F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.20510.1 Review Articles All disease begins in the (leaky) gut: role of zonulin-mediated gut permeability in the pathogenesis of some chronic inflammatory diseases

[version 1; peer review: 3 approved]

 Fasano Alessio Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Resources Visualization Writing – Original Draft Preparation https://orcid.org/0000-0002-2134-0261 a 1 2 Mucosal Immunology and Biology Research Center, Center for Celiac Research and Treatment and Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital for Children, Boston, Massachusetts, USA European Biomedical Research Institute of Salerno, Salerno, Italy afasano@mgh.harvard.edu

Competing interests: The author is co-founder and stock holder of Alba Therapeutics, a company developing treatments complementary to the gluten-free diet by exploiting gut permeability.

 31 1 2020 2020 9 F1000 Faculty Rev-69 22 1 2020 Copyright: © 2020 Fasano A 2020 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Improved hygiene leading to reduced exposure to microorganisms has been implicated as one possible cause for the recent “epidemic” of chronic inflammatory diseases (CIDs) in industrialized countries. That is the essence of the hygiene hypothesis that argues that rising incidence of CIDs may be, at least in part, the result of lifestyle and environmental changes that have made us too “clean” for our own good, so causing changes in our microbiota. Apart from genetic makeup and exposure to environmental triggers, inappropriate increase in intestinal permeability (which may be influenced by the composition of the gut microbiota), a “hyper-belligerent” immune system responsible for the tolerance–immune response balance, and the composition of gut microbiome and its epigenetic influence on the host genomic expression have been identified as three additional elements in causing CIDs. During the past decade, a growing number of publications have focused on human genetics, the gut microbiome, and proteomics, suggesting that loss of mucosal barrier function, particularly in the gastrointestinal tract, may substantially affect antigen trafficking, ultimately influencing the close bidirectional interaction between gut microbiome and our immune system. This cross-talk is highly influential in shaping the host gut immune system function and ultimately shifting genetic predisposition to clinical outcome. This observation led to a re-visitation of the possible causes of CIDs epidemics, suggesting a key pathogenic role of gut permeability. Pre-clinical and clinical studies have shown that the zonulin family, a group of proteins modulating gut permeability, is implicated in a variety of CIDs, including autoimmune, infective, metabolic, and tumoral diseases. These data offer novel therapeutic targets for a variety of CIDs in which the zonulin pathway is implicated in their pathogenesis.

 Chronic inflammatory diseases Gut permeability microbiome zonulin National Institutes of Health DK104344 NIHU19AI082655 The author declared that the following grants were involved in generating some of the data presented in this manuscript: National Institutes of Health (NIH) NIH DK104344 and NIH U19AI082655. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Editorial Note on the Review Process

F1000 Faculty Reviews are commissioned from members of the prestigious F1000 Faculty and are edited as a service to readers. In order to make these reviews as comprehensive and accessible as possible, the referees provide input before publication and only the final, revised version is published. The referees who approved the final version are listed with their names and affiliations but without their reports on earlier versions (any comments will already have been addressed in the published version).

The referees who approved this article are:

Arul Jayaraman, Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX, USA

No competing interests were disclosed.

Michael Maes, Department of Psychiatry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

No competing interests were disclosed.

Xin M Luo, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

No competing interests were disclosed.

 Introduction

Twenty-five hundred years ago, when Hippocrates stated that “All disease begins in the gut”, he had an incredible intuition that only recently has been fully appreciated because of new insights into the pathogenesis of many chronic inflammatory diseases (CIDs) afflicting humankind. Until 30 years ago, when the Human Genome Project was still in its planning stage, the general hypothesis was that genetic predisposition and exposure to an environmental trigger were both necessary and sufficient to develop CIDs, including infectious, allergic, neuroinflammatory/neurodegenerative, autoimmune diseases, and cancer. However, the epidemiological observation showing a major surge of CIDs during the past four decades in the Western hemisphere coincident with the declining rate of infectious diseases was at odds with the gene/environment paradigm 1, 2 . This generated the hygiene hypothesis supporting the notion that we had made ourselves too clean for our own good and that people embracing a Western lifestyle would slowly die of CIDs instead of rapidly succumbing to infectious diseases as still is happening in developing countries.

What we learned when the Human Genome Project was completed is that we are genetically much more rudimentary than we had previously thought. The premise of “one gene, one protein, one disease” cannot explain the complexity of the balance between health and disease and, most definitively, the CIDs epidemics. Twenty-three thousand genes are insufficient to explain all the permutations of human pathophysiology, including if and when and why we develop diseases. Rather, it is the interplay between us as individuals and the environment in which we live that dictates our clinical destiny. This interplay is physically and mechanistically regulated by biological interfaces that divide our body from the surrounding environment. At about 8 to 9 meters in length, the human intestine provides the largest interface between our body and the outside world. Tightly packed single layers of epithelial cells cover the external surfaces of our intestinal mucosa and negotiate the interaction with the surrounding environment. Although this enormous mucosal interface (200 m 2) is not apparently visible, it plays a pivotal role through its dynamic interactions with a variety of factors coming from our surrounding environment, including microorganisms, nutrients, pollutants, and other materials. Intestinal permeability, together with luminal antigen (Ag) sampling by enterocytes via the transcellular pathway and dendritic cells, regulates molecular trafficking between the intestinal lumen and the submucosa, leading to either tolerance or immune response to non-self Ag 3– 5 ( Figure 1). Intercellular tight junctions (TJs) tightly regulate paracellular Ag trafficking. TJs are extremely dynamic structures that operate in several key functions of the intestinal epithelium under both physiological and pathological circumstances 6– 8 . TJs, the most apical junctional complex of the paracellular pathway that segregates the apical and basolateral cellular compartment, were previously believed to be impermeable and static, so forming a sealing barrier. This paradigm was subverted in 1993 by the discovery of zonula occludens 1 (ZO-1) as the first component of the TJ complex 9 now being comprised of more than 150 proteins, including occludin 10 , claudins 11 , junctional adhesion molecules (JAMs) 12 , tricellulin 13 , and angulins 14 . However, despite major progress in our knowledge on the composition and function of the intercellular TJ, the mechanisms by which they are regulated are still incompletely understood. One of the major breakthroughs in understanding the role of gut permeability in health and disease has been the discovery of zonulin, the only physiologic intestinal permeability modulator described so far 15, 16 . Therefore, this article will focus mainly on the body of literature published on zonulin as a biomarker of gut permeability to outline the pathogenic role of a leaky gut in a variety of CIDs. However, it should be pointed out that zonulin is not involved in all CIDs and that not all CIDs have been linked to increased gut permeability.

Proposed chain of events leading to chronic inflammatory disease.

