, and post-natal arrangement of the intestinal mucosa suggests an
CONTACT Alessio Fasano, M.D. [email protected] Massachusetts General Hospital For Children, 175 Cambridge St., Boston, MA 02114, USA.
© 2016 Taylor & Francis
e1251384-2 C. STURGEON AND A. FASANO
Figure 1. Proposed effect of environmental stimuli causing changes in microbiome composition leading to CIDs. Pre-, peri-, and/or post-
natal environmental factor can affect microbiota composition causing loss of barrier function, increased antigen trafficking, and altered
immune response in genetically susceptible individuals eventually leading to the onset of CID.
intimate cross talk between epithelial cells and the Farquhar and Paladeand, and are comprised of
underneath immune system for the coordinated sur- transmembrane proteins including occludin,15 clau-
veillance of the content of the intestinal lumen. The dins,16 junctional adhesion molecules (JAM),17 tri-
intestinal mucosa is charged with task of maintaining cellulin,18 and angulins.19 These transmembrane
the balance between the absorption of nutrients and proteins interact between themselves (both homo-
ions, the secretion of fluids, and the protection from philic and heterophilic interactions) and with intra-
microorganisms, toxins, and dietary antigens present cellular scaffolding proteins, including zonula
in the lumen. The epithelial cells are held together by occludins (ZOs), which are anchored to the actin
tight junctions, adherens junctions and desmo- cytoskeleton. The interaction of occludins, claudins,
somes.13 Historically, tight junctions were thought to JAMs and tricellulin between cells and with ZOs
be an impermeable barrier blocking paracellular pas- maintain the integrity of the tight junction and
sage of macromolecules. We now know that tight control the passage of molecules through the para-
junctions are dynamic structures involved in both cellular space.
physiologic and pathologic regulation of intestinal epi-
thelial antigen tafficking.14
Regulation of intestinal tight junctions
Regulation of tight junctions is essential in maintaining
Structure of tight junctions
barrier homeostasis, both in between body compart-
Tight junctions are the most apical junctional ments and between body and external environment.
complex connecting both neighboring epithelial Most of the research on intercellular tight junction reg-
and endothelial cells, first described in 1963 by ulation has been focused on cytokine-mediated
TISSUE BARRIERS e1251384-3
dysfunction in the context of established chronic Role of intestinal permeability in disease

inflammation, particularly those affecting the intestinal
mucosa. TNF-a and IFN-g have been extensively stud- A large number of CID have been described to have
ied for their effects on the tight junction barrier in the alterations in intestinal permeability including IBD,33
gut. The effect of TNF-a on the intestinal barrier has CD,12,34-36 IBS,37 multiple sclerosis (MS),38 rheuma-
been associated to IBD,20 graft-versus-host disease,21 toid arthritis (RA),33 type-1-diabetes (T1D),39
and celiac disease (CD).22 In patients with Crohn’s dis- asthma,40,41 necrotizing enterocolitis42-44 and autism
ease (CrD) anti-TNF treatment is able to correct bar- spectrum disorders (ASD).45 Interestingly, less than
rier disruption seen in the colon.20 10% of subjects with compatible genetic makeup
The mechanism of TNF-a barrier disruption has advance to clinical disease, suggesting that environ-
been shown to be mediated by myosin-light-chain ment stimuli play a key role in determining those that
kinase (MLCK). MLCK activation alone has been progress to disease. Since the intestinal epithelium is
shown to decrease tight junction permeability both in the largest mucosal surface that provides an interface
vitro and in vivo.23,24 IFN-g increases intestinal per- between host and environment, inappropriate antigen
meability through changes in expression and localiza- trafficking through the intestinal mucosa may be
tion of tight junction proteins as well as involved.
rearrangement of the cytoskeleton.25 Under normal physiological conditions, the major-
Pattern recognition receptors (PRRs) are impor- ity (»90%) of antigens that pass through the intestinal
tant in the early innate immune response in the epithelium travel through the transcellular pathway.
