Composition Of Tight Junctions Research Articles

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

(1) Background: Atopic dermatitis is one of the most common inflammatory skin diseases characterized by T helper (Th) 2 and Th22 cells producing interleukin (IL)-4/IL-13 and IL-22, respectively. The specific contribution of each cytokine to the impairment of the physical and the immune barrier via Toll-like receptors (TLRs) is poorly addressed concerning the epidermal compartment of the skin. (2) Methods: The effect of IL-4, IL-13, IL-22, and the master cytokine IL-23 is evaluated in a 3D model of normal human skin biopsies (n = 7) at the air-liquid interface for 24 and 48 h. We investigated by immunofluorescence the expressions of (i) claudin-1, zonula occludens (ZO)-1 filaggrin, involucrin for the physical barrier and (ii) TLR2, 4, 7, 9, human beta-defensin 2 (hBD-2) for the immune barrier. (3) Results: Th2 cytokines induce spongiosis and fail in impairing tight junction composition, while IL-22 reduces and IL-23 induces claudin-1 expression. IL-4 and IL-13 affect the TLR-mediated barrier largely than IL-22 and IL-23. IL-4 early inhibits hBD-2 expression, while IL-22 and IL-23 induce its distribution. (4) Conclusions: This experimental approach looks to the pathogenesis of AD through molecular epidermal proteins rather than cytokines only and paves the way for tailored patient therapy.

Structure Composition and Intracellular Transport of Clathrin-Mediated Intestinal Transmembrane Tight Junction Protein.

Tight junctions (TJs) are located in the apical region of the junctions between epithelial cells and are widely found in organs such as the brain, retina, intestinal epithelium, and endothelial system. As a mechanical barrier of the intestinal mucosa, TJs can not only maintain the integrity of intestinal epithelial cells but also maintain intestinal mucosal permeability by regulating the entry of ions and molecules into paracellular channels. Therefore, the formation disorder or integrity destruction of TJs can induce damage to the intestinal epithelial barrier, ultimately leading to the occurrence of various gastrointestinal diseases, such as inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), and irritable bowel syndrome (IBS). However, a large number of studies have shown that TJs protein transport disorder from the endoplasmic reticulum to the apical membrane can lead to TJs formation disorder, in addition to disruption of TJs integrity caused by external pathological factors and reduction of TJs protein synthesis. In this review, we focus on the structural composition of TJs, the formation of clathrin-coated vesicles containing transmembrane TJs from the Golgi apparatus, and the transport process from the Golgi apparatus to the plasma membrane via microtubules and finally fusion with the plasma membrane. At present, the mechanism of the intracellular transport of TJ proteins remains unclear. More studies are needed in the future to focus on the sorting of TJs protein vesicles, regulation of transport processes, and recycling of TJ proteins, etc.

Human duodenal organoid-derived monolayers serve as a suitable barrier model for duodenal tissue.

Usually, duodenal barriers are investigated using intestinal cell lines like Caco-2, which in contrast to native tissue are limited in cell-type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three-dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid-derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco-2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco-2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco-2. Transcriptomic data showed that Caco-2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco-2 cells differ mostly in transcellular properties, ODMs reflect trans- and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue-specific investigations.

Barrier Perturbation in Porcine Peyer's Patches by Tumor Necrosis Factor is Associated With a Dysregulation of Claudins.

The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense.

Paracellular intestinal permeability of chickens induced by DON and/or C. jejuni is associated with alterations in tight junction mRNA expression