( 1) Under physiological circumstances there is a tightly control of mucosal antigen trafficking (antigen sampling) that, in concert with specific immune cells and chemokine and cytokine mediators lead to anergy and therefore to mucosal tolerance. ( 2) Gut dysbiosis causes inappropriate production of increased amount of zonulin with subsequent functional loss of gut barrier function, followed by microbiota-derived antigen and endotoxin trafficking from the lumen to the lamina propria triggering innate and immunoregulatory responses causing a pro-inflammatory micromilieu. ( 3) If this process continues, an adaptive immune response is mounted, causing production of pro-inflammatory cytokines, including interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) that cause further opening of the paracellular pathway to the passage of antigens, creating a vicious cycle. ( 4) Ultimately, these processes lead to break of tolerance with subsequent onset of chronic inflammatory disease whose nature is influenced by the specific host genetic background that dictates which organ or tissue will be targeted by the inflammatory process. DC, dendritic cell; IL-10, interleukin 10; TGF-β, transforming growth factor beta; Tregs, regulatory T cells. This figure was re-used from Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers. doi:10.1080/21688370.2016.1251384 with permissions 17 .

 The zonulin pathway and its activation

Zonulin is composed of a family of related proteins 17– 19 whose first member, pre-haptoglobin 2 (HP2), the precursor of HP2, was identified almost two decades ago 20, 21 . Haptoglobins evolved from a complement-associated protein (mannose-binding lectin-associated serine protease, or MASP) that lost its protease function because of mutations in the catalytic domain to then acquire new functions, including the capability to modulate intercellular TJs 18 . The frequent zonulin polymorphisms secondary to high mutation rate during evolution led to a family of structurally and functionally related zonulins, including pre-HP2 and properdin, another member of the MASP family 22 .

Among the several potential intestinal luminal stimuli that can stimulate zonulin release, small exposure to large amounts of bacteria (bacteria overgrowth) and gluten, the protein causing celiac disease (CD), have been identified as the two most powerful triggers 23, 24 . Zonulin secretion has been shown to be MyD88-dependent 25 and is followed by an increase in gut permeability secondary to the disassembly of the protein ZO-1 from the tight junctional complex 26 . Gliadin triggers zonulin release through the CXCR3 receptor activated by its engagement to MyD88 with a subsequent increase in gut permeability 27 , suggesting that gluten is misinterpreted by the zonulin pathway as a potential harmful component of a microorganism. Taken together, these data suggest that the activation of the zonulin pathway may represent a defensive mechanism that “flushes out” microorganisms, contributing to the innate immune response of the host against changes in microbiome ecosystem, specifically bacterial colonization of the small intestine (small intestinal bacterial overgrowth) or changes in its composition (dysbiosis) or both. These findings are in line with the growing evidence on the role of changes in gut microbiome composition and function in causing functional changes in gut permeability, with subsequent increased Ag trafficking and break of tolerance leading to CID in genetically susceptible individuals ( Figure 1).

 Pathological consequences of the activation of the zonulin pathway

Modulation of intestinal permeability, including the activation of the zonulin pathway, is part of the physiological machinery to maintain mucosal homeostasis and therefore does not always translate in clinical pathological outcomes. Indeed, studies on transgenic mice with constitutive activation of myosin light-chain kinase (MLCK), an intracellular mediator of TJ disassembly, showed increased intestinal permeability without signs of overt disease 28 . Similarly, mice lacking JAM-A, a key TJ structural component, or the muscle myosin IIA heavy chain (NM IIA) showed increased intestinal permeability but only low-grade colonic inflammation and normal epithelial architecture 29, 30 . Finally, zonulin transgenic mice constitutively producing high levels of zonulin showed increased gut permeability but not pathologic phenotype 31 . Interestingly, zonulin transgenic, JAM-A −/−, and NM IIA −/− mice all showed increased susceptibility to chemical-induced colitis 29– 31 . Together, these data suggest that gut permeability may contribute to the development of several CIDs provided that additional genetic components regulating immune response and an imbalanced microbiome are coexistent. Indeed, there is growing evidence that the additional and mutually influenced elements of the triad of gut permeability, immune system, and gut microbiome—together with genetic predisposition and exposure to environmental triggers—make the “perfect storm” for CIDs development.

 Challenges in measuring zonulins with currently commercially available enzyme-linked immunosorbent assays

Several groups, including ours, have been questioning what exactly the commercially available zonulin enzyme-linked immunosorbent assay (ELISA) kits measure 22, 32 . Using different approaches, both we 22 and others 32 identified complement C3 as the top match protein identified by the antibodies used for the ELISA. However, complement C3 is most likely an unspecific product overshadowing the real targets. Indeed, the respective ELISA kits did not detect any complement C proteins obtained from different suppliers when tested under native and denatured conditions, as well as when C3 was spiked in serum 22 . Also, the same kit did not detect recombinant zonulin, mature HP1, or mature HP2 22 . Given the additional mass spectrometric hits reported 22, 32 , a few proteins stand out, although, without further validation, these data need to be interpreted with caution, since only more abundant proteins may be identified by mass spectrometric analysis, while the protein(s) of interest belonging to the zonulin family may be in low abundance in serum samples and therefore not identifiable with this approach. Keeping this in mind and the fact that the protein(s) of interest should be in the roughly 50-kDa range, combined Western blot analysis and ELISA test confirmed that the polyclonal antibodies raised against the zonulin-derived synthetic peptide AT1001 and used in the Immundiagnostik kit (Immundiagnostik AG, Bensheim, Germany) detect properdin among other proteins 22 . However, when purified proteins/peptides, including the AT1001 peptide used to raise the polyclonal antibodies (internal control), were tested by ELISA, they were highly underestimated by the test. One possible explanation for these results is that zonulin as both pre-HP2 and properdin is not the main target detected by the ELISA; however, the fact that even AT1001 was underestimated seems to suggest that this hypothesis cannot entirely explain these results. Alternatively, it is possible that tertiary and quaternary (multimers) structure arrangements present in sera samples but not in recombinant proteins are necessary in order to properly detect any zonulin member by this ELISA. Given these results, it is likely that the commercially available ELISAs detect one or more members of the zonulin family that have not been discovered yet.

 Alterative tests to measure gut permeability

With the appreciation that gut microbiome composition/function can affect intestinal permeability and vice versa and that loss of gut barrier function allows passage of endotoxin from gut lumen to systemic circulation, there are additional tools to be considered to monitor intestinal permeability. The presence of cytotoxic bacterial products in serum can be evaluated by using IgA/IgM responses to sonicated samples of common Gram-negative gut commensal bacteria, and assays of serum lipopolysaccharides (LPSs) and other bacterial toxins, including cytolethal distending toxin subunit B, provide good methods to screen for increased gut permeability in combination with IgM levels to zonulin and measuring gut dysbiosis 33 .

 Role of zonulin pathway in specific chronic inflammatory disease

Zonulin has been implicated in many CIDs ( Table 1). Independent from the CIDs considered, the steps leading to break of tolerance and subsequent development of CID seem to be similar ( Figure 1). Below, we will review some of the CIDs that have been associated with dysregulation of the zonulin pathway, and a more complete list of diseases and related references is presented in Table 1.