intestine. Toll-like receptors (TLRs) are a class of The transcellular pathway is regulated and leads to
transmembrane PRRs that are important for micro- lysosomal degradation of antigens into small non-
bial recognition and control of immune responses. immunogenic peptides. The remaining »10% of pro-
TLR2 is one member of the TLR family, which rec- teins cross the epithelium through the paracellular
ognizes conserved patterns on both gram-negative pathway as full intact proteins or partially digested
and gram-positive bacteria. TLR2 is expressed on peptides as a tightly regulated antigen trafficking
many cell types through the intestine including epi- through intestinal tight junction modulation which
thelial cells.26 Stimulation of TLR2 in vitro leads to antigenic tolerance.12
increased trans epithelial electrical resistance The role of epithelial cells in maintaining mucosal
through PKC activation and translocation of ZO-1 homeostasis was postulated by Hermiston and Gor-
to the tight junction complex.27 ZO-1 is controlled don using chimeric mice with a defective cadherin.46
by the PI3K/Akt pathway in a MYD88 dependent These mice developed profound epithelial defects
manner.27 Additional studies provide evidence on including incomplete cell polarization, inappropriate
the protective effect of TLR2 against DSS-induced actin cytoskeleton distribution, increased migration
colitis. The TLR2 stimulation did not cause of enterocytes along crypt-villous axis and prema-
increase in FITC-dextran passage or redistribution ture apoptosis.46 These mice went on to develop an
of ZO-1 away from the tight junction.28 inflammatory bowel disease resembling CrD without
Proteinase activated receptor (PARs) are a family additional external stimuli.47 Additional studies
of g-protein-couple-receptors that are activated by show transgenic mice with constitutively active
proteolytic cleavage of their N-terminus revealing a MLCK show increased intestinal permeability due
tether ligand. PAR2 is found on both the apical to tight junction disassembly.24 Although, these
and baso-lateral side of enterocytes.29 Stimulation mice show increased permeability they do not mani-
of basolateral PAR2 results in increase permeability fest any signs of overt disease.24 The increased per-
through redistribution of ZO-1, occludins, and F- meability observed in these mice is considered
actin.30 In addition, Coelho et al. demonstrated similar to the barrier dysfunction seen in healthy
that in vivo the apical stimulation with a PAR2 relatives of patients with CrD, CD, and T1D.
activating peptide (SLIGRL) causes a dose depen- Experiments performed on JAM-A knockout mice
dent increase in intestinal permeability.31 Stimula- revealed that these animals have increased intestinal
tion of PAR1 has also been shown to increase permeability but only low grade colonic inflamma-
intestinal permeability.32 tion and normal epithelial architecture.48 Similar
results were obtained with an intestinal specific non Zonulin as a master regulator of intercellular
muscle myosin IIA heavy chain knockout mice (NM tight junction in health and disease
IIA cKO).49 Both JAM-A¡/¡ and NM IIA cKO
mice also show increased susceptibility to DSS Research while developing a vaccine for Vibrio chol-
induced colitis. Together these data suggest intesti- era, led our group to the discovery of zonula occludens
nal permeability may contribute to the development toxins (Zot), an enterotoxin which is able to reversibly
of several CID, provided that additional genetic open intracellular tight junctions.53 Subsequent
traits regulating immune response and exposure to research led to the appreciation of the complexity of
an environmental trigger are present. This hypothe- the signaling cascades triggered by Zot involved in its
sis is consistent with a case report of a healthy first regulation of the paracellular pathway.
degree relative of a CrD patient who displayed signs Zot causes polymerization of actin of targeted cells
of increased intestinal permeability 8 y prior to her leading to disassembly of tight junction complexes
own development of CrD.50 through a protein kinase C (PKC)-dependent mecha-
Additional experiments on transgenic mice with nism.54 Immunofluorescent studies have shown that
constitutively active MLCK shed additional light on the Zot is able to interact with epithelial cells along the GI
role of increase permeability on disease development. tract with the highest binding in the jejunum and dis-
The MLCK transgenic mice were crossed with recombi- tal ileum and also decreasing along the villous to crypt
nation-activation gene (rag)-1 knockout mice (which axis.55 These binding studies confirm data on the
lack mature B and T cells). The Rag-1¡/¡ and consti- regional effect of Zot along the intestine.
tutively active MLCK mice as well as their Rag1¡/¡ Given the complexity of the intracellular signaling
with normal MLCK received CD4CCD45RBhi naive T activated by Zot leading to tight junction modulation,
