Regulation Of Epithelial Barrier Function Research Articles

Acidosis regulation of epithelial barrier function and immune signaling

A common but often underappreciated aspect of the inflammatory microenvironment is extracellular acidification. As with other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients. Colonic pH as low as 4 have been reported in IBD patients. In contrast, the pH of a healthy colon is between a pH of 7.0‐7.4. Recently, we identified an adaptive tissue response in intestinal epithelial cells (IEC) which buffered extracellular acidification during neutrophil transepithelial migration, suggesting pH homeostasis is an important aspect of acute inflammation. Extensions of these studies identified a previously undescribed CREB‐dependent pathway for sensing extracellular pH and a unique acidosis gene signature in IEC. The full impact of acidosis on IEC function remains unclear. To better understand the impact of chronic extracellular acidification on IEC function, we investigated changes in IEC immune signaling and barrier function in the setting of acute and chronic exposure to acidosis. We initially examined barrier function in T84 IEC cultured under acidic conditions and observed a significant decrease in barrier function, as determined by transepithelial electrical resistance (TER) and paracellular flux, in cells exposed to acidosis in a pH range of 6.0 – 7.4. Interestingly, following acute acidification, treatment with low pH for less than 8hr, barrier function recovered within 16hr. However, following chronic acidification, treatment with low pH beyond 16hr resulted in an appreciable barrier dysfunction for several days. We extended these studies to examine expression and localization of tight‐junction proteins in IEC exposed to acidosis and observed a time and dose‐dependent decrease in ZO‐1 and E‐cadherin expression, with almost complete loss of expression after 72hr at a pH of 6.0. Likewise, expression and localization of claudins 1, 2, and 4 decreased over time. In parallel to a loss of barrier function, IEC exposed to chronic acidosis revealed a prominent increase in the expression of inflammatory markers including several chemokines (IL‐8, CCL20, TNF) as well as anti‐microbial peptides (mucins and beta‐defensins).Thus, chronic extracellular acidosis induces the loss of IEC tight junction function, while inducing the expression of several pro‐inflammatory chemokines and the production of various anti‐microbial peptides. These results identify extracellular acidification as significant signaling mechanism which induces a loss of epithelial features and the acquisition of a more immune‐like phenotype. Furthering our understandings of the mechanisms involved in acidosis induced phenotype switching in IEC has the potential to uncover novel therapeutic drug targets.

Interleukin-28A induces epithelial barrier dysfunction in CD patient-derived intestinal organoids.

Intestinal barrier dysfunction is a pathogenic hallmark in Crohn's disease (CD). Identifying key players that regulate intestinal barrier may provide novel leads for therapeutic intervention. Interleukin-28A (IL-28A) is a newly identified IL-10/interferon cytokine family member, with its most implicated function being antiviral and anti-proliferative properties. However, the role and underlying mechanisms of IL-28A in the regulation of epithelial barrier in CD remain so far unexplored. IL-28A levels were measured in the plasma and biopsies of CD patients and healthy subjects. CD patient-derived intestinal organoids were characterized by differentiation gene markers and then exposed to TNF-α, IFN-γ, IL-1β or LPS, or IL-28A with or without GLPG0634 (filgotinib). Epithelial permeability was assessed by FITC-D4 flux. Expression of junctional components was analyzed by qRT-PCR, immunofluorescence staining, or Western blotting. JAK-STAT activity was analyzed by Western blotting. IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IL-28A and its receptor complex IL-28AR/IL-10R2 were detected in CD patient-derived intestinal organoids and showed a selective response to IFN-γ exposure. IL-28A triggered epithelial barrier disruption and accompanied by reduced ZO-1 and E-cadherin expression. This effect was mediated by JAK-STAT1 pathway. Pre-incubation with the JAK1 inhibitor filgotinib ameliorated the barrier dysfunction induced by IL-28A. These results identified IL-28A as a novel regulator of epithelial barrier function and could be a putative target for CD treatment. We provide novel basic evidence that restoring intestinal barrier is a potential mechanism that contributes to the clinical benefits of JAK1 inhibitor in patients with CD.NEW & NOTEWORTHY IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IFN-γ exposure stimulated IL-28A expression in intestinal organoids. Partially mimicking the effect of IFN-γ, IL-28A impaired epithelial barrier function and disrupted junctional components through the activation of JAK-STAT1 signaling, whereas JAK1 inhibitor ameliorated the above-mentioned effects of IL-28A. These findings highlight the newly identified cytokine IL-28A as a novel contributor to CD pathogenesis and could be a putative target for CD treatment. We also provide new evidence for potential applications of JAK inhibition in CD therapy.