Chronic inflammatory diseases in which zonulin has been linked as a biomarker of gut permeability.
Disease Model References
Aging Human 37, 38
Ankylosis spondylitis Human 39
Attention deficit hyperactivity disorder Human 40
Autism Human 41, 42
Celiac disease Human 15– 20, 23– 27, 43– 48
Chronic fatigue syndrome/myalgic encephalomyelitis Human 49
Colitis – inflammatory bowel diseases Human 50, 51
Colitis Mouse 52
Environmental enteric dysfunction Human 53
Gestational diabetes Human 54, 55
Glioma Human 56
Glioma Cell 57
Insulin resistance Human 58
Irritable bowel syndrome Human 59, 60
Hyperlipidemia Human 61
HIV Human 62– 66
Major depressive disorders Human 67, 68
Multiple sclerosis Mouse 69
Multiple sclerosis Human 70
Necrotizing enterocolitis Rat 71
Necrotizing enterocolitis Human 72
Non-alcoholic fatty liver disease Human 73– 77
Non-celiac gluten sensitivity Human 53, 78
Obesity Human 79– 87
Schizophrenia Human 41, 88, 89
Sepsis Human 90
Type 1 diabetes Rat 91
Type 1 diabetes Human 92, 93
Type 2 diabetes Human 94, 95
Aging

Aging is the result of a constellation of cumulative changes that are deleterious, progressive, universal, and thus far irreversible. Aging damage can occur at the molecular (DNA, proteins, and lipids), cellular, or organ level (or a combination of these). Recent scientific successes in rejuvenation and extending a lifespan of model animals give hope to achieve negligible senescence, reverse aging or at least significantly delay it. Many non-mutually excluding theories have been formulated to model the senescence process, including the free radical theory 34 , the cellular senescence and apoptotic theory 35 , the immune system theory of aging 36 , and (pertinent to this article) the intestinal permeability and aging theory. Indeed, several reports in both animal models and humans link gut permeability to non-infective chronic inflammation and metabolic changes typical of the senescence process. In fruit fly, the increase in intestinal permeability leads to systemic metabolic defects and immune changes previously linked to aging and is the best predictor of imminent death, even more than the actual age of the insect 96 . In humans, it has been reported that zonulin serum concentration is higher in older adults, is positively associated with concentrations of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and intriguingly is negatively correlated with skeletal muscle strength and habitual physical activity 37 . These results suggest that zonulin-dependent gut permeability is associated with both systemic inflammation and two key indices of physical frailty associated with aging. These results have been corroborated by more recent data generated in disease-free ultra-centenarians showing lower levels of serum zonulin and endotoxemia as instigator of inflammation compared with young patients with acute myocardial infarction 38 , supporting the notion that, as in the fruit fly, gut permeability may impact lifespan expectations.

 Autoimmune disorders

Celiac disease. Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten-containing grains in genetically susceptible individuals and can be reversed when gluten is eliminated from the diet. As mentioned above, indigestible fragments of gluten are able to bind CXCR3 and release zonulin 27 . CD has been used as a model disorder to study the effect of zonulin since its involvement in the development and pathogenesis of the disease has been well documented 15– 20, 23– 27 . Even if gluten can trigger zonulin release in both healthy individuals and CD subjects, the amount and duration of zonulin produced are much higher in the latter group, leading to a significant increase in gut permeability, as shown by the capability of the zonulin inhibitor AT1001 (now named larazotide acetate) to prevent the zonulin permeating activity both in ex vivo models 43, 44 and in a transgenic animal model of CD in which it prevented gluten-dependent inflammation and intestinal damage 38 . Larazotide acetate has been tested in patients with CD, showing good safety and efficacy in preventing gluten-dependent inflammation 45– 48 , and is now in phase III clinical trial.

Type 1 diabetes. Type 1 diabetes (T1D) is an autoimmune condition caused by the destruction of the insulin-producing β cells of the pancreas, and the pathogenesis of this disease is still not fully understood. Several studies, in both animal models and T1D patients, have shown increased intestinal permeability to precede the development of T1D 97, 98 . In a recent elegant study, it was demonstrated that loss of gut barrier integrity was actually the causal factor for the microbiota-mediated T1D 99 in susceptible mice, further supporting the critical role of the gut barrier–microbiome–immune system triad in the pathogenesis of CID. BioBreeding diabetes-prone rats, which spontaneously develop T1D, have increased small intestinal permeability which precedes the loss of tolerance to glucose by at least one month 100 . Oral administration of the zonulin blocker AT1001 in these rats corrected the gut barrier defect and reduced the incidence of diabetes, suggesting a mechanistic role of the zonulin-dependent gut barrier modulation in the pathogenesis of T1D 91 . The involvement of zonulin in T1D was confirmed in human studies showing that about 50% of patients with T1D have increased serum zonulin levels, some of them showing these changes in the pre-diabetic phase of the disease 92 . Interestingly, a subset (about 25%) of healthy first-degree relatives of patients with T1D also showed increased serum zonulin 92 . Similar data were generated in children at risk of T1D in which zonulin correlated with Glo-3A antibodies (a potential biomarker of the disease) in cases (at-risk children in the pre-clinical phase [positive auto-antibodies] or overt T1D) but not in controls (at-risk children negative for auto-antibodies) 93 . Combined, these data suggest that zonulin may play a role in the pathogenesis of T1D in a subset of patients.

Inflammatory bowel disease. Increased intestinal permeability has been shown to play a crucial role in the pathogenesis of inflammatory bowel diseases (IBDs) 101– 105 . Arrieta et al., using the IL-10 knockout colitis mouse model, showed increased small intestinal permeability that preceded the onset of the overt colitis that can be ameliorated by oral treatment with zonulin inhibitor AT-1001 52 . In humans, serum and fecal zonulin were found to be elevated in patients with active Crohn’s disease but not with ulcerative colitis 50 . In a more recent study, serum zonulin concentration was found to be higher in both diseases, and an inverse correlation was observed between serum zonulin concentration and disease duration 51 .

Multiple sclerosis. In the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis (MS), zonulin-dependent increased intestinal permeability was shown during the pre-clinical phase of neurological symptoms, suggesting a role for zonulin in disease development 69 .

It has been reported that patients affected by MS show increased permeability of both the blood–brain barrier (BBB) and the intestine. A recent report showed that zonulin concentrations were significantly higher in MS patients showing a compromised BBB as shown by magnetic resonance imaging 70 . Interestingly, baseline zonulin concentrations were associated with 1-year disease progression in progressive MS and closely mirror BBB breakdown in relapsing remitting MS. Considering these results, the authors concluded that zonulin may be responsible for the breakdown of both the intestinal barrier and the BBB in gut dysbiosis, thereby explaining how the gut–brain axis modulates neuroinflammation in MS 70 .

Ankylosing spondylitis. Ankylosing spondylitis (AS) is an inflammatory, autoimmune condition that typically begins in young adulthood but often gets overlooked or is incorrectly diagnosed as pain from a previous injury or aging. It is an underdiagnosed form of arthritis that creates inflammation in the spinal joints and causes chronic back pain and stiffness. Dysbiosis has recently been demonstrated in patients with AS but its implications in the modulation of intestinal immune responses have never been studied. By analyzing ileal biopsies from patients with AS, Ciccia et al. showed that the presence of adherent and invasive bacteria in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation 39 . Impairment of the gut vascular barrier was also present in AS, accompanied by significant upregulation of zonulin, and was associated with high serum levels of LPS, LPS-binding protein, intestinal fatty acid–binding protein (iFABP), and zonulin 39 . In in vitro studies, zonulin altered endothelial TJs while its epithelial release was modulated by isolated AS ileal bacteria. Furthermore, they provided evidence that bacterial products and zonulin influence monocyte behavior. Considering these results, the authors concluded that bacterial ileitis, increased zonulin expression, and damaged intestinal mucosal epithelial and endothelial barriers characterize the gut of patients with AS and are associated with increased blood levels of zonulin and bacterial products.

