Zonulin, regulation of tight junctions, and autoimmune diseases

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Barriers and Channels Formed by Tight Junction Proteins

Zonulin, regulation of tight junctions, and autoimmunediseases

Alessio FasanoMucosal Biology Research Center and Center for Celiac Research, University of Maryland School of Medicine, Baltimore,Maryland

Address for correspondence: Alessio Fasano, M.D., Mucosal Biology Research Center, University of Maryland School ofMedicine, 20 Penn Street HSF II Building, Room S345, Baltimore, MD 21201. [email protected]

Recent studies indicate that besides digestion and absorption of nutrients and water and electrolytes homeostasis,another key function of the intestine is to regulate the trafficking of environmental antigens across the host mucosalbarrier. Intestinal tight junctions (TJs) create gradients for the optimal absorption and transport of nutrients andcontrol the balance between tolerance and immunity to nonself antigens. To meet diverse physiological challenges,intestinal epithelial TJs must be modified rapidly and in a coordinated fashion by regulatory systems that orchestratethe state of assembly of the TJ multiprotein network. While considerable knowledge exists about TJ ultrastructure,relatively little is known about their physiological and pathophysiological regulation. Our discovery of zonulin, theonly known physiologic modulator of intercellular TJs described so far, has increased our understanding of theintricate mechanisms that regulate the intestinal epithelial paracellular pathway and has led us to appreciate that itsupregulation in genetically susceptible individuals leads to autoimmune diseases.

Keywords: tight junctions; intestine; autoimmunity; zonulin

Introduction

Improved hygiene leading to reduced exposure tomicroorganisms has been implicated as one possi-ble cause for the “epidemic” of immune-mediateddiseases, particularly autoimmune diseases, in in-dustrialized countries during the past 3–4 decades.1

Collectively, autoimmune diseases are highly preva-lent in the United States, affecting between 14.7 and23.5 million people—up to 8% of the population.2

The social and financial burdens imposed by thesechronic, debilitating diseases include poor qualityof life, high health care costs, and substantial loss ofproductivity. For example, in 2002, the average an-nual medical costs for treating type 1 diabetes (T1D)in the United States were an estimated $6.7 billion.3

In less than a decade, these costs jumped to $14.4billion.4 Apart from genetic makeup and exposureto environmental triggers, a third key element, i.e.,increased intestinal permeability, which may be in-fluenced by the composition of the gut microbiota,has been proposed in the pathogenesis of these dis-

eases.5–8 Intestinal permeability, together with anti-gen (Ag) sampling by enterocytes and lamina pro-pria dendritic cells, regulates molecular traffickingbetween the intestinal lumen and the submucosa,leading to either tolerance or immunity to nonselfAg.9–11 However, the dimensions of the paracellularspace (10–15 A) suggest that solutes with a molec-ular radius exceeding 15 A (∼3.5 kDa) (includingproteins) are normally excluded and taken up by thetranscellular pathway. Intercellular tight junctions(TJs) tightly regulate paracellular Ag trafficking. TJsare extremely dynamic structures that operate inseveral key functions of the intestinal epitheliumunder both physiological and pathological circum-stances.6 However, despite major progress in ourknowledge on the composition and function of theintercellular TJs, the mechanism(s) by which theyare regulated is(are) still incompletely understood.

In the past decade, we have focused our re-search effort on the discovery and characterizationof zonulin as the only human protein discovered todate that is known to reversibly regulate intestinal

doi: 10.1111/j.1749-6632.2012.06538.xAnn. N.Y. Acad. Sci. 1258 (2012) 25–33 c© 2012 New York Academy of Sciences. 25

Zonulin, regulation of tight junctions Fasano

permeability by modulating intercellular TJs.12–14

Zonulin expression is augmented in autoimmuneconditions associated with TJ dysfunction, includ-ing celiac disease (CD) and T1D.13,15 Both animalstudies16 and human trials17 using the zonulin syn-thetic peptide inhibitor AT1001 (now named Lara-zotide acetate) have established that zonulin is inte-grally involved in the pathogenesis of autoimmunediseases. Zonulin can be used as a biomarker of im-paired gut barrier function for several autoimmune,neurodegenerative, and tumoral diseases,18 and canbe a potential therapeutic target for the treatmentof these devastating conditions.