Exopolysaccharides from Lactobacillus plantarum NCU116 Regulate Intestinal Barrier Function via STAT3 Signaling Pathway.

Lactic acid bacteria (LAB) and their exopolysaccharides (EPS) are recognized to promote intestinal barrier function by mechanisms that remain incompletely understood. Herein, we sought to identify the roles of exopolysaccharides from Lactobacillus plantarum NCU116 (EPS116) in intestinal barrier function. Our data showed that EPS116 attenuated dextran sodium sulfate (DSS) induced colitis and promoted epithelial barrier function and the expression of tight junction (TJ) proteins in vivo and in vitro. Moreover, chromatin immunoprecipitation data showed that EPS116 facilitated STAT3 (signal transducer and activator of transcription 3) binding to the promoter of occludin and ZO-1. Furthermore, knockdown of STAT3 in Caco-2 cell with EPS116 treatment led to decreased expression of occludin and ZO-1 and increased intestinal permeability, suggesting that the regulation of epithelial barrier function by EPS116 should be STAT3 dependent. Thus, our data revealed a novel mechanism that EPS116 inhibited intestinal inflammation via regulating intestinal epithelial barrier function.

Microbiota Composition and the Integration of Exogenous and Endogenous Signals in Reactive Nasal Inflammation.

The prevalence of reactive nasal inflammatory conditions, for example, allergic rhinitis and chronic rhinosinusitis, is steadily increasing in parallel with significant environmental changes worldwide. Allergens and as yet undefined environmental agents may trigger these conditions via the involvement of host intrinsic factors, including the innate and adaptive immune system, the nasal epithelium, and the nasal nervous system. The critical role of the nasal microbiota in coordinating these components has emerged in recent studies documenting a significant association between microbial composition and the onset and progression of allergic or nonallergic inflammation. It is now clear that the local microbiota is a major player in the development of the mucosa-associated lymphoid tissue and in the regulation of such adaptive responses as IgA production and the function of effector and regulatory T cells. Microbial components also play a major role in the regulation of epithelial barrier functions, including mucus production and the control of paracellular transport across tight junctions. Bacterial components, including lipopolysaccharide, have also been shown to induce or amplify neuroinflammatory responses by engaging specific nociceptors. Finally, bacterial products may promote tissue remodeling processes, including nasal polyp formation, by interacting with formyl peptide receptors and inducing the expression of angiogenic factors and matrix-degrading enzymes.