 Metabolic disorders

Obesity. Obesity and its complications, including high cholesterol, type 2 diabetes (T2D), coronary heart disease, high blood pressure, and stroke, have been shown to be associated with chronic inflammation 106– 108 and frequently linked to alteration of the zonulin pathway, and more than 30 articles have been published on this topic. Several of these studies have shown elevated serum zonulin levels increased in obese versus non-obese subjects 79– 86 , and there is evidence of a correlation between total bacteria and serum zonulin levels, suggesting that the gut microbiota may cause increased zonulin levels, and subsequent abnormal gut permeability to endotoxin and ultimately micro-inflammation has been reported in obesity 106 . A recent report also showed that zonulin serum correlates with total calorie, protein, carbohydrate, sodium, and vitamin B 12 intake in obese women, and Ruminococcaceae and Faecalibacterium were more abundant in the low-zonulin group, suggesting that butyrate-producing gut bacteria such as Faecalibacteria could decrease gut permeability by decreasing zonulin levels and lower inflammation 87 .

Other metabolic disorders. Additional evidence suggests that zonulin is associated not only with obesity but also with its metabolic complications, including insulin resistance 58 , non-alcoholic fatty liver disease 73– 77 , gestational diabetes 54, 55 , hyperlipidemia 61 , and T2D 94, 95 .

 Intestinal diseases

Irritable bowel syndrome. Increased gut permeability has also been linked to the pathogenesis of irritable bowel syndrome (IBS) 59 . Specifically, patients with diarrhea-associated IBS showed increased serum zonulin levels 60 and involvement of the protease-activated receptor 2 (PAR2) 109, 110 , the zonulin target receptor 23 .

Non-celiac gluten sensitivity. Non-celiac gluten sensitivity (NCGS) is a clinical entity triggered by gluten as in CD but without autoimmune enteropathy 111 . It has been shown that patients with NCGS may have increased serum zonulin levels and increased intestinal permeability following gluten exposure 60, 78 .

Environmental enteric dysfunction. Environmental enteric dysfunction (EED) is a chronic disease affecting mainly the proximal intestine. It is characterized by loss of barrier function, bacterial overgrowth in the small intestine, and low-grade intestinal inflammation leading to small intestinal villous atrophy that, in some aspects, resembles CD enteropathy. The potential developmental consequences of EE/EED can be devastating to the full physical and neurocognitive development in one third of the world’s children growing up in impoverished areas. It was recently reported that serum zonulin levels and other markers of barrier dysfunction were correlated with stunted growth in patients with EED 53 .

 Cancer

There is growing evidence in the literature that Ag trafficking can also be involved in the immune component leading to the onset of a variety of cancers. Here are reported those conditions in which zonulin as a biomarker of epithelial and endothelial permeability has been associated with cancer.

Glioma. Zonulin has also been shown to be involved brain tumors, mainly gliomas 56, 57 . Increased zonulin expression of zonulin in gliomas correlated with the degree of malignancy and degradation of the BBB 56 . In vitro studies on a glioma cell line showed that zonulin was expressed in high amounts compared with non-glioma control cells 57 . Additionally, zonulin has been shown to induce transmigration of neuronal progenitor cells across the BBB 57 .

Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) globally ranks fifth for incidence and third for mortality among all malignant tumors. Although some progress has been made in exploring the pathological mechanisms and interventions of chronic liver diseases, there are still no effective biomarkers for the prediction and prevention of the progression of chronic liver diseases. Wang et al. recently reported that serum zonulin levels were significantly higher in patients with HCC compared with patients with liver cirrhosis or chronic hepatitis B or healthy subjects 112 . Moreover, the zonulin levels were increased in the advanced stage of liver cirrhosis and HCC.

 Neuroinflammatory diseases

Loss of gut barrier function (evaluated by serum IgG/IgA/IgM responses to occludin and zonulin and IgA responses to actomyosin) with subsequent increased serum levels of microbiota-derived molecules (assayed by testing serum lipopolysaccharides and bacterial toxins, including cytolethal distending toxin) and activation of the immune system (increased cytokines production) leading to neuroinflammation has been described in many neuroimmune disorders, including chronic fatigue syndrome, autism spectrum disorder (ASD), major depressive disorders (MDDs), and schizophrenia 32 .

Autism spectrum disorder. Increased serum zonulin that positively correlated with the Childhood Autism Rating Scale score has been reported in children with ASD 41 . In autism, as in other neuroinflammatory disorders, changes in zonulin-mediated gut permeability lead to pro-inflammatory status characterized by increased levels of mucosal pro-inflammatory cytokines (IL-5, IL-15, and IL-17) and decreased anti-inflammatory cytokines (transforming growth factor beta 1, or TGFβ1) detected mainly in ASD children experiencing gastrointestinal (GI) symptoms 42 .

Schizophrenia. Increased plasma IgA/IgM responses to Gram-negative bacteria have been reported in deficit schizophrenia 112 indicating leaky gut and gut dysbiosis. These results were confirmed more recently by Maes et al., who reported that the ratio of IgM to zonulin + occludin/talin + actin + viculin was significantly greater in patients with deficit schizophrenia than in those with non-deficit schizophrenia and higher in patients with schizophrenia than in controls and was significantly associated with increased IgA responses to Gram-negative bacteria 88 . IgM responses to zonulin were positively associated with schizophrenia (versus controls), whereas IgM to occludin was significantly associated with deficit schizophrenia (versus non-deficit schizophrenia and controls) 88 . The results show an upregulated paracellular pathway with breakdown of the tight and adherens junctions and increased bacterial translocation in deficit schizophrenia, suggesting their mechanistic role in causing neuroinflammation typical of the disease. These data were recently confirmed by our group 89 .

Major depressive disorders. Gut dysbiosis consistent with pathophysiological gut metagenomic signatures (upregulation LPS biosynthesis genes and deleterious metabolism of mood neurotransmitter pathways and host intestinal protective glycosaminoglycan mucins) different when compared with normal controls has been described in MDD 67 . Parallel to these changes, subjects affected by MDD showed increased plasma levels of LPS, zonulin, and FABP2 67 . Additionally, it has been demonstrated that, in patients with MDD, zonulin-mediated increased gut permeability causing increased bacterial translocation leads to marked alteration in circulating monocytes, with an expansion of the intermediate subset with increased frequency of IL-1β– and IL-6–producing cells. These changes are associated with a systemic pro-inflammatory state characterized by the enhanced serum TNF-α and IL-1β levels compared with those in the healthy controls 68 .