Toxins, antigens, and harmful pathogens continuously challenge the intestinal mucosa. Therefore, regulation of the intestinal barrier is crucial for the maintenance of mucosal homeostasis and gut health. Intercellular complexes, namely, tight junctions (TJs), regulate paracellular permeability. TJs are mainly composed of claudins (CLDN), occludin (OCLN), tight junction associated MARVEL-domain proteins (TAMPS), the scaffolding zonula occludens (ZO) proteins and junction-adhesion molecules (JAMs). Different studies have shown that a Campylobacter infection can lead to a phenomenon so-called “leaky gut”, including the translocation of luminal bacteria to the underlying tissue and internal organs. Based on the effects of C. jejuni on the chicken gut, we hypothesize that impacts on TJ proteins play a crucial role in the destructive effects of the intestinal barrier. Likewise, the mycotoxin deoxynivalenol (DON) can also alter gut permeability in chickens. Albeit DON and C. jejuni are widely distributed, no data are available on their effect on the tight junctions’ barrier in the broiler intestine and consequences for permeability. Therefore, the aim of this study was to analyze the interaction between DON and C. jejuni on the gut barrier by linking permeability with gene expression of TJ proteins and to determine the relationships between the measurements. Following oral infection of birds with C. jejuni NCTC 12744 at 14 days of age, we demonstrate that the co-exposure with DON has considerable consequences on gut permeability as well as on gut TJ mRNA expression. Co-exposure of DON and C. jejuni enhanced the negative effect on paracellular permeability of the intestine, which was also noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain time points in both jejunum and caecum. Furthermore, the increased paracellular permeability was associated with significant changes in TJ mRNA expression in the small and large intestine. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni resulted in a decreased barrier function via up-regulation of pore-forming tight junctions (CLDN7 and CLDN10), as well as the cytosolic TJ protein occludin (OCLN) that can shift to various paracellular locations and are therefore able to alter the epithelial permeability. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni affects the paracellular permeability of the gut by altering the tight junction proteins. Furthermore, analysing of correlations between TJs revealed that the mRNA expression levels of most tight junctions were correlated with each other in both jejunum and caecum. Finally, the findings indicate that the molecular composition of tight junctions can be used as a marker for gut health and integrity.

Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis

The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

Claudin-9 constitutes tight junctions of folliculo-stellate cells in the anterior pituitary gland

The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept.

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Inhibiting Connexin43/Zonula Occludens‐1 Interactions Improves Alcoholic Lung Barrier Function in vivo

Chronic alcohol consumption is well known to contribute to an array of health complications and increases the incidence and severity of acute respiratory distress syndrome (ARDS), an acute lung injury marked by severe pulmonary edema and impairment of normal gas exchange. Despite advancements in understanding its pathogenesis, the mortality rate for ARDS remains over 40% and there are currently no effective pharmacological treatments for ARDS. Chronic alcohol consumption contributes to the pathogenesis of ARDS by altering the tight junction composition of the pulmonary epithelium which is essential to maintain normal barrier function. In this study, we investigate the use of a pharmacological peptide, aCT1, which mimics the cytoplasmic carboxy-terminal PDZ binding motif of the gap junction protein Connexin43 (Cx43) to disrupt endogenous Cx43 from binding scaffold proteins, including the tight junction protein Zonula Occludens-1 (ZO-1). By inhibiting ZO-1 binding to Cx43, we hypothesize that more ZO-1 is available to associate with tight junction proteins, including claudins, to enhance epithelial barrier function. We have previously shown that chronic alcohol use exacerbates acute endotoxin-mediated lung injury by compromising both the alveolar and pulmonary capillary epithelial barriers. Here, we tested whether treatment with aCT1 strengthens alveolar barrier function and mitigates the effects of alcohol-mediated impairment of tight junctions. Male C57BL/6J mice were pair-fed 20% ethanol or water for 14 weeks to establish a chronic alcohol model in vivo. Mice were sedated and given an intratracheal instillation of 5mg/kg of the bacterial endotoxin lipopolysaccharide (LPS) derived from E. coli O55:B5 to induce a second, direct insult in the alcohol-primed lung to impact the lung epithelium. Mice were administered via nebulizer 1h post-LPS instillation either 5mg/kg of the aCT1 peptide or a scrambled control. Evans Blue (EB) dye was injected into mouse tail veins 24h after LPS instillation. To measure alveolar flooding, bronchoalveolar lavage fluid (BAL) was collected 1h later and EB in BAL was measured and normalized to serum EB. We found that administration of aCT1 decreased the leakage of EB dye from the blood into the alveolar air space in both water-fed and alcohol-fed mice, thus improving lung barrier function. This improvement in pulmonary leak suggests that aCT1 has the capacity to mitigate ARDS. Further investigation will determine whether aCT1 has a positive impact on short-term mortality rates due to endotoxemia and whether it can improve pulmonary barrier dysfunction due to other causes of acute lung injury.

