Clostridium difficile toxin B-induced colonic inflammation is mediated by the FOXO3/PPM1B pathway in fetal human colon epithelial cells

Qingqing Xu; Ying Li; Yuejuan Zheng; Yijian Chen; Xiaogang Xu; Minggui Wang

Clostridium difficile toxin B-induced colonic inflammation is mediated by the FOXO3/PPM1B pathway in fetal human colon epithelial cells

Am J Transl Res. 2020 Oct 15;12(10):6204-6219. eCollection 2020.

Authors

Qingqing Xu^{1

2}, Ying Li^{1

2}, Yuejuan Zheng³, Yijian Chen^{1

2}, Xiaogang Xu^{1

2}, Minggui Wang^{1

2}

Affiliations

¹ Institute of Antibiotics, Huashan Hospital, Fudan University Shanghai 200040, China.
² Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commision Shanghai 200040, China.
³ Department of Immunology and Microbiology, Shanghai University of Traditional Chinese Medicine Shanghai 201203, China.

PMID: 33194024
PMCID: PMC7653611

Abstract

Clostridium difficile (C. difficile) toxin B (TcdB) is as an inflammatory enterotoxin that accounts for manifestations of widespread healthcare-associated C. difficile infection, including colonic inflammation. The present work explored the molecular mechanism by which TcdB activates innate immunity and stimulates pro-inflammatory cytokine release. Fetal human colon epithelial cells (FHCs) were treated with recombinant TcdB protein. Cell growth inhibition and apoptosis were measured with Cell Counting Kit-8 and Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit, respectively. Flow cytometry analysis was also performed. Inflammatory cytokine induction was determined with enzykeme-linked immunosorbent assay analyses. Protein expression was assessed by western blot analysis. Gene overexpression and knockdown were performed with lentiviral transduction. Real-time quantitative polymerase chain reaction was used to examine gene expression. Dual-luciferase reporter assays and chromatin immunoprecipitation were implemented to explore transcriptional regulation. Mouse colon tissues were analyzed with hematoxylin and eosin staining. The results show that TcdB-induced cell growth and apoptosis and enhanced expression of interleukin-6 and tumor necrosis factor alpha in FHCs. We identified protein phosphatase magnesium-dependent 1B (PPM1B) as the key mediator promoting the phosphorylation of nuclear factor-κB p65, which accounted for the increase in pro-inflammatory cytokines. The findings demonstrate that PPM1B expression is directly regulated by the AKT/FOXO3 signaling pathway in FHCs. We confirmed the molecular mechanism with in vivo studies using a mouse model infected with C. difficile and treated with a phosphoinositide 3-kinase/AKT signaling inhibitor. In conclusion, TcdB induces inflammation in human colon epithelial cells by regulating the AKT/FOXO3/PPM1B pathway.

Keywords: Clostridium difficile toxin B (TcdB); Forkhead box O3 (FOXO3); human colon epithelial cell; protein phosphatase magnesium-dependent 1B (PPM1B).