Intestinal Epithelial Junction Research Articles

L. plantarum and L. casei synergistically mitigate Gastro-Intestinal Acute Radiation Syndrome (GI-ARS)

Introduction: Incidental or accidental acute exposure to ionizing radiation causes radiation toxicity, for which there are no known medical countermeasures (MCMs). Ionizing radiation alters host gut epithelium as well as the gut microbiome. We sought to investigate if radiation induced gut dysbiosis occurred as a result of depletion of gut-protective bacteria, and if probiotic supplementation of such bacteria could help mitigate GI-ARS post ionizing radiation exposure. Methods: Adult C57BL/6 mice were subjected to total body irradiation, and a defined concentration of probiotics (either individually or combined) were given 24 h after radiation exposure. Mice were sacrificed 24 hours post treatment (i.e., 48 h post irradiation). Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by RT-PCR for cytokine expression. Systemic inflammation was evaluated by ELISA for plasma cytokines. Results and conclusion: Our data show that radiation altered the taxonomic composition of the microbiota compared to age-matched sham treated mice. Shannon index analysis showed a significant decrease in α-diversity in irradiated mice. Bray-Curtis analysis indicated significant differences in microbiota composition between Sham and IR groups. E. coli abundance was increased whereas Lactobacillus abundance was decreased, particularly L. plantarum and L. casei. Probiotic supplementation with individual Lactobacillus species each mitigated radiation induced intestinal barrier dysfunction, gut specific as well as systemic inflammatory cytokine expression and endotoxemia. Our preliminary data further indicates that L. plantarum affects Epidermal Growth Factor Receptor (EGFR)-mediated signaling whereas L. casei was EGFR-independent. Interestingly, combined supplementation in a 1:1 ratio further enhanced recovery compared to adding the individual effects. Individual supplementation of L. plantarum and L. casei to irradiated mice reduced intestinal (ileal) permeability by 2-fold and 3-fold respectively, whereas combined supplementation reduced ileal permeability almost 8-fold compared to irradiated but untreated mice. This data indicates that L. plantarum and L. casei mitigate GI-ARS by distinct cellular mechanisms. Furthermore, data show that a mixture of these probiotic species is more effective in radio-mitigation than individually. Further mechanistic analysis will be required to identify cross-talking targets between mechanisms of these probiotics and synergistic radio-mitigation. In conclusion, Lactobacillus species are potential MCMs for the treatment of GI-ARS. Funding acknowledgements: NIH/NIAID, UO1-AI170019, UO1-AI172991. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3213: A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers

Abstract Targeting immune checkpoints like programmed death receptor 1 (PD-1) with monoclonal antibodies has achieved durable responses in a range of cancers, however, immune-related adverse events may occur in a subset of patients. As such, we developed a bispecific antibody targeting cadherin-17 (CDH17) and PD-1 with a hope to rejuvenate specifically the exhausted T cells within tumor microenvironment. In healthy individuals, CDH17 is expressed at a restricted level in intestinal epithelial junction that is not accessible to biologics, however, in gastrointestinal cancers, CDH17 is overexpressed and can be targeted by antibodies. Our new CDH17/PD-1 bispecific antibody would reactivate anti-tumor immunity with reduced risk of on-target off-tumor toxicity. Different bispecific antibody formats were designed, cloned, and expressed. Strong binders were first identified using CDH17 and PD-1 ELISA. The binding affinities to CDH17 and PD-1 of the selected candidate were examined using biolayer interferometry. The effect of these bispecific antibodies on blocking PD-1/PD-L1 axis in T cells was demonstrated in a cell-based reporter assay. T-cell mediated killing on CDH17-positive GI cancer cells and induction of T cell activation markers upon antibody treatment were also studied. A total of 7 CDH17/PD-1 bispecific antibodies of different formats were screened. Of these candidates, one, named ARB204, was selected because of its potent binding to both antigens in ELISA (i.e., EC50 values: CDH17, 1.6nM; PD-1, 0.4nM) and biolayer interferometry (i.e., KD values: CDH17, 2.4E-09M; PD-1, 6.02E-11M). ARB204 blocked PD-1/PD-L1 axis in the reporter assay with EC50 value 4.4nM. Notably, ARB204 redirected T cells to eradicate CDH17-positive pancreatic AsPC1 cells with an EC50 value of 3.5nM. No cytotoxicity against CDH17-negative cancer cells were seen. ARB204 effectively bound CDH17 on GI cancer cells and PD-1 on T cells. This bispecific design allowed selective T-cell mediated killing on cancer cells expressing CDH17, mitigating the risk of off-tumor toxicity. ARB204 warrants further studies on its immune checkpoint inhibition in animal models. Citation Format: Kwan Wa Wong, Po Yee Wong, Kronos Chow, Chui Yee O, Dennis Wong, John Moon Luk, Kwong Fai Wong. A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3213.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

TRPV6 deficiency attenuates stress and corticosterone-mediated exacerbation of alcohol-induced gut barrier dysfunction and systemic inflammation.