cells from wild-type mice. While both groups of mice it was hypothesized that the toxin may mimic an
went on to develop colitis, the colitis in mice with con- endogenous protein which is able to regulate the epi-
stitutively active MLCK was accelerated and more clini- thelial tight junctions. The combination of Ussing
cally severe.24 chamber experiments and anti-Zot antibodies led to
Intestinal barrier disruption has been shown to the identification a »47 kDa human analog to Zot,
have a role in disease development, but it’s also named zonulin.35 Ex vivo studies show endogenous
been shown not be sufficient for disease develop- human zonulin is able to increase permeability in
ment. Another key piece of the puzzle seems to be both the jejunum and ileum.56
the involvement of the mucosal immune system. It Studies on human sera from CD patients, who have
has been reported that barrier dysfunction can increased zonulin levels35 as determined by ELISA
influence the immunoregulatory process of the measurement using polyclonal zonulin cross reacting
mucosa.51 Induction of mucosal erosion and barrier anti-Zot antibodies,57 revealed that zonulin is pre-
dysfunction through intrarectal ethanol was fol- haptoglobin(Hp)-2, the pro-protein of Hp2 before
lowed by increased IFN-g and IL-10 producing enzymatic cleavage into its mature form. Recombinant
mononuclear cells and CD4CCD25C, latency-associ- zonulin produced by expressing the HP2 cDNA in a
ated peptide (LAP) expressing T cells in the lamina baculovirus system, was detected by the anti-Zot anti-
propria. The ethanol administration and subse- bodies57 and showed the expected permeating effect
quent presence of the LAPC T-cells protected on gut mucosa when tested ex vivo in C57BL/6 small
against trinitrobenzene sulphonic acid (TNBS) acid intestine. When mice were gavaged with recombinant
induced colitis. The induction of LAPC T cells was zonulin and subjected to sucrose and lactulose/manni-
dependent on CD11cCDCs, TL2, and normal tol tests they showed increased gastroduodenal and
microbiome.51 Interestingly, CD11cC DCs are able small intestine permeability measured within 24 hours
to interact with epithelial cells and increase their of zonulin exposure, which returned to baseline level
ability to induce T-regulatory cells.52 These obser- after 48 hours.57 To confirm the increase in permeabil-
vations suggest a key role for the interaction ity was specific to zonulin (pre-Hp2), recombinant
between the epithelial cells, immune cells, and the zonulin was subject to proteolytic cleavage, resulting
luminal microenvironment in the maintenance of in the mature a and b chains of Hp. The effects of
intestinal homeostasis. zonulin observed in both the in vivo and ex vivo
experiments failed to cause changes in permeability protein. The discovery of zonulin as pre-Hp2 added a
after proteolytic cleavage.57 Together these results mechanistic mean to the elevated Hp levels in the
confirmed zonulin to be pre-Hp2 and when cleaved in course of inflammation.
its mature Hp2 form, loses its effect on paracellular Additionally, the distribution of Hp genotypes in a
permeability. wide variety of disease has been extensively studied. In
2007 Carter and Worwood reviewed all the disorders
in which Hp was reported to be associated. Of the 23
Haptoglobin
disorders that were linked to Hp, 11 were shown to be
Haptoglobin is an ancient protein which has been more common in patients with the Hp2-1 or Hp2-2
genetically mapped to first appear »450 million years genotype.64 The Hp2-2 phenotype has also been asso-
ago in bony fish.58 Wicher and Fries suggested that ciated with worse prognosis of infectious diseases such
Hp evolved from the complement-associated protein as HIV65 and tuberculosis.66 Hp2 has also been shown
mannose-binding-lectin-associated serine proteinase to been associated with autoimmune disorders,
(MASP). Hp’s primary function is to bind free hemo- CD57,67 and CrD,68,69 neurological disorders, epi-
globin (Hb) in order to prevent the oxidative stress lepsy70 schizophrenia,71 complications in diabetes
caused by free intravascular Hb. The Hp-Hb complex (including diabetic nephropathy72 and diabetic reti-
is cleared through binding of the scavenger receptor nopathy73), and Chagas’ disease.74,75 This association
CD163 on monocytes/macrophages.59 of Hp2 with CID was postulated to be related to the
In humans, Hp is found in 2 genetic variants, HP1 less efficient capability of Hp2 to bind Hb, which will
and HP2. The Human HP1 gene is homologous to the cause oxidative stress from free Hb, and therefore
HP gene found in other mammals. Genetically human inflammation. While this could be a plausible hypoth-
HP1 is made up of 5 exons and 4 introns. HP2 arose esis, increased hemolysis and subsequent oxidative tis-
from an uneven crossover that occurred »2 million sue damage have never been reported in these
years ago causing the duplication of exons 3 and 4 of diseases.
