Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn’s disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.

Macrophages are critical mediators of intestinal defense and homeostasis. However, uncontrolled pro-inflammatory macrophage activity may contribute to chronic conditions such as inflammatory bowel disease. Currently, the regulatory feedback mechanisms restraining pro-inflammatory cytokine production in activated macrophages are not well understood. The ErbB4 receptor tyrosine kinase is induced on macrophages by pro-inflammatory stimulation, and chronic ErbB4 activation with its ligand NRG4 drives macrophage apoptosis after 2 days. However, the impact of endogenous NRG4/ErbB4 signaling on macrophage function remains untested. Using bone-marrow derived ErbB4-null or NRG4-null macrophages, we tested the hypothesis that NRG4/ErbB4 signaling inhibits pro-inflammatory cytokine production. We found that deletion of either the receptor or its ligand resulted in elevated pro-inflammatory cytokine expression in classically (IFNɣ/LPS)-activated cells, compared to activated cells generated from wild type littermates. NRG4 was induced by IFNɣ/LPS activation in wild type macrophages, and exogenous treatment with NRG4 led to a reduction in Tnf, Cxcl1, and Il1b expression within 24 hours. RNA sequencing of ErbB4myeKO macrophages showed elevated expression of major regulators of inflammatory skewing (Sik2) and cytokine transport (Trim16). In vivo, ErbB4myeKO mice subjected to acute DSS colitis showed exaggerated disease, and ErbB4 myeloid knockout in the IL10-KO chronic colitis model accelerated disease onset. Taken together, these findings demonstrate that NRG4/ErbB4 signaling in macrophages restrains the pro-inflammatory tone of these cells, and is an important limiting regulator of colitis severity. These results highlight a previously unknown feedback mechanism by which growth factor signaling in immune cells prevents runaway inflammation and chronic disease.

Background and Purpose Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function. Experimental Approach Monolayers of colonic epithelial cell lines (Caco‐2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR‐4 and PKC isoforms. Key Results Apically applied RGal (1000 μg ml−1) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)‐dextran flux in Caco‐2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco‐2 cells in a calcium switch assay. RGal also reversed the barrier‐damaging effects of inflammatory cytokines on FITC‐dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4‐expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ. Conclusion and Implications RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.

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