Chronic stress is co-morbid with alcohol use disorder that feedback on one another, thus impeding recovery from both disorders. Stress and the stress hormone corticosterone aggravate alcohol-induced intestinal permeability and liver damage. However, the mechanisms involved in compounding tissue injury by stress/corticosterone and alcohol are poorly defined. Here we explored the involvement of the TRPV6 channel in stress (or corticosterone) 3and alcohol-induced intestinal epithelial permeability, microbiota dysbiosis, and systemic inflammation. Chronic alcohol feeding was performed on adult wild-type and Trpv6-/- mice with or without corticosterone treatment or chronic restraint stress (CRS). The barrier function was determined by evaluating inulin permeability in vivo and assessing tight junction (TJ) and adherens junction (AJ) integrity by immunofluorescence microscopy. The gut microbiota composition was evaluated by 16S rRNA sequencing and metagenomic analyses. Systemic responses were assessed by evaluating endotoxemia, systemic inflammation, and liver damage. Corticosterone and CRS disrupted TJ and AJ, increased intestinal mucosal permeability, and caused endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. Corticosterone and CRS synergistically potentiated the alcohol-induced breakdown of intestinal epithelial junctions, mucosal barrier impairment, endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. TRPV6 deficiency also blocked the effects of CRS and CRS-mediated potentiation of alcohol-induced dysbiosis of gut microbiota. These findings indicate an essential role of TRPV6 in stress, corticosterone, and alcohol-induced intestinal permeability, microbiota dysbiosis, endotoxemia, systemic inflammation, and liver injury. This study identifies TRPV6 as a potential therapeutic target for developing treatment strategies for stress and alcohol-associated comorbidity.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator.

BackgroundGut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation (EBT), resulting in serious complications, such as systemic inflammatory response syndrome, sepsis and multiple organ failure. Cystic fibrosis transmembrane conductance regulator (CFTR) is known to be downregulated by hypoxia and modulate junctional complexes, which are crucial structures maintaining the intestinal barrier. This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn, as well as the signaling pathways involved in these processes.MethodsAn in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30% total body surface area full-thickness dermal burn were established. DF 508 mice (mice with F508del CFTR gene mutation) were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function. QRT-PCR, western blot, ELISA, TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins, as well as the function of tight junctions.ResultsOur data indicated that, in Caco-2 cells, the hypoxia condition significantly reduced CFTR expression; activated extracellular signal-regulated kinase and nuclear factor-κB signaling; elevated secretion of inflammatory factors (tumor necrosis factor-α, interleukin-1β and interleukin-8); downregulated zonula occludens-1, occludin and E-cadherin expression; decreased transepithelial electrical resistance values; and led to a cellular mislocation of ZO-1. More importantly, knockdown of CFTR caused similar alterations. The upregulation of inflammatory factors and downregulation of tight junction proteins (ZO-1 and occludin) induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition. In support of the in vitro data, exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo. EBT occurred in DF508 mice (mice with the F508del CFTR gene mutation), accompanied by augmented tumor necrosis factor-α, interleukin-1β and interleukin-8 levels in the ileum compared to wildtype mice. In addition, vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury.ConclusionsCollectively, the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.