Colonic bile acids regulate epithelial wound healing

BackgroundEpithelial restitution is an essential process for maintenance of intestinal barrier function. While previously published data have shown bile acids to be involved in regulation of epithelial barrier function in in vivo models of colonic inflammation, their roles in regulating restitution are not yet known. Here, we investigated the effects of bile acids on epithelial restitution and the molecular pathways involved in colonic epithelial healing.MethodsWounds were created by pinch biopsy in C5BL/6 mice during colonic endoscopy. Animals received daily topical application of deoxycholic acid (DCA; 150 μM) or the “therapeutic” bile acid, ursodeoxycholic acid (UDCA; 150 μM). Images of the wounds were taken at day 2 and day 6 post wounding and the area of the wounds was measured by Image J ®software. T84 colonic epithelial cells, grown as monolayers on transparent permeable supports, were wounded at T=0. Cells were treated with DCA (50 – 150 μM), UDCA (50 – 150 μM, a farnesoid X receptor (FXR) agonist, GW4064 (10 μM), or the cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers, CFTR(inh)‐172 (10 μM) or GlyH‐101 (25 μM). Restitution was measured as wound area after 48 h and expressed as % T=0 wound area. HEK‐293 cells were transfected with vectors expressing FXR and the luciferase gene under control of the CFTR promoter. Protein expression was assessed by western blotting, mRNA levels were assessed by q‐PCR and cell migration by Boyden chamber assay.ResultsWound healing was significantly delayed in animals treated with DCA compared to control (Day 6 Wound Area −0.41 ± 0.15 mm2, p= 0.0287, N=5). DCA also inhibited wound closure in cultured epithelial monolayers and attenuated cell migration in Boyden chamber assays, without significantly altering cell proliferation. Nuclear accumulation of FXR was detected upon DCA stimulation, while activation of FXR by GW4064 inhibited epithelial wound closure to 35.1 ± 1.1%, compared to 51.6 ± 5 % in vehicle‐treated controls (n = 5; p < 0.05) and decreased T84 cell migration in Boyden chambers to 57 ± 9.6 % (n = 3; p <0.05) of that in controls. To address the mechanism by which DCA exerts its action on wound healing, levels of CFTR expression was measured. Both, DCA and GW4064, attenuated CFTR mRNA levels, along with total and surface CFTR protein expression, whereas promoter/reporter assays revealed that transcriptional downregulation of CFTR by DCA is mediated in an FXR specific manner. Furthermore, inhibition of CFTR activity with either CFTR(inh)‐172 or GlyH‐101 mimicked the effects of FXR on wound healing. In contrast, UDCA (50 – 150 μM) enhanced wound healing in both in vitro and in vivo models and prevented the effects of DCA in cell migration and restitution assays.ConclusionsIn conclusion, these studies suggest that bile acids are important regulators of epithelial wound healing and are therefore good targets for the development of new drugs to modulate intestinal barrier function in disease treatment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract TP402: The Cerebral Cavernous Malformation Disease Causing Gene Krit1 Participates in Epithelial Barrier Maintenance and Regulation

Background: Cerebral cavernous malformation (CCM) patients develop mulberry-like microvessels in the brain. These lesions can bleed, causing clinical symptoms. Germline mutations of CCM genes have been identified in familial form of CCM, and decreased endothelial CCM protein expression leads to RhoA-Rho kinase (ROCK) dependent actin cytoskeleton reorganization and increased endothelial permeability. It is not known if CCM mutations, which occur in all cells of a familial CCM patient, can impact barrier function of other body cavity lining cells, such as epithelial cells. Understanding how CCM protein function in epithelia may help us identify risks of CCM patients to epithelium-related diseases. Aim: To determine if and how the CCM gene KRIT1/CCM1 affects epithelial barrier maintenance and regulation. Methods: KRIT1 knockdown (KD) intestinal epithelial Caco-2 cells were generated by stably transfecting an anti-KRIT1 siRNA expressing plasmid. Epithelial barrier function, measured as transepithelial resistance (TER) and size selective cation permeability was determined by current clamp. Protein expression and localization was determined by western blot and immunofluorescent microscopy, respectively. Results: KRIT1 KD intestinal epithelial Caco-2 cell monolayers have decreased TER and selectively increased permeability to small cations, along with decreased claudin-1expression. ROCK inhibition (Y-27632) and myosin motor inhibition (blebbistatin) both decreased TER in control Caco-2 monolayers, but not in KRIT1 KD monolayers, indicating KRIT1 is important for myosin regulation of epithelial barrier function. Tumor necrosis factor (TNF), a cytokine that decreases epithelial barrier function through inducing myosin contraction, caused more severe barrier loss in KRIT1 KD monolayers relative to control monolayers, which is blocked by pan-caspase inhibition (zVAD), indicating KRIT1 loss promotes TNF-induced barrier loss through inducing cell death. Conclusion: KRIT1 regulates epithelial barrier by affecting tight junction protein expression and impacting myosin- and apoptosis- dependent processes. These changes in epithelial function may increase familial CCM patients’ susceptibility to intestinal and other diseases.

Smad nuclear interacting protein 1 (SNIP1) inhibits intestinal inflammation through regulation of epithelial barrier function.