Chronic fatigue syndrome or myalgic encephalomyelitis. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is an illness characterized by profound and pervasive fatigue in addition to a heterogeneous constellation of symptoms. The etiology of this condition remains unknown; however, it has been suggested that enteric dysbiosis is implicated in the pathogenesis of CFS/ME 49 . In a systemic review, the microbiome composition of patients with CFS/ME was compared with that of healthy controls, showing statistically differences in some studies but with inconsistent findings in the studies considered 49 . Nevertheless, evidence of loss of barrier function in CFS/ME 32 is consistent with the overall theme of mutual influence between gut dysbiosis and intestinal barrier function.

 Conclusions

Besides genetic predisposition and exposure to environmental triggers, the pathogenesis of a variety of CIDs seems to involve mutually influenced changes in gut permeability/Ag trafficking, immune activation, and changes in composition/function of the gut microbiome. Zonulin is a modulator of both epithelial and endothelial barrier functions and its role in health and disease remains an object of active research. Gut dysbiosis may cause the release of zonulin leading to the passage of luminal contents across the epithelial barrier causing the release of pro-inflammatory cytokines that themselves cause increased permeability establishing a vicious loop leading to massive influx of dietary and microbial Ags triggering the activation of T cells. Depending on the host genetic makeup, activated T cells may remain within the GI tract, causing CID of the gut (IBD, IBS, CD, and EED), or migrate to several different organs to cause systemic CID. The effect of the zonulin inhibitor larazotide acetate in mitigating inflammation both in animal models and in human clinical trials not only confirms the pathogenic role of zonulin in many CIDs but also opens the possibility of targeting gut permeability in a variety of CIDs in which a pathogenic role for zonulin has been hypothesized or proven.

 Abbreviations

Ag, antigen; AS, ankylosing spondylitis; ASD, autism spectrum disorder; BBB, blood–brain barrier; CD, celiac disease; CFS/ME, chronic fatigue syndrome/myalgic encephalomyelitis; CID, chronic inflammatory disease; EED, environmental enteric dysfunction; ELISA, enzyme-linked immunosorbent assay; GI, gastrointestinal; HCC, hepatocellular carcinoma; HP, haptoglobin; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; IL, interleukin; JAM, junctional adhesion molecule; LPS, lipopolysaccharide; MASP, mannose-binding lectin-associated serine protease; MDD, major depressive disorder; MS, multiple sclerosis; NCGS, non-celiac gluten sensitivity; T1D, type 1 diabetes; T2D, type 2 diabetes; TJ, tight junction; TNF-α, tumor necrosis factor alpha; ZO-1, zonula occludens 1

 Ege MJ : The Hygiene Hypothesis in the Age of the Microbiome. Ann Am Thorac Soc. 2017;14(Supplement_5):S348–S353. 29161087 10.1513/AnnalsATS.201702-139AW Haahtela T : A biodiversity hypothesis. Allergy. 2019;74(8):1445–56. 30835837 10.1111/all.13763

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Chieppa M Rescigno M Huang AY : Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med. 2006;203(13):2841–52. 17145958 10.1084/jem.20061884 2118178

F1000 Recommendation

Mowat AM Millington OR Chirdo FG : Anatomical and cellular basis of immunity and tolerance in the intestine. J Pediatr Gastroenterol Nutr. 2004;39 Suppl 3:S723–4. 15167360 10.1097/00005176-200406003-00003 Rescigno M Lopatin U Chieppa M : Interactions among dendritic cells, macrophages, and epithelial cells in the gut: implications for immune tolerance. Curr Opin Immunol. 2008;20(6):669–75. 18852045 10.1016/j.coi.2008.09.007 Arrieta MC Bistritz L Meddings JB : Alterations in intestinal permeability. Gut. 2006;55(1):1512–20. 16966705 10.1136/gut.2005.085373 1856434 Fasano A Shea-Donohue T : Mechanisms of disease: the role of intestinal barrier function in the pathogenesis of gastrointestinal autoimmune diseases. Nat Clin Pract Gastroenterol Hepatol. 2005;2(9):416–22. 16265432 10.1038/ncpgasthep0259 Turner JR : Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9(11):799–809. 19855405 10.1038/nri2653 Itoh M Nagafuchi A Yonemura S : The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol. 1993;121(3):491–502. 8486731 10.1083/jcb.121.3.491 2119563 Furuse M Hirase T Itoh M : Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123(6 Pt 2):1777–88. 8276896 10.1083/jcb.123.6.1777 2290891 Furuse M Fujita K Hiiragi T : Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141(7):1539–50. 9647647 10.1083/jcb.141.7.1539 2132999 Martìn-Padura I Lostaglio S Schneemann M : Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998;142(1):117–27. 9660867 10.1083/jcb.142.1.117 2133024 Ikenouchi J Furuse M Furuse K : Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol. 2005;171(6):939–45. 16365161 10.1083/jcb.200510043 2171318

F1000 Recommendation

Higashi T Tokuda S Kitajiri S : Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis. J Cell Sci. 2013;126(Pt 4):966–77. 23239027 10.1242/jcs.116442

F1000 Recommendation

Fasano A Not T Wang W : Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 2000;355(9214):1518–9. 10801176 10.1016/S0140-6736(00)02169-3 Wang W Uzzau S Goldblum SE : Human zonulin, a potential modulator of intestinal tight junctions. J Cell Sci. 2000;113 Pt 24:4435–40. 11082037 Sturgeon C Fasano A : Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers. 2016;4(4):e1251384. 28123927 10.1080/21688370.2016.1251384 5214347 Fasano A : Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiol Rev. 2011;91(1):151–75. 21248165 10.1152/physrev.00003.2008 Valitutti F Fasano A : Breaking Down Barriers: How Understanding Celiac Disease Pathogenesis Informed the Development of Novel Treatments. Dig Dis Sci. 2019;64(7):1748–58. 31076989 10.1007/s10620-019-05646-y 6586517 Tripathi A Lammers KM Goldblum S : Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc Natl Acad Sci U S A. 2009;106(39):16799–804. 19805376 10.1073/pnas.0906773106 2744629 Rittirsch D Flierl MA Nadeau BA : Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system. Am J Physiol Lung Cell Mol Physiol. 2013;304(12):L863–72. 23564505 10.1152/ajplung.00196.2012 3680747 Scheffler L Crane A Heyne H : Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Front Endocrinol (Lausanne). 2018;9:22. 29459849 10.3389/fendo.2018.00022 5807381 El Asmar R Panigrahi P Bamford P : Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology. 2002;123(5):1607–15. 12404235 10.1053/gast.2002.36578 Drago S El Asmar R Di Pierro M : Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scand J Gastroenterol. 2006;41(4):408–19. 16635908 10.1080/00365520500235334 Thomas KE Sapone A Fasano A : Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in Celiac disease. J Immunol. 2006;176(4):2512–21. 16456012 10.4049/jimmunol.176.4.2512 Clemente MG De Virgiliis S Kang JS : Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function. Gut. 2003;52(2):218–23. 12524403 10.1136/gut.52.2.218 1774976 Lammers KM Lu R Brownley J : Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008;135(1):194–204.e3. 18485912 10.1053/j.gastro.2008.03.023 2653457

F1000 Recommendation

Su L Le Shen, Clayburgh DR : Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis. Gastroenterology. 2009;136(2):551–63. 19027740 10.1053/j.gastro.2008.10.081 2712351 Laukoetter MG Nava P Lee WY : JAM-A regulates permeability and inflammation in the intestine in vivo. J Exp Med. 2007;204(13):3067–76. 18039951 10.1084/jem.20071416 2150975