Abstract 419: PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer

Abstract Compromised barrier function of colon epithelial tissue is associated with inflammatory bowel diseases (IBDs), and increases the risk of colorectal cancer (CRC) through a process of tumor-elicited inflammation (TEI). Apical tight junction (TJ) proteins, including ZO-1, occludin and specific claudins, are critical in the maintenance of epithelial barrier function and control of paracellular permeability. Changes in TJ composition in the kidney are strongly linked to the etiology of autosomal dominant polycystic kidney disease (ADPKD), with ADPKD-inducing mutations in PKD1 causing non-leaky barriers that can withstand high hydrostatic pressure within renal cysts a hallmark of this common (~1 in 600) inherited disease. Intriguingly, a large population study suggested decreased incidence of CRC in ADPKD patients, while other studies implicated PKD1 in control of epithelial-mesenchymal transition (EMT) and invasion. Based on these data, we hypothesized that loss of PKD1 might hinder susceptibility to ulcerative colitis and CRC, based on reorganization of TJs and limitation of TEI in the colon. We directly assessed PKD1 control of epithelial barrier function using conditional Pkd1fl/fl mice in which tamoxifen induces Cre-lox dependent targeted inactivation of Pkd1 gene in the colon. We compared 10-12 week old wt or induced Pkd1fl/fl mice treated for 5 days with 2.5% DSS in drinking water to induce colitis, to a non-DSS control group. For wt mice, DSS increased orally gavaged FITC-dextran in the serum versus vehicle-treated mice, and histopathological assessment indicated significant damage to colon. In contrast, DSS did not increase FITC-dextran in the serum of Pkd1−/- mice, and less DSS-induced tissue damage was observed, suggesting reinforced colon barrier function. Further, based on immunofluorescence (IF) analysis of tissue sections, the claudins CLDN4 and CLDN7, associated with barrier impermeability, were strongly elevated in the colonic epithelium of Pkd1fl/fl versus wt mice, with consistently greater localization to cell junctions in Pkd1fl/fl versus wt mice. Using a Cdx2-ERT2/Cre model for tamoxifen-induced loss of Apc and/or Pkd1 in the colon, we compared tumorigenesis in the Apcfl/fl, Apcfl/fl Pkd1fl/fl, and Apcfl/flPkd1fl/+ genotypes. There was a highly significant increase in the number and size of Apcfl/fl-induced tumors based on retention of Pkd1. Loss of Pkd1 substantially increased CLDN4 in normal and tumor tissue, and decreased TNFα expression in tumors, suggesting reduced inflammation. Analysis in organoids recapitulated observed effects of Pkd1, indicating cell-autonomous effect. Epistasis experiments suggest activation of the tumor suppressor CFTR as a mechanism by which loss of PKD1 limits EMT and tumor growth; we found chemical activators of CFTR selectively limited the growth of PKD1-positive organoids. Better understanding of the role of PKD1 and its effectors may suggest therapeutic strategies, and inform genetic counseling, for IBD and CRC. Citation Format: Anna S. Nikonova, Anna A. Kiseleva, Alexander Deneka, Rossella Tricarico, Sergei Grivennikov, Erica Golemis. PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 419.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2–0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Abstract 3459: PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer

