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

TL;DR

This study shows that TLR4 contributes to intestinal inflammation and cell death caused by Clostridioides difficile toxins, and blocking TLR4 reduces these effects.

Contribution

The study reveals TLR4's role in C. difficile-induced inflammation and cell death in enteric glial cells, and demonstrates that inhibiting TLR4 mitigates these responses.

Findings

TLR4 expression increases in C. difficile-infected intestinal tissue and toxin-exposed enteric glial cells.

Blocking TLR4 reduces NFκB p65 nuclear translocation and TNF-α expression but does not affect IL-6 upregulation.

TLR4 inhibition mitigates toxin-induced cell death in enteric glial cells.

Abstract

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.

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