F1000 Recommendation

Naydenov NG Feygin A Wang D : Nonmuscle Myosin IIA Regulates Intestinal Epithelial Barrier in vivo and Plays a Protective Role During Experimental Colitis. Sci Rep. 2016;6:24161. 27063635 10.1038/srep24161 4827066 Sturgeon C Lan J Fasano A : Zonulin transgenic mice show altered gut permeability and increased morbidity/mortality in the DSS colitis model. Ann N Y Acad Sci. 2017;1397(1):130–42. 28423466 10.1111/nyas.13343 5479715 Ajamian M Steer D Rosella G : Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems. PLoS One. 2019;14(1):e0210728. 30640940 10.1371/journal.pone.0210728 6331146

F1000 Recommendation

Simeonova D Ivanovska M Murdjeva M : Recognizing the Leaky Gut as a Trans-diagnostic Target for Neuroimmune Disorders Using Clinical Chemistry and Molecular Immunology Assays. Curr Top Med Chem. 2018;18(19):1641–55. 30430944 10.2174/1568026618666181115100610

F1000 Recommendation

Dodig S Čepelak I Pavić I : Hallmarks of senescence and aging. Biochem Med (Zagreb). 2019;29(3):030501. 31379458 10.11613/BM.2019.030501 6610675

F1000 Recommendation

Soto-Gamez A Quax WJ Demaria M : Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. J Mol Biol. 2019;431(15):2629–43. 31153901 10.1016/j.jmb.2019.05.036

F1000 Recommendation

Prieto LI Baker DJ : Cellular Senescence and the Immune System in Cancer. Gerontology. 2019;65(5):505–12. 31212284 10.1159/000500683 6703936

F1000 Recommendation

Qi Y Goel R Kim S : Intestinal Permeability Biomarker Zonulin is Elevated in Healthy Aging. J Am Med Dir Assoc. 2017;18(9):810.e1–810.e4. 28676292 10.1016/j.jamda.2017.05.018 5581307

F1000 Recommendation

Carrera-Bastos P Picazo Ó Fontes-Villalba M : Serum Zonulin and Endotoxin Levels in Exceptional Longevity versus Precocious Myocardial Infarction. Aging Dis. 2018;9(2):317–321. 29896420 10.14336/AD.2017.0630 5963352

F1000 Recommendation

Ciccia F Guggino G Rizzo A : Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis. Ann Rheum Dis. 2017;76(6):1123–32. 28069576 10.1136/annrheumdis-2016-210000 6599509 Özyurt G Öztürk Y Appak YÇ : Increased zonulin is associated with hyperactivity and social dysfunctions in children with attention deficit hyperactivity disorder. Compr Psychiatry. 2018;87:138–42. 30414552 10.1016/j.comppsych.2018.10.006

F1000 Recommendation

Esnafoglu E Cırrık S Ayyıldız SN : Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects. J Pediatr. 2017;188:240–4. 28502607 10.1016/j.jpeds.2017.04.004

F1000 Recommendation

Rose DR Yang H Serena G : Differential immune responses and microbiota profiles in children with autism spectrum disorders and co-morbid gastrointestinal symptoms. Brain Behav Immun. 2018;70:354–68. 29571898 10.1016/j.bbi.2018.03.025 5953830 Gopalakrishnan S Tripathi A Tamiz AP : Larazotide acetate promotes tight junction assembly in epithelial cells. Peptides. 2012;35(1):95–101. 22401910 10.1016/j.peptides.2012.02.016 Gopalakrishnan S Durai M Kitchens K : Larazotide acetate regulates epithelial tight junctions in vitro and in vivo. Peptides. 2012;35(1):86–94. 22401908 10.1016/j.peptides.2012.02.015 Paterson BM Lammers KM Arrieta MC : The safety, tolerance, pharmacokinetic and pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of concept study. Aliment Pharmacol Ther. 2007;26(5):757–66. 17697209 10.1111/j.1365-2036.2007.03413.x

F1000 Recommendation

Kelly CP Green PH Murray JA : Larazotide acetate in patients with coeliac disease undergoing a gluten challenge: a randomised placebo-controlled study. Aliment Pharmacol Ther. 2013;37(2):252–62. 23163616 10.1111/apt.12147

F1000 Recommendation

Leffler DA Kelly CP Abdallah HZ : A randomized, double-blind study of larazotide acetate to prevent the activation of celiac disease during gluten challenge. Am J Gastroenterol. 2012;107(10):1554–62. 22825365 10.1038/ajg.2012.211 3463856 Leffler DA Kelly CP Green PH : Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomized controlled trial. Gastroenterology. 2015;148(7):1311–9.e6. 25683116 10.1053/j.gastro.2015.02.008 4446229 Du Preez S Corbitt M Cabanas H : A systematic review of enteric dysbiosis in chronic fatigue syndrome/myalgic encephalomyelitis. Syst Rev. 2018;7(1):241. 30572962 10.1186/s13643-018-0909-0 6302292

F1000 Recommendation

Malíčková K Francová I Lukáš M : Fecal zonulin is elevated in Crohn's disease and in cigarette smokers. Pract Lab Med. 2017;9:39–44. 29034305 10.1016/j.plabm.2017.09.001 5633835

F1000 Recommendation

Caviglia GP Dughera F Ribaldone DG : Serum zonulin in patients with inflammatory bowel disease: a pilot study. Minerva Med. 2019;110(2):95–100. 30160088 10.23736/S0026-4806.18.05787-7

F1000 Recommendation

Arrieta MC Madsen K Doyle J : Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse. Gut. 2009;58(1):41–8. 18829978 10.1136/gut.2008.150888 2597688 Guerrant RL Leite AM Pinkerton R : Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil. PLoS One. 2016;11(9):e0158772. 27690129 10.1371/journal.pone.0158772 5045163 Mokkala K Tertti K Rönnemaa T : Evaluation of serum zonulin for use as an early predictor for gestational diabetes. Nutr Diabetes. 2017;7(3):e253. 28319108 10.1038/nutd.2017.9 5380900 Demir E Ozkan H Seckin KD : Plasma Zonulin Levels as a Non-Invasive Biomarker of Intestinal Permeability in Women with Gestational Diabetes Mellitus. Biomolecules. 2019;9(1): pii: E24. 30641999 10.3390/biom9010024 6359541

