Maintenance Of Epithelial Barrier Function Research Articles

The preclinical discovery and development of zolbetuximab for the treatment of gastric cancer

ABSTRACT Introduction Gastric cancer remains a formidable challenge in oncology with high mortality rates and few advancements in treatment. Claudin-18.2 (CLDN18.2) is a tight junction protein primarily expressed in the stomach and is frequently overexpressed in certain subsets of gastric cancers. Targeting CLDN18.2 with monoclonal antibodies, such as zolbetuximab (IMAB362), has shown promising efficacy results in combination with chemotherapy. Areas covered The molecular cell biology of CLDN18.2 is discussed along with studies demonstrating the utility of CLDN18.2 expression as a biomarker and therapeutic target. Important clinical studies are reviewed, including Phase III trials, SPOTLIGHT and GLOW, which demonstrate the efficacy of zolbetuximab in combination with chemotherapy in patients with CLDN18.2-positive advanced gastric cancer. Expert opinion CLDN18.2 is involved in gastric differentiation through maintenance of epithelial barrier function and coordination of signaling pathways, and its expression in gastric cancers reflects a ‘gastric differentiation’ program. Targeting Claudin-18.2 represents the first gastric cancer specific ‘targeted’ treatment. Further studies are needed to determine its role within current gastric cancer treatment sequencing, including HER2-targeted therapies and immunotherapies. Management strategies will also be needed to better mitigate zolbetuximab-related treatment side effects, including gastrointestinal (GI) toxicities.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Poly(D,l-lactide-co-glycolide) particles are metabolised by the gut microbiome and elevate short chain fatty acids

The production of short chain fatty acids (SCFAs) by the colonic microbiome has numerous benefits for human health, including maintenance of epithelial barrier function, suppression of colitis, and protection against carcinogenesis. Despite the therapeutic potential, there is currently no optimal approach for elevating the colonic microbiome's synthesis of SCFAs. In this study, poly(D,l-lactide-co-glycolide) (PLGA) was investigated for this application, as it was hypothesised that the colonic microbiota would metabolise PLGA to its lactate monomers, which would promote the resident microbiota's synthesis of SCFAs. Two grades of spray dried PLGA, alongside a lactate bolus control, were screened in an advanced model of the human colon, known as the M-SHIME® system. Whilst the high molecular weight (Mw) grade of PLGA was stable in the presence of the microbiota sourced from three healthy humans, the low Mw PLGA (PLGA 2) was found to be metabolised. This microbial degradation led to sustained release of lactate over 48 h and increased concentrations of the SCFAs propionate and butyrate. Further, microbial synthesis of harmful ammonium was significantly reduced compared to untreated controls. Interestingly, both types of PLGA were found to influence the composition of the luminal and mucosal microbiota in a donor-specific manner. An in vitro model of an inflamed colonic epithelium also showed the polymer to affect the expression of pro- and anti-inflammatory markers, such as interleukins 8 and 10. The findings of this study reveal PLGA's sensitivity to enzymatic metabolism in the gut, which could be harnessed for therapeutic elevation of colonic SCFAs.

Clipping EpCAM to release Claudin-7 for the greater good of the epithelial barrier.

Maintenance of epithelial barrier function requires dynamic repair and remodeling of tight junctions. In this issue, Higashi et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202204079) demonstrate that the proteolytic cleavage of EpCAM by membrane-anchored serine proteinases releases Claudin-7 to join tight junctions, suggesting a novel mechanism that couples sensing with repair of damaged tight junctions.

Ganglioside Alters Phospholipase Trafficking, Inhibits NF-κB Assembly, and Protects Tight Junction Integrity.

Background and Aims: Dietary gangliosides are present in human milk and consumed in low amounts from organ meats. Clinical and animal studies indicate that dietary gangliosides attenuate signaling processes that are a hallmark of inflammatory bowel disease (IBD). Gangliosides decrease pro-inflammatory markers, improve intestinal permeability, and reduce symptoms characteristic in patients with IBD. The objective of this study was to examine mechanisms by which dietary gangliosides exert beneficial effects on intestinal health.Methods: Studies were conducted in vitro using CaCo-2 intestinal epithelial cells. Gangliosides were extracted from milk powder and incubated with differentiated CaCo-2 cells after exposure to pro-inflammatory stimuli. Gut barrier integrity was assessed by electron microscopy, epithelial barrier function was examined by measuring transepithelial electric resistance, and content of HBD-2, IL-23, NF-κB, and sPLA2 was assessed by ELISA.Results: Ganglioside attenuated the decrease in integrity of tight junctions induced by pro-inflammatory stimuli and improved epithelial barrier function (P < 0.05). Ganglioside decreased the basolateral secretion of sPLA2 (P ≤ 0.05), lowered HBD-2 and IL-23 levels (P ≤ 0.05), and inhibited NF-κB activation (P ≤ 0.05).Conclusions: In summary, the present study indicates that ganglioside GD3 improves intestinal integrity by altering sPLA2 trafficking, and the production of pro-inflammatory mediators is mitigated by decreasing assembly of the NF-κB complex. Dietary gangliosides may have promising potential beneficial effects in IBD as decreased inflammatory signaling, improved intestinal integrity, and maintenance of epithelial barrier function have been demonstrated in vitro.

