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The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri. Author summary: The small intestinal tuft cell is emerging as a versatile sentinel cell type in a variety of infections involving protozoans, nematodes, and certain bacteria. Whether the tuft cell responds to enteric cestode infections and the extent of its involvement in mediating the anti-helminth response is not yet characterized. Using mice infected with Hymenolepis diminuta, we show that tuft cells help accelerate expulsion of this lumen-dwelling cestode, by coordinating local events that form an integral part of the classical "weep and sweep" anti-helminthic response. We further show that although infection with H. diminuta induces both tuft cell hyperplasia as well as protection from subsequent infection with the nematode Heligmosmoides bakeri, the tuft cell is not solely responsible for mediating systemic and other local Th2 responses against H. diminuta. Our work reveals subtle roles for the tuft cell in initiating and coordinating host anti-parasitic activity in cestode infections. At the same time, we show that redundancies exist in the host's mucosal arsenal that ultimately mediate worm expulsion in tuft cell deficient mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer. NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.