Abstract Compromised integrity of the epithelial barrier function of colon tissue associated with inflammatory bowel diseases (IBDs) or arising from tumor-elicited inflammation (TEI) are associated with and support formation of colorectal cancer (CRC). Apical tight junction (TJ) proteins, including multiple specific claudins, are critical in regulating TJ barrier function and paracellular permeability. Changes in TJ composition in the kidney are strongly linked to the etiology of autosomal dominant polycystic kidney disease (ADPKD), with ADPKD-inducing mutations in PKD1 causing non-leaky barriers that can withstand high hydrostatic pressure within renal cysts a hallmark of this common (1 in 500) inherited disease. Intriguingly, a large population study has found a decreased incidence of CRC in ADPKD patients. Based on these and other suggestive data, we hypothesized that loss of PKD1 would hinder development of ulcerative colitis and initiation of colitis associated cancer, based on reorganizing TJs and TEI in the colon. To evaluate the impact of Pkd1 loss on colon barrier function, we treated 10-12 week old wt or Pkd1fl/fl mice with tamoxifen-induced, Cre expression from the CreERT2 cassette for 5 days with 2.5% DSS in drinking water to induce acute colitis, compared to a no-DSS control group. For the wtcohort, treatment with DSS increased orally gavaged FITC-dextran detectable in serum versus controls. In contrast, no Pkd1-/- mice showed a similar DSS-dependent response to FITC-dextran, suggesting reinforced colon barrier function; further, histopathological assessment confirmed less DSS-induced damage in Pkd1fl/fl mice. Further, based on immunofluorescence (IF) analysis of tissue sections, claudins 4 and 7 associated with increased barrier function) strongly elevated in the colonic epithelium of Pkd1fl/fl versus wt mice, with the expressed proteins having consistently greater localization to cell junctions in Pkd1fl/fl versus wt mice. We also investigated the formation of CRC tumors, using a CDX2-ERT2/Cre model for tamoxifen-induced loss of Apc and/or Pkd1 in the colon to compare tumorigenesis in the Apcfl/fl, Apcfl/fl Pkd1fl/fl, and Apcfl/flPkd1fl/+ genotypes. We observed a highly significant progressive increase in the number and size of Apc mutation-induced tumors based on retention of the Pkd1 gene. Preliminary qRT-PCR analysis of these tumors shows that a Pkd1fl/fl genotype substantially increases CLDN4 in normal and tumor tissue, and decreases TNFα expression in tumors, suggesting reduced inflammation. This work and extended mechanistic characterization identifies PKD1 as a vital regulator of signaling systems already associated with colitis and CRC. Understanding the role of PKD1 and its effectors may provide useful information to genetic counselors assessing risk of IBD and CRC, and suggest therapeutic strategies. Citation Format: Anna S. Nikonova, Anna Kiseleva, Ilya Serebriiskii, Sergei Grivennikov, Erica A. Golemis. PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3459.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

Mammalian small intestinal (SI) tight junctions (TJ) link epithelial cells to one another and function as aperm selective barrier, strictly modulating the passage of ions and macromolecules through the “pore” and “leak” pathways, respectively, thereby preventing absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. SI epithelial permeability is ascribed primarily to properties of TJ between adjoiningenterocytes (ENT), because there is almost no information on TJ composition and paracellular permeability of non‐enterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. In the current study we directed murine intestinal crypts to form specialized organoids highly enrichedin stem cells (ISC), absorptive ENTs, secretory goblet (GOB) or Paneth cells(PAN). Morphological and morphometriccharacteristics of these cells in organoids were similar to those in vivo. Expression of certain TJ genes varied with cell type. Occludin and tricellulin levels were high in both ISC and PAN organoids, while claudin‐1, junctional adhesion molecule‐1, and cingulin were greatest mainly in PAN. Claudin‐2 was greatest in ISC and claudin‐7 expression was greatest in ENT organoids. In contrast, claudin‐15, zonula occludens 1 (ZO‐1) and e‐cadherin distribution were relatively homogeneous among cell types. Immunofluorescent staining showed that e‐cadherin and claudin‐7 marked mainly the basolateral membrane, while claudin‐2, ZO‐1 and occludin resided apically. Remarkably, organoids enrichedin ENTs and GOBs, respectively were 3.7‐fold and 3.4‐fold more permeable to 4 kda dextran thanthose enriched in ISCs (P<0.05). Similarly, ENT and GOB were 2.9 and2.6‐fold more permeable to 10 kda dextran compared to ISC organoids. The trend was similar (>2‐fold)for ENT and GOB vs. PAN organoids for both 4 and 10 kda dextran flux (P<0.05). As was expected, for all cell types 10 kda dextran flux was significantly lower than 4 kda (P < 0.05). A time course of dextran flux in ENT and PAN organoids revealed that 4 kda dextran entered the lumen of ENT by 10 min and highest intensity was observed by 30 min (P<0.05). In contrast, dextran flux was nearly absent in PAN organoids, regardless of time point. Additionally, the TJ regulator larazotide prevented the ~10‐fold increases in dextran flux induced by the TJ disrupter AT1002 in both ISC and ENT organoids (P < 0.05), indicating that AT1002 non‐selectively increases permeability. Lastly, dedifferentiation of mature ENTs results inreacquisition of ISC‐like characteristics in TJ composition and dextranpermeability, suggesting that post‐differentiation properties of TJs are not hard wired. In conclusion, differentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellularpathway of epithelia.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 (RPF) and 1456673 (RPF) and by NIH Grant R01‐DK‐102934 (NG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

BackgroundMammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia.ResultsHere we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired.ConclusionsDifferentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level.In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.

Heterogeneity of tight junctions in the thick ascending limb.

Renal tubular transport mechanisms are optimized to be energy efficient and tailored to local gradients and transport rates. The combined transcellular action of ion channels, transporters, and pumps, together with the paracellular pathway, enables kidney function. Monogenetic diseases and mouse models indicate that both trans- and paracellular proteins can become disease-causing candidates and may be targets for future therapeutic approaches. Recent advances in tight junction research have provided new insights into their structure, function, and regulation. The thick ascending limb (TAL) is a nephron segment with specific requirements for the paracellular pathway. It has to fuel the generation of the corticomedullary concentration gradient, to be watertight, and to provide a highly selective permeability for Na+ and divalent cations. Tight junction composition and function in the TAL is organized along the corticomedullary axis. Even on the level of a seemingly homogeneous tubular epithelium like the TAL, there is a separation of tight junction protein expression in the strands between the respective tricellular nexus of the junctional network. Here, we highlight some new insights from our recent work and that of others in this context. In addition, we provide some perspectives for the further study of paracellular transport mechanisms.

Intestinal Epithelial TCPTP Deficiency Reduces Expression of the Tight Junction Protein Tricellulin through MEK/ERK Signaling

Genome‐wide association studies identified protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) as a susceptibility gene for inflammatory bowel disease (IBD), Diabetes and Celiac disease, while loss of the PTPN2 protein product T‐cell protein tyrosine phosphatase (TCPTP) compromises intestinal epithelial barrier function. The protein composition of tight junctions (TJ) determines the tightness of the barrier and alterations in select TJ proteins leads to increased paracellular permeability. Tricellulin (also called marvelD2) is a recently discovered tight junction (TJ) protein, which is preferentially localized at tricellular contacts. Tricellulin plays an important role in the maintenance of intestinal epithelial barrier function by restricting paracellular permeability to macromolecules. The aim of the present study was to identify the molecular mechanism underlying barrier defects in TCPTP‐deficient intestinal epithelial cells (IEC). For this purpose, we developed Caco2‐BBe and HT‐29 intestinal epithelial cell lines (IECs) expressing short hairpin RNA for TCPTP using a lentiviral system to stably knock down TCPTP expression (TCPTP‐KD). Western blot analysis indicated that TCPTP expression was significantly reduced (by 45 ± 4%, p<0.01, n=3) in TCPTP‐KD cells. FD4 permeability was increased in KD cells (p<0.001; n=3) compared to control cells. Western blot and immunofluorescence studies revealed that the expression of tricellulin expression was decreased (WB: 44 ± 8.8%, p<0.05; n=3) in unchallenged TCPTP‐KD cells compared to control shRNA transfected cells. This decrease was further enhanced by challenge with the pro‐inflammatory cytokine interferon‐gamma (IFN‐g) (n=2). In previous studies, we observed increased basal phosphorylation of the TCPTP substrate ERK1/2 in TCPTP‐KD cells. Moreover, an inverse relationship exists between mitogen‐activated protein kinase kinase kinase 1 (MEKK1) that lies upstream of ERK1/2, and MarvelD3, a member of the same protein family as tricellulin. To investigate whether tricellulin loss in TCPTP‐KD cells occurs through MEK/ERK signaling, TCPTP‐KD IEC were treated with the ERK1/2 inhibitor U0126 (10mM) for 14 hours. ERK inhibition rescued tricellulin expression in TCPTP‐KD cells. In conclusion, we report that tricellulin expression is down regulated, at least in part, by the MEK/ERK pathway and that decreased tricellulin levels could contribute to the increased macromolecule permeability in cells lacking TCPTP.Support or Funding InformationNIH‐R01‐DK‐091281 and Crohn's & Colitis Foundation of America(CCFA) to Declan F. McCole