F1000 Recommendation

Skardelly M Armbruster FP Meixensberger J : Expression of Zonulin, c-kit, and Glial Fibrillary Acidic Protein in Human Gliomas. Transl Oncol. 2009;2(3):117–20. 19701495 10.1593/tlo.09115 2730142 Díaz-Coránguez M Segovia J López-Ornelas A : Transmigration of neural stem cells across the blood brain barrier induced by glioma cells. PLoS One. 2013;8(4):e60655. 23637756 10.1371/journal.pone.0060655 3618035 Moreno-Navarrete JM Sabater M Ortega F : Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance. PLoS One. 2012;7(5):e37160. 22629362 10.1371/journal.pone.0037160 3356365 Camilleri M Gorman H : Intestinal permeability and irritable bowel syndrome. Neurogastroenterol Motil. 2007;19(7):545–52. 17593135 10.1111/j.1365-2982.2007.00925.x Barbaro MR Cremon C Caio G : The role of zonulin in non-celiac gluten sensitivity and irritable bowel syndrome. United Euro Gastroenterol J. 2015;3:A87. Reference Source Ohlsson B Orho-Melander M Nilsson P : Higher Levels of Serum Zonulin May Rather Be Associated with Increased Risk of Obesity and Hyperlipidemia, Than with Gastrointestinal Symptoms or Disease Manifestations. Int J Mol Sci. 2017;18(3): pii: E582. 28282855 10.3390/ijms18030582 5372598 Funderburg N Boucher M Sattar A : Rosuvastatin Decreases Intestinal Fatty Acid Binding Protein (I-FABP), but Does Not Alter Zonulin or Lipopolysaccharide Binding Protein (LBP) Levels, in HIV-Infected Subjects on Antiretroviral Therapy. Pathog Immun. 2016;1(1):118–128. 27500282 10.20411/pai.v1i1.124 4972492 Koay WLA Lindsey JC Uprety P : Intestinal Integrity Biomarkers in Early Antiretroviral-Treated Perinatally HIV-1-Infected Infants. J Infect Dis. 2018;218(7):1085–9. 29762690 10.1093/infdis/jiy271 6107740 Utay NS Somasunderam A Hinkle JE : Serum Bovine Immunoglobulins Improve Inflammation and Gut Barrier Function in Persons with HIV and Enteropathy on Suppressive ART. Pathog Immun. 2019;4(1):124–46. 31139758 10.20411/pai.v4i1.276 6508431

F1000 Recommendation

Pastor L Langhorst J Schröder D : Different pattern of stool and plasma gastrointestinal damage biomarkers during primary and chronic HIV infection. PLoS One. 2019;14(6):e0218000. 31185037 10.1371/journal.pone.0218000 6559643

F1000 Recommendation

Dirajlal-Fargo S El-Kamari V Weiner L : Altered intestinal permeability and fungal translocation in Ugandan children with HIV. Clin Infect Dis. 2019;pii: ciz561. 31260509 10.1093/cid/ciz561

F1000 Recommendation

Stevens BR Goel R Seungbum K : Increased human intestinal barrier permeability plasma biomarkers zonulin and FABP2 correlated with plasma LPS and altered gut microbiome in anxiety or depression. Gut. 2018;67(8):1555–1557. 28814485 10.1136/gutjnl-2017-314759 5851874

F1000 Recommendation

Alvarez-Mon MA Gómez AM Orozco A : Abnormal Distribution and Function of Circulating Monocytes and Enhanced Bacterial Translocation in Major Depressive Disorder. Front Psychiatry. 2019;10:812. 31803077 10.3389/fpsyt.2019.00812 6873610

F1000 Recommendation

Nouri M Bredberg A Weström B : Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells. PLoS One. 2014;9(9):e106335. 25184418 10.1371/journal.pone.0106335 4153638 Camara-Lemarroy CR Silva C Greenfield J : Biomarkers of intestinal barrier function in multiple sclerosis are associated with disease activity. Mult Scler. 2019:1352458519863133. 31317818 10.1177/1352458519863133

F1000 Recommendation

Ling X Linglong P Weixia D : Protective Effects of Bifidobacterium on Intestinal Barrier Function in LPS-Induced Enterocyte Barrier Injury of Caco-2 Monolayers and in a Rat NEC Model. PLoS One. 2016;11(8):e0161635. 27551722 10.1371/journal.pone.0161635 4995054 Tarko A Suchojad A Michalec M : Zonulin: A Potential Marker of Intestine Injury in Newborns. Dis Markers. 2017;2017:2413437. 28769143 10.1155/2017/2413437 5523403 Pacifico L Bonci E Marandola L : Increased circulating zonulin in children with biopsy-proven nonalcoholic fatty liver disease. World J Gastroenterol. 2014;20(45):17107–14. 25493023 10.3748/wjg.v20.i45.17107 4258579 Cakir M Aksel İşbilen A Eyüpoğlu İ : Effects of long-term synbiotic supplementation in addition to lifestyle changes in children with obesity-related non-alcoholic fatty liver disease. Turk J Gastroenterol. 2017;28(5):377–83. 28797988 10.5152/tjg.2017.17084 Kim JH Heo JS Baek KS : Zonulin level, a marker of intestinal permeability, is increased in association with liver enzymes in young adolescents. Clin Chim Acta. 2018;481:218–24. 29544751 10.1016/j.cca.2018.03.005

F1000 Recommendation

Kim AS Ko HJ : Plasma concentrations of zonulin are elevated in obese men with fatty liver disease. Diabetes Metab Syndr Obes. 2018;11:149–57. 29719415 10.2147/DMSO.S163062 5922244

F1000 Recommendation

Kwon JH Lee HG Seo KH : Combination of Whole Grapeseed Flour and Newly Isolated Kefir Lactic Acid Bacteria Reduces High-Fat-Induced Hepatic Steatosis. Mol Nutr Food Res. 2019;63(4):e1801040. 30537110 10.1002/mnfr.201801040 Volta U Caio G Tovoli F : Non-celiac gluten sensitivity: Questions still to be answered despite increasing awareness. Cell Mol Immunol. 2013;10(5):383–92. 23934026 10.1038/cmi.2013.28 4003198 Zak-Gołąb A Kocełak P Aptekorz M : Gut microbiota, microinflammation, metabolic profile, and zonulin concentration in obese and normal weight subjects. Int J Endocrinol. 2013;2013:674106. 23970898 10.1155/2013/674106 3732644 Kuzma JN Cromer G Hagman DK : No differential effect of beverages sweetened with fructose, high-fructose corn syrup, or glucose on systemic or adipose tissue inflammation in normal-weight to obese adults: a randomized controlled trial. Am J Clin Nutr. 2016;104(2):306–14. 27357093 10.3945/ajcn.115.129650 4962158 Mokkala K Röytiö H Munukka E : Gut Microbiota Richness and Composition and Dietary Intake of Overweight Pregnant Women Are Related to Serum Zonulin Concentration, a Marker for Intestinal Permeability. J Nutr. 2016;146(9):1694–700. 27466607 10.3945/jn.116.235358 Stenman LK Lehtinen MJ Meland N : Probiotic With or Without Fiber Controls Body Fat Mass, Associated With Serum Zonulin, in Overweight and Obese Adults—Randomized Controlled Trial. EBioMedicine. 2016;13:190–200. 27810310 10.1016/j.ebiom.2016.10.036 5264483 Gil-Cardoso K Ginés I Pinent M : Effects of flavonoids on intestinal inflammation, barrier integrity and changes in gut microbiota during diet-induced obesity. Nutr Res Rev. 2016;29(2):234–48. 27841104 10.1017/S0954422416000159 Küme T Acar S Tuhan H : The Relationship between Serum Zonulin Level and Clinical and Laboratory Parameters of Childhood Obesity. J Clin Res Pediatr Endocrinol. 2017;9(1):31–8. 28008865 10.4274/jcrpe.3682 5363162 Houttu N Mokkala K Laitinen K : Overweight and obesity status in pregnant women are related to intestinal microbiota and serum metabolic and inflammatory profiles. Clin Nutr. 2018;37(6 Pt A):1955–66. 29338886 10.1016/j.clnu.2017.12.013

F1000 Recommendation

Kuzma JN Hagman DK Cromer G : Intraindividual Variation in Markers of Intestinal Permeability and Adipose Tissue Inflammation in Healthy Normal-Weight to Obese Adults. Cancer Epidemiol Biomarkers Prev. 2019;28(3):610–5. 30573517 10.1158/1055-9965.EPI-18-0641 6401228

F1000 Recommendation

Mörkl S Lackner S Meinitzer A : Gut microbiota, dietary intakes and intestinal permeability reflected by serum zonulin in women. Eur J Nutr. 2018;57(8):2985–97. 30043185 10.1007/s00394-018-1784-0 6267414

F1000 Recommendation

Maes M Sirivichayakul S Kanchanatawan B : Upregulation of the Intestinal Paracellular Pathway with Breakdown of Tight and Adherens Junctions in Deficit Schizophrenia. Mol Neurobiol. 2019;56(10):7056–73. 30972627 10.1007/s12035-019-1578-2

F1000 Recommendation

Barber GS Sturgeon C Fasano A : Elevated zonulin, a measure of tight-junction permeability, may be implicated in schizophrenia. Schizophr Res. 2019;211:111–2. 31307857 10.1016/j.schres.2019.07.006 Klaus DA Motal MC Burger-Klepp U : Increased plasma zonulin in patients with sepsis. Biochem Med (Zagreb). 2013;23(1):107–11. 23457771 10.11613/bm.2013.013 3900088 Watts T Berti I Sapone A : Role of the intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-prone rats. Proc Natl Acad Sci U S A. 2005;102(8):2916–21. 15710870 10.1073/pnas.0500178102 549484

F1000 Recommendation

Sapone A de Magistris L Pietzak M : Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes. 2006;55(5):1443–9. 16644703 10.2337/db05-1593 Simpson M Mojibian M Barriga K : An exploration of Glo-3A antibody levels in children at increased risk for type 1 diabetes mellitus. Pediatr Diabetes. 2009;10(8):563–72. 19622083 10.1111/j.1399-5448.2009.00541.x 2814050 Zhang D Zhang L Zheng Y : Circulating zonulin levels in newly diagnosed Chinese type 2 diabetes patients. Diabetes Res Clin Pract. 2014;106(2):312–8. 25238913 10.1016/j.diabres.2014.08.017 Jayashree B Bibin YS Prabhu D : Increased circulatory levels of lipopolysaccharide (LPS) and zonulin signify novel biomarkers of proinflammation in patients with type 2 diabetes. Mol Cell Biochem. 2014;388(1–2):203–10. 24347174 10.1007/s11010-013-1911-4 Rera M Clark RI Walker DW : Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc Natl Acad Sci U S A. 2012;109(52):21528–33. 23236133 10.1073/pnas.1215849110 3535647

F1000 Recommendation

Carratù R Secondulfo M de Magistris L : Altered intestinal permeability to mannitol in diabetes mellitus type I. J Pediatr Gastroenterol Nutr. 1999;28(3):264–9. 10067726 10.1097/00005176-199903000-00010 De Magistris L Secondulfo M Iafusco D : Altered mannitol absorption in diabetic children. Ital J Gastroenterol. 1996;28(6):367. 8891852 Sorini C Cosorich I Lo Conte M : Loss of gut barrier integrity triggers activation of islet-reactive T cells and autoimmune diabetes. Proc Natl Acad Sci U S A. 2019;116(30):15140–9. 31182588 10.1073/pnas.1814558116 6660755

F1000 Recommendation

Meddings JB Jarand J Urbanski SJ : Increased gastrointestinal permeability is an early lesion in the spontaneously diabetic BB rat. Am J Physiol. 1999;276(4):G951–7. 10198339 10.1152/ajpgi.1999.276.4.G951 Buhner S Buning C Genschel J : Genetic basis for increased intestinal permeability in families with Crohn's disease: role of CARD15 3020insC mutation? Gut. 2006;55(3):342–7. 16000642 10.1136/gut.2005.065557 1856071

F1000 Recommendation

Büning C Geissler N Prager M : Increased small intestinal permeability in ulcerative colitis: rather genetic than environmental and a risk factor for extensive disease? Inflamm Bowel Dis. 2012;18(10):1932–9. 22344959 10.1002/ibd.22909 Peeters M Geypens B Claus D : Clustering of increased small intestinal permeability in families with Crohn's disease. Gastroenterology. 1997;113(3):802–7. 9287971 10.1016/s0016-5085(97)70174-4 Teahon K Smethurst P Levi AJ : Intestinal permeability in patients with Crohn's disease and their first degree relatives. Gut. 1992;33(3):320–3. 1568650 10.1136/gut.33.3.320 1373820 Wyatt J Vogelsang H Hübl W : Intestinal permeability and the prediction of relapse in Crohn's disease. Lancet. 1993;341(8858):1437–9. 8099141 10.1016/0140-6736(93)90882-h Olszanecka-Glinianowicz M Chudek J Kocełak P : Body fat changes and activity of tumor necrosis factor α system--a 5-year follow-up study. Metabolism. 2011;60(4):531–6. 20580040 10.1016/j.metabol.2010.04.023 Olszanecka-Glinianowicz M Zahorska-Markiewicz B Janowska J : Serum concentrations of nitric oxide, tumor necrosis factor (TNF)-alpha and TNF soluble receptors in women with overweight and obesity. Metabolism. 2004;53(10):1268–73. 15375781 10.1016/j.metabol.2004.07.001 Zahorska-Markiewicz B Janowska J Olszanecka-Glinianowicz M : Serum concentrations of TNF-alpha and soluble TNF-alpha receptors in obesity. Int J Obes Relat Metab Disord. 2000;24(11):1392–5. 11126333 10.1038/sj.ijo.0801398 Linsalata M Riezzo G D’Attoma B : Noninvasive biomarkers of gut barrier function identify two subtypes of patients suffering from diarrhoea predominant-IBS: a case-control study. BMC Gastroenterol. 2018;18(1):167. 30400824 10.1186/s12876-018-0888-6 6219148

F1000 Recommendation

Singh P Silvester J Chen X : Serum zonulin is elevated in IBS and correlates with stool frequency in IBS-D. United European Gastroenterol J. 2019;7(5):709–15. 31210949 10.1177/2050640619826419 6545708

F1000 Recommendation

Bueno L : Protease activated receptor 2: a new target for IBS treatment. Eur Rev Med Pharmacol Sci. 2008;12 Suppl 1:95–102. 18924448 Wang X Li Mm Niu Y : Serum Zonulin in HBV-Associated Chronic Hepatitis, Liver Cirrhosis, and Hepatocellular Carcinoma. Dis Markers. 2019;2019:5945721. 31485278 10.1155/2019/5945721 6710742

F1000 Recommendation