HP1 giving rise to a gene with 7 exons and 6 introns.60
Hp is translated as a pro-protein before enzymatic
Zonulin signaling
cleavage into an a and b chain by C1r-like protein in
the endoplasmic reticulum (ER).61 The b chain Sequential and structural analysis of zonulin revealed
(»35kDa) is conserved in Hp1 and Hp2 and contains an epidermal growth factor (EGF)-like motif. It was
an inactive chymotrypsin-like serine protease domain, therefore hypothesized that zonulin may disassemble
while the a chain exists in 2 forms a-1 (9kDa) and TJ through EGF activation, since it has been described
a-2 (18kDa), corresponding to the HP1 and HP2 gene EGF can modulate the actin cytoskeleton,76,77 similar
respectively, and contain a complement control to the effects seen with zonulin.56,78 In vitro studies in
domain.62,63 These 2 a chains give rise to 3 different Caco-2 cells showed zonulin caused EGFR phosphory-
possible genetic combinations in humans, Hp1-1 lation and subsequent increases in permeability which
(Hp1homozygous), Hp2-1(Hp2-1 heterozygous), and was blocked by an EGFR inhibitor.57 To confirm the
Hp2-2 (Hp2 homozygous). After pre-Hp is cleaved, effect was due to zonulin and not mature Hp2, trypsin
the a and b chains form polymers which are the digested zonulin was tested and showed no EGFR acti-
mature functional form of Hp. The a and b chains vation.57 Additionally, it was shown that EGFR activa-
form disulfide bridges and heterodimerize. The heter- tion was dependent on PAR2 as demonstrated both in
odimer a and b pair is able to then form polymers Caco2 cells in which the receptor was silenced, and in
with other a and b pairs. PAR2¡/¡ mice.57 Zonulin contains a PAR2 activating
Before the discovery of zonulin as pre-Hp2, no bio- peptide-like sequence in its b-chain (FCAGMS) very
logical function had been described for either form of similar to the PAR2 Zot activating peptide AT1002
Hp precursors, as they are cleaved in the endoplasmic (FCIGRL). It had been reported previously that several
reticulum and minimal pre-Hp is found circulating in GPCRs including PAR2 are able to transactivate
the plasma. Interestingly, Hp was historically used EGFR.79
clinically as a marker of general inflammation, similar The signaling pathways triggered by Zot and zonu-
to C-reactive protein today, as it is an acute phase lin leading to tight junction disassembly have been
Figure 2. Mechanism of gliadin- and bacteria-induced zonulin release and subsequent increase in intestinal permeability. Gliadin spe-
cific peptides or bacteria (1) cause a CXCR-3-mediated, MyD88-dependent zonulin release (2). Zonulin transactivates EGFR through
PAR2 leading to PCK-a dependent tight junction disassembly (3). Increased intestinal permeability leads to paracellular passage of non-
self antigens (4) into the lamina propria where they are able to interact with the immune system.
extensively studied and resulted being similar enteric pathogens, including commensal Eschericha
(Fig 2).80 Indeed, as shown with zonulin, Zot also coli, lab E. coli, virulent E. coli, and Salmonella typhi
binds to PAR2 through its AT1002 active domain gen- have been shown to cause a release of zonulin from
erated during Zot trafficking in V. cholera.81 AT1002 the intestine when applied to the apical surface.84 Fol-
(FCIGRL) structurally resembles the PAR2 activating lowing the release of zonulin, the intestine showed
peptide tethering motif (SLIGRL) and causes increased permeability and disassembly of ZO-1 from
increased permeability through displacement of ZO-1 the tight junction complex.84
and occludin from the cell junctions that occurred Gliadin is the other trigger that has been
only if PAR2 was expressed in the target cells described to release zonulin.85,86 Gliadin, only when
(Fig 2).82 The displacement of ZO-1 and occludin was applied to the apical surface, caused a release of
shown to be secondary to PCKa-dependent phos- zonulin, and subsequent increase in permeability, in
phorylation of ZO-1, causing decreased tight junction both cell culture models and ex vivo studies of intes-
protein-protein interactions, and of myosin-1C that, tinal tissue.87,88 The increase in permeability, but
together with the cytoskeletal rearrangement, tempo- not the release of zonulin was blocked with pretreat-
rarily removes ZO-1 and occludin from the junctional ment of the zonulin inhibitor AT-1001.85 Lammers
complex (Fig 2).82 While ZO-1 displacement per se is et al. described that specific non-digestible gliadin
not sufficient to cause a barrier defect,83 the combina- peptides are able to bind the CXCR3 receptor on
tion with other intracellular signaling events affecting the apical surface of enterocytes with subsequent
TJ, including occludin displacement, actin polymeri- MyD88-dependent zonulin release.88 The CXCR3
zation, and myosin-1C phosphorylation54,82 may con- receptor is also overexpressed on the apical surface
tribute to a more profound rearrangement of the of CD patients,88 which may explain the increased
junctional complex that ultimately cause transient TJ levels of zonulin detected in intestinal explant
disassembly. obtained from CD patients when exposed to glia-
din.34 While the full signaling cascade following gli-
adin binding to CXCR3 leading to release of zonulin
Zonulin release
is not completely understood, it has been shown to
The two major triggers of zonulin release that have be dependent on MyD88, a key adapter molecule in
been described so far are bacteria and gliadin. It is well the TLR signaling pathway88 (Fig 2). Gliadin is also
described that many enteric pathogens are able to pro- able to cause a release of zonulin and pro-inflamma-
duce enterotoxins that affect the intestinal tight junc- tory cytokines from macrophages similar to the
tion of the host. In addition to enteroxins, several response seen after bacterial exposure.86 The zonulin
Table 1. List of CIDs in which zonulin has been implicated.

Disease Category Disease Model Disease Association References
2,12,34,35,57,78,85,86,88,90-92
Autoimmune Celiac Disease Human Specific role in pathogenesis
3,11,103,106-108
Type-1-Diabetes Human, Rat Possible role in pathogenesis
9
Inflammatory Bowel Disease/Colitis Mouse Possible role in pathogenesis
12,114
Multiple sclerosis / EAE Human, Mouse Possible role in pathogenesis
119-121
Metabolic Disorders Obesity/Insulin resistance Human Upregulated
121,122
Type-2-Diabetes Human Upregulated
125,126
Polycystic ovary syndrome Human Upregulated
129
Lung Disease Acute Lung Injury Mouse Possible role in pathogenesis
12
Asthma Human Possible role in pathogenesis
133
Heart Disease Coronary artery disease Human Upregulated
134,135
Neurological Disease Glioma Human, Cell culture Upregulated
137,138
Systemic Infectious Diseases Septicemia Human Upregulated
142,143
HIV Human Downregulated
144
Intestinal Diseases Irritable Bowel Syndrome Human Upregulated
149
Non-Celiac gluten sensitivity Human Upregulated
150
Environmental Enteropathy Human Associated
153
Necrotizing Enterocolitis Rat, Cell Culture Upregulated
release from macrophages is also MyD88 dependent, (Fig 3). Under physiological circumstances there is a
but TLR2 and TLR4 independent.88 tightly control of mucosal antigen trafficking (anti-
gen sampling) that, in concert with specific immune
cells and chemokine and cytokine mediators lead to
Role of zonulin in specific diseases
anergy and, therefore, to mucosal tolerance (Fig 3).
Zonulin has been implicated in many CIDs The inappropriate production of increased amount
(Table 1). Independent from the CID considered, of zonulin causes a functional loss of barrier func-
the steps leading to break of tolerance and subse- tion, with subsequent inappropriate and uncon-
quent development of CID seem to be similar trolled antigen trafficking instigating an innate
Figure 3. Proposed mechanism of zonulin in causing loss of barrier function leading to development of CID. Normal barrier trafficking of
non-self antigens (antigen sampling), together with specific gut-associated lamina propria cells and cytokines micro milieu leads to
mucosal tolerance (1). Environmental stimuli cause microbiome imbalance triggering zonulin release (2) leading to increased antigen
influx from gut lumen to the lamina propria (3). Antigens in the lamina propria activate the immune system causing IFN-g and TNF-a
release further exacerbating the increased gut permeability and immune response (4). This leads to a vicious cycle which causes break
of tolerance and ultimately, onset of CIDs in genetically predisposed individuals.
immune response by the submucosal immune com- Gluten is a complex molecule consisting of gliadin
partment. If this process continues, an adaptive and glutenins. Gliadin is a subunit of the protein glu-
immune response is mounted causing production of ten which is found in wheat, rye, and barley. Through
pro-inflammatory cytokines, including IFN-g and extensive research at least 50 toxic epitopes have been
TNF-a that cause further opening of the paracellular identified. Their effects include cytotoxicity, immuno-
pathway to the passage of antigens, creating a modulatory, and barrier disruption. The a-gliadin
vicious cycle. Ultimately, these processes lead to fragment has been mapped with specific domains
break of tolerance with subsequent onset of CID exerting different effects on the body. The 31-43 pep-
(Fig 3) whose nature is influenced by the specific tide exerts a cytotoxic effect, the 57-89 (referred to as
host genetic background that dictates which organ 33mer) exerts an immunomodulatory effect, the 111-
or tissue will be targeted by the inflammatory pro- 130 and 151-170 are able to bind CXCR3 and release
cess. Below we will review the CID that have been zonulin88 and the 261-277 causes interleukin (IL)-8
associated with dysregulation of the zonulin path- release.89
way, with special emphasis on CD and T1D, the 2 CD has been used as a model disorder to study the
conditions in which the role of zonulin in disease effect of zonulin since its involvement in the develop-
pathophysiology and gut barrier disruption has been ment and pathogenesis of the disease has been well
demonstrated, while in many of the other CIDs zon- documented.2,12,34,35,57,78,85,86,88,90-92 In CD patients,
ulin has only been shown to be upregulated and the zonulin is produced after gluten ingestion as shown
mechanism has not been described. by Drago et al.34 Exposure of intestinal biopsies to
pepsin-trypsin(PT)-digested gliadin fragments in the
Ussing chamber caused an increase in zonulin release.
