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Background & Aims Communication between T cells and the intestinal epithelium is altered in many diseases, causing T-cell activation, depletion, or recruitment, and disruption of the epithelium. We hypothesize that activation of T cells regulates epithelial barrier function by targeting the assembly of the tight junction complex. Methods In a 3-dimensional and 2-dimensional co-culture model of activated T cells subjacent to the basolateral surface of an epithelial monolayer, the pore, leak, and unrestricted pathways were evaluated using transepithelial resistance and flux of fluorescently labeled tracers. T cells were acutely and chronically activated by cross-linking the T-cell receptor. Tight junction assembly and expression were measured using quantitative polymerase chain reaction, immunoblot, and immunofluorescence confocal microscopy. Results Co-culture with acutely and chronically activated T cells decreased the magnitude of ion flux through the pore pathway, which was maintained in the presence of acutely activated T cells. Chronically activated T cells after 30 hours induced a precipitous increase in the magnitude of both ion and molecular flux, resulting in an increase in the unrestricted pathway, destruction of microvilli, expansion in cell surface area, and cell death. These fluctuations in permeability were the result of changes in the assembly and expression of tight junction proteins, cell morphology, and viability. Co-culture modulated the expression of immune mediators in the epithelium and T cells. Conclusions Bidirectional communication between T cells and epithelium mediates a biphasic response in barrier integrity that is facilitated by the balance between structural proteins partitioning in the mobile lateral phase vs the tight junction complex and cell morphology., Graphical abstract

Low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl) produced by myeloperoxidase (MPO) is present in atherosclerotic lesions, where it is implicated in the propagation of inflammation and acceleration of lesion development by multiple pathways, including the induction of endothelial dysfunction. Thiocyanate (SCN-) ions are utilised by MPO to produce the oxidant hypothiocyanous acid (HOSCN), which reacts with LDL in a different manner to HOCl. Whilst the reactivity of HOCl-modified LDL has been previously studied, the role of HOSCN in the modification of LDL in vivo is poorly defined, although emerging evidence suggests that these particles have distinct biological properties. This is important because elevated plasma SCN- is linked with both the propagation and prevention of atherosclerosis. In this study, we demonstrate that both HOSCN- and HOCl-modified LDL inhibit endothelium-mediated vasorelaxation ex vivo in rat aortic ring segments. In vitro experiments with human coronary artery endothelial cells show that HOSCN-modified LDL decreases in the production of nitric oxide (NO•) and induces the loss of endothelial nitric oxide synthase (eNOS) activity. This occurs to a similar extent to that seen with HOCl-modified LDL. In each case, these effects are related to eNOS uncoupling, rather than altered expression, phosphorylation or cellular localisation. Together, these data provide new insights into role of MPO and LDL modification in the induction of endothelial dysfunction, which has implications for both the therapeutic use of SCN- within the setting of atherosclerosis and for smokers, who have elevated plasma levels of SCN-, and are more at risk of developing cardiovascular disease., Highlights • Myeloperoxidase produces HOCl and HOSCN that modify LDL in a distinct manner. • HOSCN- and HOCl-LDL inhibit endothelium-mediated vasorelaxation in aortic rings ex vivo. • HOSCN- and HOCl-LDL decrease endothelial production of nitric oxide in vitro. • Decreased eNOS activity is seen, which associated with enzyme uncoupling. • HOSCN- and HOCl-LDL induce colocalisation of eNOS and caveolin 1.

Background & Aims Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The trigger for HSC activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype. Methods EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSC activation, migration, and proliferation were assessed. Loss- and gain-of-function studies were performed to explore the potential role of peroxisome proliferator-activated receptor-γ (PPAR-γ)-targeting microRNAs (miRNAs) carried by EVs into HSC. Results Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of profibrogenic genes (collagen-I, α-smooth muscle actin, and tissue inhibitor of metalloproteinases-2), proliferation, chemotaxis, and wound-healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSCs. The hepatocyte-derived EV miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression, with miR-128-3p being the most efficiently transferred. Furthermore, loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation. Conclusions Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel antifibrotic targets for NAFLD and other fibrotic diseases.