Papel do receptor de potencial transitório vanilóide 4 (trpv4) na morte celular e na resposta inflamatória induzida pelas toxinas A e B do clostridioides difficile em células gliais entéricas

Abstract

C. difficile, a Gram-positive, spore-forming anaerobic bacterium, is considered the main cause of diarrhea associated with antibiotic use in hospitalized patients, causing C. difficile infection (CDI). The main virulence factors of C. difficile are toxins A and B (TcdA and TcdB), which are involved in the inflammatory response and cell death of the intestinal epithelium. Enteric glial cells (EGCs) play a central role in the functional homeostasis of the intestinal epithelium and in inflammatory bowel diseases (IBD) pathogenesis. The receptor potential vanilloid 4 (TRPV4) is a non-selective, Ca2+ permeable cation channel widely expressed on the cell surface of the gastrointestinal epithelium and also plays an important role in the pathophysiology of IBD. This receptor is activated by various stimuli, such as bacterial endotoxins. Knowing that TRPV4 is involved in the inflammatory process of the gastrointestinal tract, the objective of this research was to evaluate the expression of TRPV4 in EGCs and its role in the inflammatory response and cell death induced by TcdA and TcdB of C. difficile in these cells, as well as its expression in the cecum and colon of mice infected by C. difficile. To evaluate the expression and role of TRPV4, EGCs were incubated with TcdA (50ng/mL) or TcdB (1ng/mL) for 18h in the presence or absence of the TRPV4 receptor antagonist (RN-1734, 100µM). TRPV4 expression in EGCs was analyzed by qPCR, Western blotting and immunofluorescence. Its role in the inflammatory response (NFκB, TNF-α, IL-6 and IL-1β) apoptosis (phosphatidylserine binding to annexin-V, cleaved caspase-3 and bcl-2) evaluated by immunofluorescence (NFκB and TNF -α), qPCR (IL-6, IL-1β and bcl-2), luminescence assay (annexin-V) and Western blotting (cleaved caspase-3). TRPV4 expression in the cecum and colon of mice infected with C. difficile (VPI10463) was analyzed by immunohistochemistry. TcdA and TcdB increase TRPV4 gene and protein expression in EGCs. RN-1734, a TRPV4 antagonist, significantly decreased NFκB nuclear translocation in EGCs induced by both toxins. Furthermore, the antagonist decreased the protein expression of TNF-α and gene expression of IL-6 in EGCs exposed to TcdA, but did not decrease the expression induced by TcdB. However, RN-1734 did not decrease IL-1β gene expression induced by toxins. RN-1734 decreased apoptosis in EGCs, reduced caspase-3 activation and upregulated antiapopototic factor bcl-2 in toxin- induced EGCs. Mice infected with C. difficile had a significant increase in TRPV4 expression in the cecum and colon and this increase in TRPV4 was detected in epithelial cells, submucosa and myenteric plexus cells compared to uninfected. Based on the these findings, infection caused by C. difficile and the action of its toxins on CGEs induce the expression of TRPV4, which in turn intensifies the inflammatory response and induces cell death by stimulating caspase-3 activation and also by downregulating bcl-2 expression in EGCs.