Gluten-induced symptoms in diarrhea-predominant irritable bowel syndrome are associated with increased myosin light chain kinase activity and claudin-15 expression

The mechanisms underlying diarrhea-predominant irritable bowel syndrome (IBS-D) are poorly understood, but increased intestinal permeability is thought to contribute to symptoms. A recent clinical trial of gluten-free diet (GFD) demonstrated symptomatic improvement, relative to gluten-containing diet (GCD), which was associated with reduced intestinal permeability in non-celiac disease IBS-D patients. The aim of this study was to characterize intestinal epithelial tight junction composition in IBS-D before and after dietary gluten challenge. Biopsies from 27 IBS-D patients (13 GFD and 14 GCD) were examined by H&E staining and semiquantitative immunohistochemistry for phosphorylated myosin II regulatory light chain (MLC), MLC kinase, claudin-2, claudin-8 and claudin-15. Diet-induced changes were assessed and correlated with urinary mannitol excretion (after oral administration). In the small intestine, epithelial MLC phosphorylation was increased or decreased by GCD or GFD, respectively, and this correlated with increased intestinal permeability (P<0.03). Colonocyte expression of the paracellular Na+ channel claudin-15 was also markedly augmented following GCD challenge (P<0.05). Conversely, colonic claudin-2 expression correlated with reduced intestinal permeability (P<0.03). Claudin-8 expression was not affected by dietary challenge. These data show that alterations in MLC phosphorylation and claudin-15 and claudin-2 expression are associated with gluten-induced symptomatology and intestinal permeability changes in IBS-D. The results provide new insight into IBS-D mechanisms and can explain permeability responses to gluten challenge in these patients.

Claudins: vital partners in transcellular and paracellular transport coupling.

Tight junction (TJ) strands between epithelial or endothelial cells are formed by claudins, a protein family comprising up to 27 members in mammals. Although many more proteins are involved in the formation of TJ complexes, claudins are the only TJ proteins that are able to form TJ-like strands when overexpressed in cells that are normally devoid of TJs (e.g., fibroblasts). Within the paracellular cleft, the extracellular domains of claudins provide the matrix that seals the paracellular pathway. However, within this matrix, some claudins act as channels that specifically allow certain ions to cross this barrier. Barrier-forming claudins predominate in epithelia that enclose compartments containing harmful ion concentrations (e.g., H+ in the stomach, K+ in the inner ear endolymph) or high pressures (e.g., in blastocoel or brain ventricle formation during development). Here, even seemingly minor alterations in TJ composition may be detrimental to the organism. In contrast, in many transporting epithelia, channel-forming claudins are essential for transcellular and paracellular transport coupling. Mutation or knockout of channel-forming claudins in these tissues brings both transcellular and paracellular transports to a standstill. The present review will present examples to illustrate the importance of single members of the claudin family in general epithelial transport physiology.