Giardia extracellular vesicles disrupt intestinal epithelial junctions and inhibit the growth of commensal bacteria while increasing their swimming motility

IntroductionExtracellular vesicles (EVs) have emerged as important mediators of host‐parasite interactions. The protozoan parasite Giardia duodenalis, the most common cause of parasitic diarrheal diseases worldwide, produces EVs in response to changes in the environment. The exact role of EVs in the pathogenesis of giardiasis remains unclear. In this study, we investigate the effects of EVs on the intestinal barrier such as the intestinal epithelium and the gut commensal bacteria. Disruption of the intestinal barrier has been implicated in the development of post‐giardiasis gut disorders and EVs may represent a novel mechanism of this disruption.AimsThe aim of this research is to characterize Giardia EVs and investigate their effect on the host epithelium and commensal gut bacteria.MethodsG. duodenalis (isolate NF) trophozoites were either incubated with 10X bile for 1 hour or left untreated and EVs were isolated using Qiagen Exo‐Easy Maxi Kit. The concentration and size of EVs were assessed using Nanosight track analysis (NTA). Proteomic analysis of EVs was conducted using liquid chromatography with tandem mass spectrometry. The effects of Giardia EVs on the host epithelium were assessed by exposing epithelial primary cell line (SCBN) to EVs for 24 hours and examining the tight junction proteins ZO‐1 and claudin‐4 by fluorescence microscopy. Antibacterial effect of Giardia EVs was assessed on gut commensals such as Escherichia coli strain HB101 and strain K12, Enterobacter cloacae (human isolate) and Enterococcus faecalis (human isolate) by measuring bacterial kinetic growth for 12 hours. Swimming motility of non‐invasive commensals was assessed on 0.3% agar after incubating with EVs for 24 hours and the halo sizes were measured as a marker of swimming behavior.ResultsOur findings indicate that bile treated Giardia trophozoites produce more EVs as compared to untreated trophozoites. NTA revealed that Giardia EVs range around 200 nm. Proteomic analysis of EVs revealed the presence of well characterized Giardia virulence factors such as cysteine proteases, tenascins, oxidative defense enzymes, variant surface proteins as well as metabolic enzymes such as arginine deaminase and ornithine carbamoyl transferase. Additionally, exposure of SCBN cells to EVs resulted in the disruption and/or rearrangement of tight junction proteins Zo‐1 and Claudin‐4. Moreover, our results also indicate that Giardia EVs exert bacteriostatic effects on E. coli strains HB101, K12, E. cloacae and E. faecalis. Finally, Giardia EVs also significantly increased the swimming motility of non‐invasive commensals E. coli strain HB101 and E. cloacae.ConclusionOur research highlights a novel mechanism of Giardia’s interaction with the intestinal barrier. Giardia EVs contain virulence factors that disrupt the host’s intestinal epithelium and modify the growth and motility of commensal bacteria. These effects may have significant implications in disease pathophysiology.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC)

42 CFTR Mediates Enteric Bacterial Translocation Post Severe Burn via ERK and Inflammatory Signaling

Abstract Introduction This research was to investigate the role of cystic fibrosis transmembrane conductance regulator (CFTR) in mediating enteric bacterial translocation (EBT) post severe burn as well as the signaling pathways involved. Methods A hypoxia treatment model of Caco-2 in vitro and a 30% TBSA full-thickness dermal burn mice model in vivo were established to explore the effect of major burn injury on intestinal epithelial barrier and the expression of CFTR and tight junction proteins. In addition, the DF508 mice (mice with F508del CFTR gene mutation) were used to analyze bacterial counts in extraintestinal organs and the expression of ZO-1, OCLN and E-cadherin. And CFTR specific inhibitor or down-regulation of CFTR in Caco-2 cells were used to explore the effect of CFTR on tight junction of intestinal cells. Furthermore, in the models above, MAPK pathway and inflammatory factors were detected to explore the mechanism. Finally, Vitamin D3 was used to pretreat mice before severe burn or Caco-2 cells before hypoxia. Bacterial counts in extraintestinal organs and the expression of CFTR, ZO-1, OCLN and P-ERK as well as the concentrations of inflammatory factors were detected. Results Our data showed a decrease of CFTR, concomitant with activation of ERK signaling, elevation of inflammatory factors (TNF-α, IL-1β and IL-8) and declined expression of ZO-1, occludin and E-cadherin, and a cellular mislocation of ZO-1 and OCLN post hypoxia. Furthermore, either administration of CFTR specific inhibitor or down-regulation of CFTR caused similar alterations as hypoxia did. Ileum of DF508 -/- and +/- mice exhibited increased inflammation, declined ZO-1, occludin and E-cadherin and EBT compared to +/+ mice. Moreover, vitamin D3 could reverse the junctional damage induced by hypoxia, thus preventing occurrence of EBT, possibly via up-regulating CFTR. Conclusions CFTR/ERK/inflammatory factors signaling was critical in modulating intestinal epithelial junction, accordingly regulating EBT. Vitamin D3 can protect intestinal epithelial barrier after severe burn, which may be associated with the up-regulation of CFTR, the inhibition of ERK pathway and decreased pro-inflammatory cytokine. Applicability of Research to Practice This research may be used in practice in the future to regulate the metabolism after severe burns.