Smad nuclear interacting protein 1 (SNIP1) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms involved are still largely unknown. Our results demonstrated that SNIP1 was markedly decreased in intestinal epithelial cells (IEC) from IBD patients compared with healthy controls. Impaired expression of SNIP1 caused a significant decrease of transepithelial electrical resistance but an increase of fluorescein isothiocyanate-dextran flux in Caco-2 monolayers, whereas overexpression of SNIP1 reversed such effects. Overexpression of SNIP1 also inhibited the activity of NF-κB p65 and proinflammatory cytokine production (e.g., TNF-α, IL-1β, and IL-8) by IEC. Importantly, supplementation of exogenous SNIP1 significantly ameliorated intestinal mucosal inflammation in experimental colitis, characterized by less-severe intestinal epithelial barrier damage and decreased proinflammatory cytokine production. Our data thus demonstrated a novel mechanism whereby SNIP1 regulates intestinal inflammation through modulating intestinal epithelial barrier function. Targeting SNIP1 may provide a therapeutic approach for the treatment of IBD.

Vitamin D3 induces vitamin D receptor and HDAC11 binding to relieve the promoter of the tight junction proteins.

Intestinal epithelial barrier dysfunction and vitamin D (VitD)-deficiency play a critical role in a large number of diseases. The histone deacetylases (HDAC) are associated with a large number of immune diseases. This study tests a hypothesis that the interaction between VitD and HDAC is associated with the regulation of epithelial barrier functions. In this study, human intestinal epithelial cell line, T84 cells, was cultured into monolayers to be used as a model to test the epithelial barrier functions. We observed that in a VitD-deficient environment, the T84 monolayer barrier function was compromised. Exposure to calcitriol (the active form of VitD3) in the culture increased the expression of VitD receptor (VDR) in T84 cells. In a VitD-sufficient environment, VDR formed a complex with histone deacetylase-11 (HDAC11); the complex was markedly decreased in a VitD-deficient environment. We also observed that significantly more binding of HDAC11 to the promoter of the tight junction proteins inhibit the gene transcription activities of these loci in the VitD-deficient environment, which were abolished by the presence of calcitriol in the culture. In conclusion, the interaction between VDR and HDAC11 plays a crucial role in the maintenance of the epithelial barrier integrity.

Myosin IXb variants and their pivotal role in maintaining the intestinal barrier: A study in Crohn’s disease

Background. Myosin IXb (MYO9B) is involved in the regulation of epithelial barrier function. We hypothesized that MYO9B variants are associated with increased intestinal permeability measured in patients with Crohn’s disease (CD), where barrier dysfunction is crucially involved in disease development. Methods. We sequenced MYO9B and genotyped five MYO9B variants (rs1545620, rs1457092, rs2279003, rs2305764 and rs2279002) and correlated these data to measurement of intestinal permeability in German CD patients (n = 122) obtained by standard oral sugar test using the lactulose/mannitol ratio after measurement of urinary excretion. We furthermore studied MYO9B variants in three European cohorts with inflammatory bowel disease (IBD) and healthy controls : Germany (CD = 264; ulcerative colitis = 143 [UC]; HC = 372); Hungary (CD = 147; UC = 117; HC = 195), the Netherlands (CD = 157; HC = 219). Results. We found an association for four studied MYO9B variants to an increased intestinal permeability in CD patients (rs1545620, p = 0.010; rs1457092, p = 0.024; rs2279003, p = 0.003; rs2305764, p = 0.015). Furthermore, we observed significantly higher absolute values of intestinal permeability for individuals carrying risk alleles within MYO9B. Looking for an overall disease association, only the rs2305764 variant was associated with CD in the Dutch cohort (p = 0.004), but not in the German or Hungarian cohort. No association to UC or a distinct phenotype in both CD and UC patients was observed for all studied MYO9B variants. Conclusion. Our data suggest a link between MYO9B variants to an increased intestinal permeability in CD patients. This supports the influence of Myosin IXb on the integrity of the epithelial barrier. The role of MYO9B variants in the overall susceptibility to IBD, however, remains to be elucidated.

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin-Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.

Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.

JAM‐A –mediated regulation of epithelial barrier function is dependent on a complex with ZO‐2, Afadin and PDZ‐GEF1 that modulates Rap2c activity.