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.

Temporal shifts in the mycobiome structure and network architecture associated with a rat (Rattus norvegicus) deep partial-thickness cutaneous burn.

With a diverse physiological interface to colonize, mammalian skin is the first line of defense against pathogen invasion and harbors a consortium of microbes integral in maintenance of epithelial barrier function and disease prevention. While the dynamic roles of skin bacterial residents are expansively studied, contributions of fungal constituents, the mycobiome, are largely overlooked. As a result, their influence during skin injury, such as disruption of skin integrity in burn injury and impairment of host immune defense system, is not clearly delineated. Burn patients experience a high risk of developing hard-to-treat fungal infections in comparison to other hospitalized patients. To discern the changes in the mycobiome profile and network assembly during cutaneous burn-injury, a rat scald burn model was used to survey the mycobiome in healthy (n = 30) (sham-burned) and burned (n = 24) skin over an 11-day period. The healthy skin demonstrated inter-animal heterogeneity over time, while the burned skin mycobiome transitioned toward a temporally stabile community with declining inter-animal variation starting at day 3 post-burn injury. Driven primarily by a significant increase in relative abundance of Candida, fungal species richness and abundance of the burned skin decreased, especially in days 7 and 11 post-burn. The network architecture of rat skin mycobiome displayed community reorganization toward increased network fragility and decreased stability compared to the healthy rat skin fungal network. This study provides the first account of the dynamic diversity observed in the rat skin mycobiome composition, structure, and network assembly associated with postcutaneous burn injury.

Intestinal barrier dysfunction orchestrates the onset of inflammatory host-microbiome cross-talk in a human gut inflammation-on-a-chip.

The initiation of intestinal inflammation involves complex intercellular cross-talk of inflammatory cells, including the epithelial and immune cells, and the gut microbiome. This multicellular complexity has hampered the identification of the trigger that orchestrates the onset of intestinal inflammation. To identify the initiator of inflammatory host-microbiome cross-talk, we leveraged a pathomimetic "gut inflammation-on-a-chip" undergoing physiological flow and motions that recapitulates the pathophysiology of dextran sodium sulfate (DSS)-induced inflammation in murine models. DSS treatment significantly impaired, without cytotoxic damage, epithelial barrier integrity, villous microarchitecture, and mucus production, which were rapidly recovered after cessation of DSS treatment. We found that the direct contact of DSS-sensitized epithelium and immune cells elevates oxidative stress, in which the luminal microbial stimulation elicited the production of inflammatory cytokines and immune cell recruitment. In contrast, an intact intestinal barrier successfully suppressed oxidative stress and inflammatory cytokine production against the physiological level of lipopolysaccharide or nonpathogenic Escherichia coli in the presence of immune elements. Probiotic treatment effectively reduced the oxidative stress, but it failed to ameliorate the epithelial barrier dysfunction and proinflammatory response when the probiotic administration happened after the DSS-induced barrier disruption. Maintenance of epithelial barrier function was necessary and sufficient to control the physiological oxidative stress and proinflammatory cascades, suggesting that "good fences make good neighbors." Thus, the modular gut inflammation-on-a-chip identifies the mechanistic contribution of barrier dysfunction mediated by intercellular host-microbiome cross-talk to the onset of intestinal inflammation.

Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function

Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiology on allergic asthma phenotypes are currently unknown. The mitochondrial Ca2+ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca2+ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca2+ uptake and reactive oxygen species (ROS) production, preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca2+ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.

Ectodysplasin A regulates epithelial barrier function through sonic hedgehog signalling pathway

Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X‐linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive. In this study, we found tight junctional proteins ZO‐1 and claudin‐1 expression is largely reduced in epidermal, corneal and lung epithelia in Eda mutant Tabby mice at different postnatal ages. These declines are associated with tail ulceration, corneal pannus formation and lung infection. Furthermore, topical application of recombinant Eda protein markedly mitigated corneal barrier dysfunction. Using cultures of a human corneal epithelial cell line and Tabby mouse skin tissue explants, Eda up‐regulated expression of ZO‐1 and claudin‐1 through activation of the sonic hedgehog signalling pathway. We conclude that EDA gene expression contributes to the maintenance of epithelial barrier function. Such insight may help efforts to identify novel strategies for improving management of XLHED disease manifestations in a clinical setting.

Activation of muscarinic receptors prevents TNF-α-mediated intestinal epithelial barrier disruption through p38 MAPK

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.