The zonulin system

Identification of zonulin as prehaptoglobin 2Through proteomic analysis of human sera, wehave recently identified zonulin as prehaptoglobin(HP)2,19 a molecule that, to date, has only beenregarded as the inactive precursor for HP2, one ofthe two genetic variants (together with HP1) of hu-man HPs. Mature human HPs are heterodimericplasma glycoproteins composed of � and � polypep-tide chains that are covalently associated by disulfidebonds and in which only the � chain is glycosylated20

(Fig. 1). While the � chain (36 kDa) is constant, the� chain exists in two forms, i.e., �1 (∼9 kDa) and�2 (∼18 kDa). The presence of one or both of the �chains results in the three human HP phenotypes,i.e., HP1-1 homozygote, HP2-1 heterozygote, andHP2-2 homozygote.

Despite this multidomain structure, thehemoglobin only function assigned to HPs, to date,is to bind Hb to form stable HP–Hb complexesthereby preventing Hb-induced oxidative tissuedamage.21 In contrast, no function has ever beendescribed for their precursor forms. The primarytranslation product of mammalian Hp mRNA is apolypeptide that dimerizes contranslationally and isproteolitically cleaved while still in the endoplasmicreticulum.22 Conversely, zonulin is detectable inan uncleaved form in human serum, adding anextremely intriguing aspect of the multifunctionalcharacteristics of HPs. HPs are unusual secretoryproteins in that they are proteolytically processedin the endoplasmic reticulum, the subcellularfraction of which we detected the highest zonulinconcentration.23 Wicher and Fries22 found thatCr1LP mediates this cleavage in a specific manner,since the enzyme did not cleave the proform of

Figure 1. Western blotting using zonulin cross-reacting anti-Zot polyclonal antibodies on CD patient sera showed three mainpatterns: sera showing an 18 kDa immunoreactive band and a fainter 45 kDa band (lane 1), sera showing only a 9 kDa band(lane 2), and sera showing both the 18 and 9 kDa bands (lane 3). The cartoon shows the structure of both pre-HP1 and pre-HP2and their mature proteins. HPs evolved from a complement-associated protein (mannose-binding lectin-associated serine protease,MASP), with their α-chain containing a complement control protein (CCP), while the β-chain is related to chymotrypsin-like serineproteases (SP domain) containing an epidermal growth factor-like motif. The gene encoding the α2-chain of pre-HP2 originatedin India almost 2 million years ago through a chromosomal aberration (unequal crossing over) of HP1. Pre-HPs are translated assingle-chain precursor proteins. Pre-HPs may be proteolytically cleaved intracellularly into α- and β-chains that remain disulfidelinked, referred to as cleaved, two-chain, mature HPs.

26 Ann. N.Y. Acad. Sci. 1258 (2012) 25–33 c© 2012 New York Academy of Sciences.

Fasano Zonulin, regulation of tight junctions

complement C1s, a protein similar to pre-HP2,particularly around the cleavage site. Therefore, itis conceivable to hypothesize that the activity ofCr1LP modulates the zonulin pool.

Zonulin functional characterizationSince we have reported previously that the key bio-logical effect of zonulin is to affect the integrity ofintercellular TJs, we specifically focused our effortson demonstrating that the recombinant pre-HP2alters intestinal permeability. Indeed, ex vivo exper-iments showed that recombinant pre-HP2 induced atime- and dose-dependent reduction in TEER whenadded to murine small intestinal mucosa.19 Theseresults were validated independently in an in vivointestinal permeability assay in which zonulin, butnot its cleaved form, induced a significant and re-versible increase in both gastroduodenal and smallintestinal permeability.19 The evidence that zonulincleaved in its �2 and � subunits lost the permeabil-ity activity further supports the notion that pre-HP2(alias, zonulin) and mature HP2 exert two differentbiological functions most likely related to the differ-ent folding of the protein in its cleaved or uncleavedform.