Intestinal mucosal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional Adhesion Molecule A (JAM‐A) is a TJ‐associated protein known to regulate epithelial barrier, however mechanisms linking JAM‐A to regulation of permeability are incompletely understood. Proteomic, biochemical and cell‐biological approaches were used to identify candidate proteins involved in JAM‐A regulation of epithelial permeability. Our findings suggest that JAM‐A directly binds to the scaffold molecule ZO‐2 and indirectly associates with Afadin and PDZ‐GEF1 to regulate activation of the small GTPase Rap2c. A link to regulation of barrier was confirmed by siRNA‐mediated downregulation of these regulatory proteins leading to enhanced permeability. Since ZO‐2, Afadin and Rap2c have been implicated in cytoskeletal dynamics, we investigated whether JAM‐A regulation of barrier function is mediated through peri‐junctional actomyosin. While photobleaching experiments suggest that JAM‐A does not regulate actin turnover, JAM‐A deficient cells display enhanced activation of RhoA, a known inducer of actomyosin contraction. These findings suggest that JAM‐A regulates epithelial permeability through association with a multimolecular signaling complex that activates Rap2c, modulating contraction of the peri‐junctional actin cytoskeleton.

Neutrophil Interactions With ICAM-1 at the Luminal Surface of the Intestinal Epithelium Enhance Neutrophil Survival and Alter Epithelial Barrier Function

Neutrophil transepithelial migration (TEM) is a hallmark of inflammatory bowel disease (IBD), and is associated with epithelial injury and impaired barrier function. While TEM has been extensively modeled, little is known about neutrophil interactions with ligands expressed on the luminal surface of the intestinal epithelium. ICAM-1 is a well-recognized signaling receptor and endothelial ligand for migrating neutrophils. Its expression is also induced on the luminal (apical) membrane of intestinal epithelial cells (IEC) exposed to interferon γ (IFNγ), and in the colonic mucosa of individuals with active IBD. However, the role of ICAM-1 in mediating functional effects of neutrophil interactions with intestinal epithelial cells is undefined. ICAM-1 expression in cultured IECs and mouse intestinal epithelium was induced by exposure to IFNγ (100U/mL, 24h) and IFNγ+TNFα (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. We confirmed upregulation of ICAM-1 expression in IFNγ treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNγ and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority (˜60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNγ. Neutrophil interactions with apical epithelial membrane of IFNγ pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran (˜2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran (˜1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD.

Defective epithelial barrier in chronic rhinosinusitis: The regulation of tight junctions by IFN-γ and IL-4

Chronic rhinosinusitis (CRS) is a common disease with still unclear pathophysiologic mechanisms. Epithelial tight junctions (TJs) have been shown to be involved in different chronic disorders, including bronchial asthma, inflammatory bowel diseases, and skin disorders. The regulation of epithelial barrier function and TJ expression has not been extensively studied in patients with CRS and in the paranasal sinus epithelium thus far. We sought to elucidate the TJ expression pattern in the epithelium of the sinonasal mucosa and its regulation in patients with CRS. Trans-tissue resistance was measured in biopsy specimens from healthy control subjects and patients with CRS with and without nasal polyps. TJ protein expression was determined by using immunofluorescence, Western blotting, and real-time PCR. Primary epithelial cell cultures from patients with CRS and control subjects were used in air-liquid interface (ALI) cultures for the measurement of transepithelial resistance (TER) and TJ expression. The effect of IFN-γ, IL-4, and IL-17 on ALI cultures was assessed. A decreased trans-tissue resistance was found in biopsy specimens from patients with CRS with nasal polyps along with an irregular, patchy, and decreased expression of the TJ molecules occludin and zonula occludens 1. TER was reduced in ALI cultures from patients with CRS with nasal polyps. The cytokines IFN-γ and IL-4 decreased TER, whereas IL-17 did not have any influence on epithelial integrity. A defective epithelial barrier was found in patients with CRS with nasal polyps along with a decreased expression of TJ proteins. The disruption of epithelial integrity by IFN-γ and IL-4 in vitro indicates a possible role for these proinflammatory cytokines in the pathogenesis of patients with CRS.

Regulation of epithelial barrier function by Crohn's disease associated gene PTPN2

The aim of this study was to determine whether there is a synergistic disrupting effect between IFN and TNF on intestinal epithelial barrier function as well as whether this effect is influenced by the knockdown of Crohn's disease associated PTPN2 gene.