Autoimmune disorders
Interesting, zonulin release could be measured in both
Celiac disease CD in remission and healthy control patients,
CD is an autoimmune enteropathy triggered by the although in healthy controls the level of zonulin
ingestion of gluten-containing grains in genetically release was low and tightly regulated, as shown by the
susceptible individuals. CD is a complex genetic disor- short-term release, with zonulin returning to baseline
der in which many gene associations have been identi- within 20 minutes.34 Conversely, CD patients dis-
fied. The HLA status has been identified as accounting played a much more robust and prolonged zonulin
for up to 40% of the genetic load. The presence of release following gliadin stimulation,34 followed by a
either DQ2 (HLA-DQA105-DQB102) or DQ8 significant increase in gut permeability. The release of
(HLA-DQA103-DQB10302) is necessary but not zonulin and subsequent increase in intestinal perme-
sufficient to develop CD as »40% of the general popu- ability was blocked using the zonulin antagonist AT-
lation also has either DQ2 or DQ8. The ingestion of 1001.34 Also, noteworthy was the observation that CD
gluten in CD patients causes destruction of the intesti- patients in full remission without PT-gliadin stimula-
nal villi through a mechanism only partially estab- tion had constitutively increased zonulin produced by
lished. Diagnosis is based on serological screening the intestine which correlated to an increase in perme-
showing presence of auto-antibodies to tissue trans- ability compared to healthy controls.34
glutaminase enzyme, followed by an upper endoscopy AT1001 (now named Larazotide acetate), is a syn-
with duodenal biopsy showing the typical celiac auto- thetic 8 amino acid peptide that antagonize the zonu-
immune enteropathy characterized by the presence of lin pathway.93 Pre-clinical trials have shown larazotide
intraepithelial lymphocytes, crypt hyperplasia, and vil- acetate to be able to prevent the zonulin permeating
lous blunting. Current treatment options are limited activity.11,94,95 Larazotide acetate was also able to block
to dietary restriction of gluten from the diet, gluten horseradish peroxidase passage and suppress the
free diet (GFD). The GFD allows the intestinal mucosa innate immune response following gliadin challenge
to heal and villous architecture to return to normal in in HLA-HCD4/DQ8 mice,94 a double transgenic used
many cases. Although, diagnosis is based on an intesti- to study responses to gliadin before severe inflamma-
nal biopsy, CD is a systemic disease which can affect tion and intestinal damage.96-99
many different organs and cause several extra- Larazotide acetate is currently in clinical trials as a
intestinal symptoms. treatment for CD. Phase II clinical trial results have
shown that following a gluten challenge larazotide ace- The involvement of zonulin in T1D seen in BBDP rats
tate is able to decrease permeability (only in inpatient was confirmed in human studies showing »50% of T1D
setting),100 decrease GI and extra-intestinal symp- patients have increased serum zonulin levels, some of
toms,100-102 and block in increase of tTG antibod- them showing these changes in the pre-diabetic phase of
ies.101,102 Additionally, a study on patients who had the disease.3 Interestingly, a subset (»25%) of first degree
persistent symptoms despite following a strict GFD relatives of T1D patients also showed increased serum
showed larazotide was able to reduce their symp- zonulin.3 These data suggest zonulin may play a role in
toms.103 Larazotide acetate is now entering phase III the pathogenesis of T1D in a subset of patients.
clinical trials in CD patients.
Inflammatory bowel disease
Type-1-diabetes Increased intestinal permeability has been shown to
T1D is an autoimmune condition caused by the play a crucial role in the pathogenesis of both CrD
destruction of the insulin producing b-cell of the pan- and UC.109-113 Arrieta et al. used the IL-10 knockout
creas.104 The exact pathogenesis of T1D is not (IL-10¡/¡) colitis model to show a direct relationship
completely understood, however both genetic and between increases in small intestinal permeability and
environment factors seem to be at play. T1D shares a development of colitis.9 IL-10¡/¡ mice were shown
genetic association with CD with the HLA locus, spe- to have increased permeability in the small intestine
cifically HLA DQ2 and DQ8. The trigger of T1D has that preluded the development of colitis. Treatment
not been discovered but many possible environmental with the zonulin inhibitor AT-1001 caused a signifi-
factors have been scrutinized, although none have cant reduction in the severity of colitis.9 Together,
been confirmed as a clear causative agent of T1D. these experiments suggest a role for increased small
T1D has similar pathogenic challenges to other auto- intestinal permeability in causing aberrant antigen
immune disorders, as the environmental trigger must trafficking with subsequent activation of the gut
cross the intestinal barrier and interact with the immune cell that, ultimately migrate to the large intes-
immune system. tine where they cause more severe colitis. Therefore,
Gastrointestinal symptoms have been well docu- restoration of a normal small intestinal barrier func-
mented to occur in T1D patients but thought to be tion may be an effective treatment option for colitis.