Structural analysis of zonulin revealed similari-ties with several growth factors that, like zonulin,affect intercellular TJ integrity.24,25 Our data show-ing that zonulin but not its cleaved subunits ac-tivate EGFR19 and that its effect on TEER wasprevented by the EGFR tyrosine kinase inhibitorAG-147819 suggest that zonulin is properly folded toactivate EGFR and, therefore, to cause TJ disassem-bly only in its uncleaved form. Several G protein–coupled receptors (GPCR), including Proteinase Ac-tivated Receptor 2 (PAR2), transactivate EGFR.26

Zonulin prokaryotic counterpart Zot active peptideFCIGRL (AT1002) has structural similarities withPAR2-activating peptide (AP), SLIGRL, and causesPAR2-dependent changes in TEER, a finding that wehave demonstrated in WT, but not PAR2–/– mice.27

Therefore, it was not totally unexpected that exper-iments in Caco2 cell, in which PAR2 was silenced,showed decreased EGFR Y1068 phosphorylation inresponse to recombinant zonulin compatible withPAR2-dependent transactivation of EGFR.19 To fur-ther establish a role for PAR2 in EGFR activation inresponse to zonulin, we conducted small intesti-nal barrier function studies using segments iso-lated from either C57BL/6 WT or PAR2

–/– mice.

As expected, zonulin decreased TEER in intestinalsegments from C57BL/6 WT mice, while it failedto reduce TEER in small intestinal segments fromPAR2

–/– mice,19 so linking zonulin-induced PAR2-dependent transactivation of EGFR with barrierfunction modulation.

To summarize, we have reported for the firsttime the novel characterization of zonulin as pre-HP2, a multifunctional protein that, in its intactsingle chain form, regulates intestinal permeabil-ity through PAR2-mediated EGFR transactivation,while in its cleaved two-chain form acts as a Hbscavenger (Fig. 2).

Stimuli that cause zonulin release in the gutAmong the several potential intestinal luminal stim-uli that can trigger zonulin release, we identifiedsmall intestinal exposure to bacteria or gluten asthe two more powerful triggers.18,23,28 Enteric in-fections have been implicated in the pathogenesisof several pathological conditions, including aller-gic, autoimmune, and inflammatory diseases, bycausing impairment of the intestinal barrier. Wehave generated evidence that small intestines ex-posed to enteric bacteria secrete zonulin.28 This se-cretion was independent of either the animal speciesfrom which the small intestines were isolated or thevirulence of the microorganisms tested, occurredonly on the luminal aspect of the bacteria-exposedsmall intestinal mucosa, and was followed by an in-crease in intestinal permeability coincident with thedisengagement of the protein ZO-1 from the tightjunctional complex.28 This zonulin-driven openingof the paracellular pathway may represent a defen-sive mechanism, which flushes out microorganismsso contributing to the innate immune response ofthe host against bacterial colonization of the smallintestine.

Besides bacterial exposure, we have shown thatalso gliadin, a storage protein present in wheat andthat triggers CD in genetically susceptible individu-als, also affects the intestinal barrier function by re-leasing zonulin.29 This effect of gliadin is polarized,i.e., gliadin increases intestinal permeability onlywhen administered on the luminal side of the intesti-nal tissue.29 This observation led us to the identifica-tion of the chemokine receptor CXCR3 as the targetintestinal receptor for gliadin.30 Our data demon-strate that in the intestinal epithelium, CXCR3 is ex-pressed at the luminal level, is overexpressed in CD

Ann. N.Y. Acad. Sci. 1258 (2012) 25–33 c© 2012 New York Academy of Sciences. 27


Figure 2. Zonulin can activate EGFR through direct binding (1) and/or through PAR2 transactivation (2). This second mechanismcan be mediated by either Src signaling (2a) or by the release of MMPs and/or ADAMS that in turn will activate Pro-HB-EGF.When cell tryptase IV cleaves zonulin in its two subunits (so eliminating one of the three required disulfide bridges necessary forEGF activity), the molecule is not able to bind to EGFR (3), while it acquires a different function (Hb binding) and becomes aninflammatory marker.

patients, colocalizes with gliadin, and that this in-teraction coincides with recruitment of the adapterprotein, MyD88, to the receptor.28 We also demon-strated that binding of gliadin to CXCR3 is crucialfor the release of zonulin and subsequent increaseof intestinal permeability, since CXCR3-deficientmice failed to respond to gliadin challenge in termsof zonulin release and TJ disassembly.30 Using an�-gliadin synthetic peptide library, we identifiedtwo �-gliadin 20mers (QVLQQSTYQLLQELCC-QHLW and QQQQQQQQQQQQILQQILQQ) thatbind to CXCR3 and release zonulin.30

Role of zonulin in autoimmune diseases

Celiac diseaseCD is an immune-mediated chronic enteropathywith a wide range of presenting manifestations ofvariable severity. It is triggered by the ingestion ofgliadin fraction of wheat gluten and similar alcohol-soluble proteins (prolamines) of barley and rye ingenetically susceptible subjects with subsequent im-mune reaction leading to small bowel inflamma-tion and normalization of the villous architecturein response to a gluten-free diet.31 CD is a uniquemodel of autoimmunity in which, in contrast tomost other autoimmune diseases, a close geneticassociation with HLA genes, a highly specific hu-moral autoimmune response against tissue trans-glutaminase autoantigen, and, most importantly,the triggering environmental factor (gliadin), are allknown. It is the interplay between genes (both HLA

and non-HLA associated) and environment (i.e.,gluten) that leads to the intestinal damage typicalof the disease.32 Under physiological circumstances,this interplay is prevented by competent intercellu-lar TJs. Early in CD, TJs are opened33–36 and severeintestinal damage ensues.

The repertoire of gluten peptides involved inthe disease pathogenesis is greater than appreci-ated previously and may differ between childrenand adult patients.37 There are at least 50 toxicepitopes in gluten peptides exerting cytotoxic, im-munomodulatory, and gut-permeating activities.38

The effect of the permeating gliadin peptides in vivowas confirmed by the analysis of intestinal tissuesfrom patients with active CD and non-CD controlsprobed for zonulin expression.23 Quantitative im-munoblotting of intestinal tissue lysates from activeCD patients confirmed the increase in zonulin pro-tein compared to control tissues.23 Zonulin upregu-lation during the acute phase of CD was confirmedby measuring zonulin concentration in sera of 189CD patients using a sandwich ELISA (Fig. 3A).13

Type 1 diabetesThe trigger of the autoimmune destruction of pan-creatic beta cells in T1D is unknown. T1D has thesame pathogenic challenges as other autoimmunediseases: what are the environmental triggers, andhow do these triggers cross the intestinal barrierto interact with the immune system?39,40 CertainHLA class II alleles account for 40% of the genetic



Figure 3. (A) CD patients showed higher serum zonulin levels compared to both their relatives and controls. (B) Similar resultswere obtained in T1D patients. (C) Serum zonulin correlated with intestinal permeability evaluated by the LA/MA test. Thepercentage of CD patients (81%) and their relatives (50%) with elevated serum zonulin levels was higher compared to T1D patients(42%) and their relatives (29%), respectively.

susceptibility to T1D in Caucasians;41 however, themajority of individuals with these HLA alleles donot develop T1D. This supports the concept thatreaction to some environmental products triggersautoimmune destruction of beta cells and leads toT1D. Gastrointestinal symptoms in T1D have beengenerally ascribed to altered intestinal motility sec-ondary to autonomic neuropathy.42 However, morerecent studies have shown that altered intestinal per-meability occurs in T1D prior to the onset of com-plications,43 which is not the case in type 2 diabetes.This has led to the suggestion that an increased in-testinal permeability due to alteration in intestinalTJs is responsible for the onset of T1D.43,44 Thishypothesis is supported by studies performed inan animal model that develops T1D spontaneouslyand showed an increased permeability of the smallintestine (but not of the colon) of the BioBreed-ing diabetic-prone (BBDP) rats that preceded theonset of diabetes by at least a month.45 Further,histological evidence of pancreatic islet destructionwas absent at the time of increased permeabilitybut clearly present at a later time.45 Therefore, thesestudies provided evidence that increased gut perme-ability occurred before either histological or overtmanifestation of diabetes in this animal model. Weconfirmed these data by reporting in the same ratmodel that zonulin-dependent increase in intesti-nal permeability precedes the onset of T1D by 2–3weeks.16