Regulation of epithelial barrier function by the inflammatory bowel disease candidate gene, PTPN2

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has been identified as an inflammatory bowel disease (IBD) candidate gene. However, the mechanism through which mutations in the PTPN2 gene contribute to the pathogenesis of IBD has not been identified. PTPN2 acts as a negative regulator of signaling induced by the proinflammatory cytokine, interferon-gamma (IFN-γ). IFN-γ is known not only to play an important role in the pathogenesis of Crohn's disease (CD), but also to increase permeability of the intestinal epithelial barrier. We have shown that PTPN2 protects epithelial barrier function by restricting the capacity of IFN-γ to increase epithelial permeability and prevent induction of expression of the pore-forming protein, claudin-2. These data identify an important functional role for PTPN2 as a protector of the intestinal epithelial barrier and provide clues as to how PTPN2 mutations may contribute to the pathophysiology of CD.

Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.

Metabolic Stress Evokes Decreases in Epithelial Barrier Function

The epithelial lining of the gastrointestinal tract is the major interface between the external world (e.g., the gut lumen) and the body, and as such the proper maintenance and regulation of epithelial barrier function is a key determinant of digestive health and host well-being. Many enteropathies are associated with increased gut permeability, including inflammatory bowel disease (IBD). Maintaining the barrier function of the epithelium, independent of whether paracellular or transcellular permeation pathways are considered, is an energy-dependent process. Here we present an overview of the impact that metabolic stress (e.g., reductions in epithelial ATP synthesis) can have on permeability characteristics of epithelial monolayers and show that metabolic stress in the presence of a commensal flora results in a significant loss of epithelial integrity, and that this increase in epithelial permeability can be enhanced by the presence of tumor necrosis factor-alpha (TNFalpha). We speculate that the combination of these factors in vivo would result in significant perturbations in epithelial barrier function that could be of pathophysiological significance and contribute to the initiation of IBD or the induction of disease relapses.

Zonulin is not increased in the cardiac and esophageal mucosa of patients with gastroesophageal reflux disease

Human Zonulin, related to the Zonula occludens toxin of Vibrio cholerae, regulates intestinal permeability and is induced in inflammatory disorders of the lower GI tract. Gastroesophageal reflux disease (GERD) is associated with an impairment of epithelial barrier function. Here, we studied expression of zonulin in the gastroesophageal mucosa of 58 patients with typical reflux symptoms and 27 asymptomatic controls. During endoscopy, multiple biopsies from gastroesophageal mucosa were obtained for routine histopathology ( Helicobacter pylori-status, inflammation) and gene expression analysis (immunohistochemistry, ELISA). Patients with GERD presented with typical histopathological alterations like elongation of papillae ( P = 0.015), basal cell hyperplasia ( P < 0.001) and dilatation of intercellular spaces ( P = 0.002). Zonulin was found to be expressed ubiquitously in gastroesophageal mucosa. Mucosal levels in controls ranged between 2.2 and 3.7 ng/μg total protein. Mean values were significantly higher in antrum (3.3 ± 1.7 ng/μg) than cardia (2.7 ± 1.2 ng/μg) and esophagus (2.2 ± 1.3 ng/μg) ( P < 0.001), but did not differ between GERD and controls for cardia and esophageal mucosa. Immunohistochemistry revealed that predominantly epithelial cells but not stromal cells contribute to the zonulin expression in gastroesophageal mucosa. In conclusion, despite its established role for intestinal permeability, Zonulin seems not to be involved in the regulation of epithelial barrier function in relation to GERD.

Shear stress regulates aquaporin-5 and airway epithelial barrier function

As the interface with the outside world, the airway epithelial barrier is critical to lung defense. Because of respiratory efforts, the airways are exposed to shear stress; however, little is known regarding the effects of shear on epithelial function. We report that low-level shear stress enhances epithelial barrier function, an effect that requires serial activation of the transient receptor potential vanilloid (TRPV) 4 and L-type voltage-gated calcium channel (VGCC) and an increase in intracellular calcium. These changes lead to a selective decrease in aquaporin-5 (AQP5) abundance because of protein internalization and degradation. To determine whether AQP5 plays a role in mediating the shear effects on paracellular permeability, we overexpressed hAQP5 in 16HBE cells, an airway epithelial cell line without endogenous AQP5. We found that AQP5 expression was needed for shear-induced barrier enhancement. These findings have direct relevance to the regulation of epithelial barrier function, membrane permeability, and water homeostasis in the respiratory epithelia.