due to altered intestinal motility secondary to auto-
nomic neuropathy.105 Recent studies have described Multiple sclerosis
increased intestinal permeability to prelude these GI Multiple sclerosis (MS) patients show increased per-
symptoms and the development of T1D.106,107 These meability of both the blood-brain barrier (BBB) and
studies, including those performed in BioBreeding the intestine. Interestingly, patients with progressive
diabetic-prone (BBDP) rats, which spontaneously MS showed increased levels of serum zonulin, while
develop T1D, suggest a possible pathogenic role for those with relapsing-remitting MS who were in remis-
intestinal barrier defects in T1D. The BBDP rats have sion showed serum zonulin levels similar to controls.12
increase intestinal permeability in the small intestine A study focused on the experimental autoimmune
(but not in the colon) which precedes the loss of toler- encephalomyelitis (EAE) mouse model of MS has fur-
ance to glucose by at least one month.108 In addition, ther described how zonulin is involved in MS.114
histological analysis of the pancreatic islets cells at the Intestinal permeability and intestinal zonulin are
time of the loss of barrier function, was normal.108 increased during the pre-clinical phase of neurological
These studies show the loss of intestinal barrier func- symptoms, suggesting a role for zonulin in disease
tion occurs before the histological damage or loss of development.114
glucose tolerance seen in T1Ds. Subsequent experi-
ments confirmed these findings and reported the
Metabolic disorders and obesity
increased intestinal permeability was zonulin-depen-
dent.11 Furthermore, oral administration of the zonu- Obesity
lin blocker AT1001 (larazotide acetate) in the BBDP Obesity has recently been shown to be associated with
rats corrected the gut barrier defect and reduced the chronic inflammation,.115-117 In an obesity mouse
incidence of diabetes.11 model, increased intestinal permeability and
absorption of macromolecules were observed.118 Acute lung injury

Additionally, obese patients are at risk for developing Leakage of plasma contents into the lungs is observed
secondary complications to their obesity such as high in acute lung injury (ALI) and acute respiratory dis-
cholesterol, type-2-diabets (T2D), coronary heart distress syndrome.128 Zonulin has been implicated in the
ease, high blood pressure, and stroke. Three studies disassembly of lung tight junctions in ALI.129 Blocking
have shown that serum zonulin level is increased in of the zonulin pathway by AT-1001 or by zonulin
obese vs non obese subjects.119-121 Zak-Golab et al. neutralizing antibodies reduced the severity of ALI.129
have shown correlation between total bacteria and Additionally, both zonulin and its agonist peptide,
serum zonulin levels. They suggest that the gut micro- AT-1002, intensified ALI and increased lung perme-
biota may cause increased zonulin levels, with subse- ability. The mechanism is thought to be through zon-
quent abnormal gut permeability to endotoxin ulin dependent complement activation in the lung.129
(lipopolysaccharide - LPS) and, ultimately micro-
inflammation seen in obesity.120 Asthma
Evidence has also been provided suggesting that In addition to increases in lung permeability, intestinal
zonulin not only is associated to obesity, but also with permeability has also been implicated in other lung
its metabolic complications. Serum zonulin has been diseases like asthma.40,41 Preliminary data suggest that
shown to be increased in T2D patients,121,122 and it a subset of asthmatic patients have increased serum
has been suggested, through multivariate analysis, that zonulin levels and »40% have increased intestinal
the relationship between insulin sensitivity and serum permeability.12
zonulin may be modulated through IL-6.119 In it inter- These data suggest that both the lung and intestinal
esting to note that zonulin promoter is under IL-6 mucosa may be routes through which specific antigens
control123 and, therefore, zonulin modulation by IL-6 can gain access to the submucosa with subsequent
may be mechanistically related to its expression. In exposure to the immune system leading to lung
addition, serum zonulin was increased in obese chil- inflammation.
dren with non-alcoholic fatty liver disease (NAFLD)
compared to obese children without NAFLD and cor- Heart diseases
related with the severity of steatosis.124
Coronary artery disease
Coronary artery disease (CAD) is a major cause of
death throughout the world. Several studies have
Polycystic ovary syndrome shown a link between infectious pathogens and
Polycystic ovary syndrome (PCOS) is an endocrinop- CAD.130-132 Additionally, enterobacteria have been
athy in women of reproductive age due to elevated lev- detected in atherosclerotic plaque biopsies. CAD
els of androgens. It has been reported that both patients have been shown to have increased serum
genetic makeup and the environment contribute to zonulin levels and high levels of Enterobacteriaceaes
the development of PCOS.125 The majority of women in their blood.133 These data suggest that zonulin-
with PCOS are overweight and insulin resistant. It is dependent bacterial translocation may cause increase
well described that PCOS is characterized by a chronic levels of bacteria in the circulation, with subsequent
state of inflammation. Studies have suggested onset of atherosclerotic plaque leading to CAD.
increased zonulin associated to altered gut permeabil-
ity,126 as key pathogenic elements, together with intes- Neurological disorders
tinal microbiota, for development of PCOS.127
The BBB is formed by endothelial cells and separates
the circulating blood from the brain. Since zonulin
can also modulate endothelial tight junctions, it was
Lung diseases
hypothesized zonulin dysregulation may be involved
In addition to the GI tract, the lung is another mucosal in the pathogenesis of neurological disorders.
surface where altered tight junctions function can play Zonulin has also been shown to be involved brain
a role in a variety of diseases. The mechanisms of air- tumors, specifically gliomas.134,135 Skardelly et al.
way inflammation are still incompletely established. showed there was increased expression of zonulin in
gliomas which correlated with the degree of malig- serum zonulin levels.144 Interestingly, PAR2 has been
nancy and degradation of the BBB.135 In vitro studies suggested to be involved in the increased permeability
on a glioma cell line showed zonulin was expressed in detected in IBS-D patients.145 Serine-proteases, which
high amounts compared to non-glioma control activate PAR2, are found to be increased in the lumal
cells.134 Additionally, zonulin has been shown to contents of IBS-D patients but not in constipated or
induce transmigration of neuronal progenitor cells alternating IBS patients.146 Diluted fecal supernatants
across the BBB.134 of human IBS-D patients increased permeability of
mouse mucosa when added to the apical surface.
Systemic infectious diseases These changes were not detected in PAR2¡/¡
mice.147 Additionally, IBS-D patients who carry either
Septicemia the HLA-DQ2 or DQ8 genotype have increased gut
Intestinal barrier dysfunction has been implicated in permeability compared to IBS-D who do not carry
the pathogenesis and progression of septicemia. one of those HLA genotypes.148 Taken together, these
Yoseph et al. have shown in an experimental model of data suggest zonulin signaling through PAR2 may be
sepsis that tight junction proteins expression is involved in the pathogenesis of IBS-D.
altered.136 In patients with septicemia serum zonulin
levels were found to be increased.137 Post-surgical sep- Non-celiac gluten sensitivity
ticemia continues to be a common complication Non-celiac gluten sensitivity (NCGS) is a newly
despite advances in surgical techniques and periopera- described condition that clinically presents similar to
tive care. It was hypothesized that zonulin could be a CD but does not have the intestinal damage seen in
key contributor to post-surgical septicemia.138 Liu CD.149 It has been reported patients with NCGS may
et al. described how treatments with probiotics can have an increase in intestinal permeability following
decrease post-surgical septicemia and is correlated gluten exposure.36 Preliminary evidence also suggest
with decreased serum zonulin levels.138 These data increased serum zonulin levels in patients with
suggest increased release of zonulin from enterocytes NCGS.144
leads to the migration of bacteria across the epithe-
lium which can lead to septicemia. Environmental enteropathy
Environmental enteropathy (EE) is a disease of unknown
HIV etiology seen in developing countries. It is hypothesized
It is widely accepted that the intestine plays an integral that constant exposure to infectious agents in the proxi-
role in the immunopathogenesis of human immuno- mal intestine causes increased gut permeability, excess of
deficiency virus (HIV).139-141 Interestingly, it has been macromolecules and endotoxin trafficking that trigger
reported that decreased zonulin levels correlated with chronic inflammation leading to an enteropathy that
increased mortality in HIV patients.142 Additionally, structurally resembles CD. Like in CD, the enteropathy
treatment with HIV treatment drugs, maraviroc and causes decreased absorption of key nutrients eventually
raltegracir (CCR5 receptor antagonist and integrase leading to stunted growth. We have recently shown that
inhibitor), increased serum zonulin levels.143 Com- serum zonulin levels and other markers of barrier dys-
bined, these data suggest that the zonulin pathway in function are correlated with stunted growth in EE
its innate immunity function can be protective against patients.150 This data provided additional evidence that a
HIV infection. functional loss of barrier function may play a key role in
the pathophysiology and EE.
Intestinal diseases
Necrotizing enterocolitis
Irritable bowel syndrome Necrotizing enterocolitis (NEC) is a devastating disease
The pathophysiology leading to the development of that affects premature infants resulting in bacteria
irritable bowel syndrome (IBS) is unknown, but it has translocation causing local infection, inflammation
been reported that patients with IBS have increased and eventually necrosis in portions of the intestine.
gut permeability.37 Recent data show patients with Changes in tight junction protein expression and local-
diarrhea associated IBS (IBS-D) having increased ization during NEC have been described in several
studies.42-44,151,152 A recently published study impli- Disclosure of potential conflicts of interest

cated the role of zonulin in NEC. Pre-treatment of CS has no potential conflict of interest. AF is co-founder and
Caco2 cells with Bifidobacterium before LPS exposure stock holder of Alba Therapeutics.
decreased release of zonulin and preserves tight junc- Funding
tion competency.153 Ling et al. went further to confirm This work was supported by NIH grant DK048373.
these results in an in vivo rat model of NEC.153 This
study highlights a model in which changes in the References
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Zonulin, A Regulator of Epithelial and Endothelial Barrier Function

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TISSUE BARRIERS

2016, VOL. 4, NO. 4, e1251384 (19 pages)

Zonulin, a regulator of epithelial and endothelial barrier functions, and its

ABSTRACT ARTICLE HISTORY

Introduction environmental factors affecting clinical outcome has

dysfunction in the context of established chronic Role of intestinal permeability in disease

Table 1. List of CIDs in which zonulin has been implicated.

absorption of macromolecules were observed.118 Acute lung injury

studies.42-44,151,152 A recently published study impli- Disclosure of potential conﬂicts of interest

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