Several reports have linked gliadin (the envi-ronmental trigger of CD autoimmunity that also

causes zonulin release from the gut, see Refs. 23 and29) to T1D autoimmunity both in animal mod-els and in human studies. Findings from studiesusing nonobese diabetic (NOD) mice and BBDPrats have implicated wheat gliadin as a dietary dia-betogen.46,47 We have recently reported a direct linkbetween antibodies to Glo-3a (a wheat-related pro-tein), zonulin upregulation, and islet autoimmunity(IA) in children at increased risk for T1D.48

Association of HP genotypes with serumzonulin expression and clinical severity inhuman disease statesWe have recently discovered that human zonulinis identical to pre-Hp2 (see above). Two co-dominant allele variants, termed HP1 and HP2, thelatter unique to the human species, are variouslydistributed in the general population, resulting inthree phenotypes: Hp1-1 (∼20% in Western pop-ulations), Hp2-1 (∼50%), and Hp2-2 (∼30%).49

Several studies have suggested that the presence ofthe HP2 allele correlates with higher risk to developimmune-mediated diseases50,51 and that HP2 ho-mozygosis is associated with poor prognosis51 anddecreased longevity.52

Zonulin phenotype in CD and T1D and its correla-tion with intestinal permeability. Using a serumzonulin ELISA, we measured serum zonulin lev-els in both CD and T1D patients, their relatives,and age- and sex-matched healthy controls. CD pa-tients showed statistically higher serum zonulin lev-els (2.37 ± 0.17 ng/mg protein) compared to both



their relatives (1.75 ± 0.27 ng/mg protein, P = 0.05)and control subjects (0.31 ± 0.03 ng/mg protein,P < 0.00001) (Fig. 3A). A total of 81% (154/190)of CD patients and 50% of their first-degree rel-atives (33/65) had serum zonulin levels that were2 SD above the mean zonulin levels detected inage-matched healthy controls. Only 4.9% (5/101)of controls had zonulin levels 2 SD above the mean(P < 0.01). Serum zonulin was higher in CD com-pared to their relatives (P < 0.00001). Similar re-sults were obtained in T1D patients in which wedetected increased serum zonulin levels (0.83 ±0.05 ng/mg protein) compared to both their rela-tives (0.62 ± 0.07 ng/mg protein, P = 0.011) andcontrol subjects (0.21 ± 0.02 ng/mg protein, P <

0.00001; Fig. 3B).15 A total of 42% (141/339) ofT1D patients and 29% of their first-degree rela-tives (26/89) had serum zonulin levels that were2 SD above the mean zonulin levels detected in age-matched healthy controls. Only 4% (4/97) of con-trols had zonulin levels 2 SD above the mean (P <

0.01). Serum zonulin was higher in T1D comparedto their relatives (P = 0.01). To establish whetherserum zonulin levels correlated with intestinal per-meability, lactulose (LA)/mannitol (MA) urine ra-tio was determined in both a subset of T1D subjectswith documented zonulin upregulation (N = 36)and their relatives (N = 56). Intestinal permeabilitycorrelated with serum zonulin (Fig. 3C).15

HP2 (alias zonulin) allele is overrepresented inimmune-mediated diseases. To establish the dis-tribution of HP1 and HP2 genes among CD patientsand matched controls, we developed a single-stepRT-PCR protocol using specific primers in exon 2and exon 5 of HP1 corresponding to exons 2 and7 of HP2. After PCR, the amplicons were run on a1% agarose gel and read under a UV bulb. The HP1genotype ran at the predicted size of 2.5 kb while theHP2 ran at 4.3 kb. Our results showed that in CDpatients HP1-1 genotype (0 copies of the zonulingene) was decreased, while the HP2-2 genotype (2copies of zonulin gene) was increased compared tohealthy controls (Table 1). Interestingly, the percent-age of HP 1-1 CD patients (0 copies of the zonulingene) was in the same range of the percentage ofCD patients that tested negative by zonulin ELISA(see above). Similar distribution of the HP geneshave been reported by other investigators in otherimmune-mediated diseases, including Crohn’s dis-

ease,53 schizophrenia,54 and chronic kidney disease(CKD)55 (Table 1).