Exploiting the Gastric Epithelial Barrier: Helicobacter pylori's Attack on Tight and Adherens Junctions.

Highly organized intercellular tight and adherens junctions are crucial structural components for establishing and maintenance of epithelial barrier functions, which control the microbiota and protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of multiple infectious diseases as well as various cancers. The gastric pathogen Helicobacter pylori exerts an amazing set of strategies to manipulate these epithelial cell-to-cell junctions, which are implicated in changing cell polarity, migration and invasive growth as well as pro-inflammatory and proliferative responses. This chapter focuses on the H. pylori pathogenicity factors VacA, CagA, HtrA and urease, and how they can induce host cell signaling involved in altering cell-to-cell permeability. We propose a stepwise model for how H. pylori targets components of tight and adherens junctions in order to disrupt the gastric epithelial cell layer, giving fresh insights into the pathogenesis of this important bacterium.

Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer

Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.

CLMP Expression is Increased in the Intestinal Epithelium Under Inflammatory Conditions and Regulates Intercellular Adhesion, Proliferation and Migration

The intestine is lined by epithelial cells that form a dynamic physical barrier against external substances. Maintenance of this mucosal barrier requires proliferation, migration and differentiation of epithelial progenitor cells along the crypt‐luminal axis while keeping strong cell‐cell cohesion. Epithelial barrier function is created by tight junctions (TJ) and increased intestinal permeability has been linked with the pathogenesis of inflammatory bowel disease. CAR‐Like Membrane Protein (CLMP) is structurally related to the TJ transmembrane protein, Coxsackie and Adenovirus Receptor, but CLMP role in controlling intestinal epithelial barrier function in homeostasis and inflammation is not known. We observed that CLMP is distributed in intercellular junctions of murine and human intestinal epithelial cells (IECs), and its expression is increased after inflammatory cytokine exposure. CLMP overexpression in IECs, unlike its depletion, resulted in strengthened cell‐cell adhesion and increased cell migration after injury indicating a positive role of CLMP in the maintenance of epithelial barrier function and wound repair. Interestingly, CLMP overexpression led to decreased epithelial cell proliferation and inhibited the growth of subcutaneous xenografts in Rag1null mice. Our results support a role of CLMP in controlling intestinal epithelial barrier function and homeostasis. These studies will help to better understand signaling pathways that influence the intestinal epithelial barrier function during chronic inflammation.

Emerging Roles for RIPK1 and RIPK3 in Pathogen-Induced Cell Death and Host Immunity.

Receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3 ) are homologous serine-threonine kinases that were recognized for their roles in directing programmed necrotic cell death or necroptosis under a broad range of pathologic settings. Emerging evidence suggests new physiologic roles for RIPK1 and RIPK3 in mediating cell death of innate immune responses. Our review discusses current evidence on the mechanisms and the impact of RIPK1- and/or RIPK3-dependent cell death in responses to a variety of viral and bacterial pathogens. Furthermore, the discussion also summarizes emerging roles for RIPK1 and RIPK3 in other facets of host immunity, including the maintenance of epithelial barrier function and pro-inflammatory processes that may, in some cases, manifest independent of cell death. Finally, we briefly consider the therapeutic opportunities in targeting RIPK1- and RIPK3-dependent processes in infection and immunity.

Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

Targeting Mitochondria-Derived Reactive Oxygen Species to Reduce Epithelial Barrier Dysfunction and Colitis

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coliwith or withoutmitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)-induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn's disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. Invitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.

To Adhere or not to Adhere: Regulation of Self-Contact Elimination by Membrane Fusion

Mutual, homophilic cell-cell adhesion between epithelial cells is required for proper maintenance of epithelial barrier function. While opposing membranes from neighboring cells rapidly assemble junctional complexes, self-contacting membranes do not, suggesting that cells have the ability to distinguish self from neighboring cells. Using a self-contact inducing micro-fabricated substrate, we show that self-contacts of normal epithelial cells are rapidly eliminated by membrane fusion between two opposing plasma membranes of a single cell. This membrane fusion is most frequently observed in E-cadherin expressing epithelial cells, but not in fibroblasts. The efficiency of self-contact elimination depends on extracellular calcium concentration and the level of E-cadherin, suggesting that E-cadherin, while not required, enhances membrane-fusion efficiency by bringing opposing membranes into close apposition to one another. Additionally, ROCK inhibition decreases self-contact induced membrane fusion of epithelial cells, suggesting that this fusion may be mechanically regulated through the actin-myosin network. This is the first demonstration of self-contact induced membrane fusion in mammalian cells, and that membrane fusion may be a key feature of the cell self-recognition signaling pathway.

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein β-catenin. Jak3 expression in these cells was essential for AJ localization of β-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of β-catenin.

Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both?

Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes. Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain–Barré syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen.