Clostridium butyricum alleviates DSS-induced colitis by suppressing neutrophil extracellular trap formation via the IL-17 pathway.

Abstract

BACKGROUND: Neutrophil extracellular traps (NETs) play a critical role in amplifying intestinal inflammation in ulcerative colitis (UC). Clostridium butyricum (CB) has shown anti-inflammatory effects in gastrointestinal diseases; however, its impact on NETs formation and related molecular mechanisms remains unclear. METHODS: UC was induced in C57BL/6 mice by DSS, followed by CB and/or PMA administration. Colonic injury was assessed by colon length measurement, histopathology, and histological scoring. NETs formation was determined by immunofluorescence staining of Ly6G and citrullinated histone H3 (Cit-H3), and serum myeloperoxidase (MPO)/Cit-H3 levels were quantified by ELISA. In vitro, NETs release was induced in neutrophils by PMA with or without CB supernatant (CBS) administration. RNA-seq and qRT-PCR/Western blot analyses were used to explore underlying signaling pathways. IL-17A knockdown via siRNA was conducted to validate mechanistic involvement. RESULTS: CB significantly alleviated DSS-induced colitis, evidenced by reduced colon shortening, lower colon mass, and improved mucosal architecture. CB markedly suppressed NETs formation in both colonic tissue and serum. Comparative transcriptomics indicated that CBS suppresses NETs formation primarily through modulation of the IL-17 signaling pathway. DSS-induced colitis and PMA stimulation markedly increased the expression of IL-17A, IL-17RA, and p-p65 and elevated pro-inflammatory cytokines (IL-1β and TNF-α), while reducing the anti-inflammatory cytokine IL-10. CB or CBS treatment significantly reversed these pathological changes in both colon tissues and neutrophils. Importantly, IL-17A knockdown significantly reduced PMA-induced activation of the IL-17A/IL-17RA/NF-κB, and CBS treatment further enhanced these inhibitory effects under IL-17A-deficient conditions. CONCLUSION: CB protects against DSS-induced colitis by suppressing IL-17A/IL-17RA/NF-κB-mediated NETs formation in neutrophils. IL-17A knockdown confirmed IL-17A as a critical upstream regulator of NETs, establishing the IL-17A-NETs axis as a central mechanistic target of CB.