Proof of concept for the pathogenic role ofzonulin-dependent intestinal barrier defect inCD and T1DCD and T1D autoimmune models suggest thatwhen the finely tuned trafficking of macromoleculesis deregulated in genetically susceptible individu-als, autoimmune disorders can occur.56 This newparadigm subverts traditional theories underlyingthe development of autoimmunity, which are basedon molecular mimicry and/or the bystander effect,and suggests that the autoimmune process can bearrested if the interplay between genes and envi-ronmental triggers is prevented by reestablishingthe intestinal barrier function. To challenge thishypothesis, zonulin inhibitor AT1001 was used withencouraging results in the BBDP rat model ofautoimmunity.16 Besides preventing the loss of in-testinal barrier function, the appearance of autoan-tibodies, and the onset of disease, pretreatment withAT1001 protected against the insult of pancreaticislets and, therefore, of the insulitis, is responsiblefor the onset of T1D.

This proof of concept in an animal model of au-toimmunity provided the rationale to design humanclinical trials in which AT1001 was initially testedin an inpatient, double-blind, randomized placebocontrolled trial to determine its safety, tolerabil-ity, and preliminary efficacy.57 No increase in ad-verse events was recorded among patients exposedto AT-1001 compared to placebo. Following acutegluten exposure, a 70% increase in intestinal per-meability was detected in the placebo group, whileno changes were seen in the AT-1001 group.57 Af-ter gluten exposure, IFN-� levels increased in fourout of seven patients (57.1%) of the placebo-group,but only in 4 out of 14 patients (28.6%) of theAT-1001 group. Gastrointestinal symptoms weresignificantly more frequent among patients of theplacebo group compared to the AT-1001 group.57

Together, these data suggest that AT-1001 is welltolerated and appears to reduce pro-inflammatorycytokine production and gastrointestinal symp-toms in CD patients. AT1001 has now been testedin approximately 500 subjects with an excellentsafety profile and promising efficacy in protectingagainst symptoms caused by gluten exposure in CDpatients.58



Table 1. HP genotype distribution in several immune-mediated diseases

CD19 Crohn’s disease56 Schizophrenia57 CKD58

Genotype Cntr Pts Cntr Pts Cntr Pts Cntr Pts

HP 1-1 20.6 7.1 23.9 10.1 14.1 9.2 9.4 3.8

HP 1-2 43.5 35.7 44.0 46.2 46.9 38.8 46.5 43.7

HP 2-2 35.9 57.2 32.1 43.7 39.0 52.0 44.1 52.6

Concluding remarks

The gastrointestinal tract has been extensively stud-ied for its digestive and absorptive functions. A moreattentive analysis of its anatomo-functional charac-teristics, however, clearly indicates that its functionsgo well beyond the handling of nutrients and elec-trolytes. The exquisite regional-specific anatomicalarrangements of cell subtypes and the finely regu-lated cross talk between epithelial, neuroendocrine,and immune cells highlight other less-studied, yetextremely important functions of the gastrointesti-nal tract. Of particular interest is the regulationof antigen trafficking by the zonulin-dependentparacellular pathway and its activation by intesti-nal mucosa–microbiota/gluten interactions. Thesefunctions dictate the switch from tolerance to im-munity, and are likely integral mechanisms involvedin the pathogenesis of inflammatory and autoim-mune processes.

The classical paradigm of inflammatory patho-genesis involving specific genetic makeup and expo-sure to environmental triggers has been challengedrecently by the addition of a third element, the lossof intestinal barrier function. Genetic predisposi-tion, miscommunication between innate and adap-tive immunity, exposure to environmental triggers,and loss of intestinal barrier function secondaryto the activation of the zonulin pathway by food-derived environmental triggers or changes in gutmicrobiota all seem to be key ingredients involvedin the pathogenesis of inflammation, autoimmu-nity, and cancer. This new theory implies that oncethe pathologic process is activated, it is not auto-perpetuating. Rather, it can be modulated or evenreversed by preventing the continuous interplay be-tween genes and the environment. Since zonulin-dependent TJ dysfunction allows such interactions,new therapeutic strategies aimed at reestablishing

the intestinal barrier function by downregulatingthe zonulin pathway offer innovative and not yet-explored approaches for the management of thesedebilitating chronic diseases.

Acknowledgments

Work presented in this review was supported in partby the National Institutes of Health Grants DK-48373 and DK-078699 to AF.

Conflict of interest

Dr. Fasano is a stock holder of Alba Therapeutics.

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Zonulin, regulation of tight junctions, and autoimmune diseases