Severe neonatal anemia increases intestinal permeability by disrupting epithelial adherens junctions.

Anemia is a frequent diagnosis in critically ill infants, but the clinical implications of severe anemia in these patients remain unclear. In this study, we examined preweaned mice to investigate the effects of severe anemia during early infancy on gut mucosal permeability. C57BL/6 mice were subjected to timed phlebotomy between postnatal days (P) 2-10 to induce severe anemia (hematocrits 20%-24%), and intestinal permeability was tracked longitudinally between P10 and P20 as intestine-to-plasma translocation of enteral macromolecules and bacterial translocation. Epithelial junctions were evaluated by electron microscopy, polymerase chain reactions, immunohistochemistry, and/or enzyme immunoassays on intestinal tissues, Caco-2 intestinal epithelial-like cells, and colonic organoids. Preweaned mouse pups showed an age-related susceptibility to severe anemia, with increased intestinal permeability to enteral macromolecules (dextran, ovalbumin, β-lactoglobulin) and luminal bacteria. Electron micrographs showed increased paracellular permeability and ultrastructural abnormalities of the adherens junctions. These findings were explained by the loss of E-cadherin in epithelial cells, which was caused by destabilization of the E-cadherin (Cdh1) mRNA because of microRNA let-7e-5p binding to the 3'-untranslated region. Severe anemia resulted in a disproportionate and persistent increase in intestinal permeability in preweaned mice because of the disruption of epithelial adherens junctions. These changes are mediated via microRNA let-7e-mediated depletion of Cdh1 mRNA.NEW & NOTEWORTHY This research article shows that newborn infants with severe anemia show an age-related susceptibility to developing increased intestinal permeability to ingested macromolecules. This abnormal permeability develops because of abnormalities in intestinal epithelial junctions caused by a deficiency of the molecule E-cadherin in epithelial cells. The deficiency of E-cadherin is caused by destabilization of its mRNA precursor because of increased expression and binding of another molecule, the microRNA let-7e-5p, to the E-cadherin mRNA.

Bacillus thuringiensis targets the host intestinal epithelial junctions for successful infection of Caenorhabditis elegans.

Pathogenic bacteria use different strategies to infect their hosts, including the simultaneous production of pore forming toxins and several virulence factors that may synergize their pathogenic effects. However, how the pathogenic bacteria are able to break out the host intestinal barrier is poorly understood. The infectious cycle of Bacillus thuringiensis (Bt) bacterium in Caenorhabditis elegans is a powerful model system to study the early stages of the infection process. Bt produces Cry pore-forming toxins during the sporulation phase that are key virulence factors involved in its pathogenesis. In this study, we show that Bt disrupts the intestinal epithelial junctions of C. elegans at early stages of infection allowing Bt bacterium to complete its life cycle in the worm. We further confirmed that the vegetative Bt cells trigger a quorum sensing response that is activated by PlcR regulator, resulting in production of different virulence factors, such as the metalloproteinases ColB and Bmp1, that besides Cry toxins are necessary to disrupt the nematode epithelial junctions causing efficient bacterial host infection and death of the nematode. Our work provides new insights into the pathogenesis of Bt and highlights the importance of breaking down host epithelial junctions for a successful infection. A similar mechanism could be used by other pathogen-host interactions since epithelial junctions are conserved structures from insects to mammals.

Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation

The actin cytoskeleton is a crucial regulator of the intestinal mucosal barrier, controlling the assembly and function of epithelial adherens and tight junctions (AJs and TJs). Junction-associated actin filaments are dynamic structures that undergo constant turnover. Members of the actin-depolymerizing factor (ADF) and cofilin protein family play key roles in actin dynamics by mediating filament severing and polymerization. We examined the roles of ADF and cofilin-1 in regulating the structure and functions of AJs and TJs in the intestinal epithelium. Knockdown of either ADF or cofilin-1 by RNA interference increased the paracellular permeability of human colonic epithelial cell monolayers to small ions. Additionally, cofilin-1, but not ADF, depletion increased epithelial permeability to large molecules. Loss of either ADF or cofilin-1 did not affect the steady-state morphology of AJs and TJs but attenuated de novo junctional assembly. The observed defects in AJ and TJ formation were accompanied by delayed assembly of the perijunctional filamentous actin belt. A total loss of ADF expression in mice did not result in a defective mucosal barrier or in spontaneous gut inflammation. However, ADF-null mice demonstrated increased intestinal permeability and exaggerated inflammation during dextran sodium sulfate-induced colitis. Our findings demonstrate novel roles for ADF and cofilin-1 in regulating the remodeling and permeability of epithelial junctions, as well as the role of ADF in limiting the severity of intestinal inflammation.

