Role of TLR4 in Enteric Glia Response to Clostridioides Difficile Toxins: Insights From In Vivo and In Vitro Studies

Clostridioides difficile (C. difficile ) is a Gram-positive anaerobic bacillus that causes intestinal disorders. Toll-like receptor 4 (TLR4) plays a key role in innate immunity. This study examines the role of TLR4 in the response to C. difficile toxins, which induce cell death and inflammatory responses in enteric glial cells (EGCs). Male C57BL/6 mice were infected with C. difficile, and cecum samples were analysed 3 days post-infection for TLR4 expression. In vitro, EGCs were exposed to C. difficile toxins with or without C34, a TLR4 antagonist, or pre-exposed to TLR4-specific 21-nt small interfering RNAs (siRNA). TLR4 expression was assessed by immunocytochemistry, immunofluorescence, qPCR, and Western blotting. NFκB p65, TNF-α, IL-6, cleaved caspase-3, and phosphatidylserine binding to annexin-V were evaluated. TLR4 expression increased in infected intestinal tissue and toxin-exposed EGCs. TLR4 antagonist or TLR4 knockdown reduced NFκB p65 nuclear translocation and TNF-α expression but did not affect IL-6 upregulation. Additionally, TLR4 antagonist or TLR4 knockdown mitigated toxin-induced cell death, as shown by decreased cleaved caspase-3 and phosphatidylserine binding. These findings suggest that TLR4 contributes to C. difficile pathogenesis and that its inhibition reduces inflammation and prevents cell death in EGCs.