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.

Disruption of the epithelial barrier upon Shigella flexneri infection

Shigella is one of the most communicable bacterial pathogens and causes the disease shigellosis. Shigella invades the colonic mucosa from the basolateral surface, but how Shigella gets to the basolateral domain is incompletely understood. We have shown that S. flexneri alters the cell‐cell junction complex facilitating paracellular passage of the organism across the epithelial barrier. To understand the mechanism by which this bacterium alters the intestinal epithelial junctions, we focused on SepA, a bacterial protein associated with tissue invasion and destruction upon infection. To test the hypothesis that SepA is involved in altering the intestinal epithelial barrier function, polarized monolayers of human intestinal epithelia or xenotransplanted human intestine were infected with wild‐type S. flexneri or an isogenic SepA mutant strain. We found that absence of SepA lead to a less severe disturbance on the intestinal barrier, which correlate with not only a decrease in bacterial invasion, but also significant reduction of neutrophils transepithelial migration. Functionally, we found SepA to elicit cleavage of the intracellular domain of E‐cadherin, which we speculate contributes to the observed cell‐cell junction permeability, and in turns permits S. flexneri access to the basolateral domain to invade and trigger the intense inflammatory response, a hallmark of this disease.NIH

Berberine Ameliorates Intestinal Epithelial Tight-Junction Damage and Down-regulates Myosin Light Chain Kinase Pathways in a Mouse Model of Endotoxinemia

This study aimed to examine the protective effect of berberine in endotoxin-induced intestinal tight-junction injury in a mice model of endotoxinemia. Endotoxinemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Mice were randomized to 5 groups: control mice, berberine-treated mice, lipopolysaccharide (LPS)-injected mice, mice pretreated with berberine, and mice administered berberine following LPS injection. Samples were collected 12 h after LPS treatment. Ileal mucosal permeability to fluorescein isothiocyanate dextran assay indicated that berberine reduced the permeability of the gut barrier in endotoxinemia. Transmission electron microscopy revealed that pretreatement with berberine partly prevented ultrastructural disruption of tight junctions by LPS. Immunofluorescence and Western blot analysis were performed, and the results demonstrated that pretreatement with berberine partially reversed the redistribution of tight-junction proteins in colon epithelium and in membrane microdomains. Our data also indicated that pretreatement with berberine could suppress translocation, from cytoplasm to the nucleus, of nuclear factor-κB and myosin light chain kinase activation in the intestinal epithelium. Pretreatement with berberine attenuates disruption of tight junctions in intestinal epithelium in a mice model of endotoxinemia. This may possibly have been mediated through down-regulation of the nuclear factor-κB and myosin light chain kinase pathway.

Intestinal epithelial barrier integrity requires Desmoglein2‐mediated adhesion

The integrity of intercellular junctions in intestinal epithelium is crucial for sealing the intestinal barrier. While specific roles of tight and adherens junctions are well known, the contribution of desmosomal adhesion for maintaining the intestinal epithelial barrier has not been addressed. We generated a desmoglein 2 antibody directed against the extracellular domain (Dsg2 ED) to test whether impaired Dsg2‐mediated adhesion affects intestinal barrier functions in vitro. This antibody was capable to block Dsg2 interaction in cell‐free atomic force microscopy experiments. For in vitro studies of the intestinal barrier we used Caco2 cells following differentiation into enterocyte‐like epithelial monolayers. Application of Dsg2 ED to Caco2 monolayers increased cell dissociation compared to controls. Under similar conditions Dsg2 antibody significantly decreased transepithelial electrical resistance. As revealed by immunostaining, this was due to Dsg2 ED antibody‐induced loss of tight junction proteins from the cell borders. Similar results were obtained using a commercial monoclonal antibody against the extracellular domain of Dsg2 to Caco2 monolayers. Antibody‐induced effects were blocked by absorption experiments. Our data indicate that Dsg2‐mediated adhesion affects tight junction integrity and is required to maintain intestinal epithelial barrier properties.