【Cited FineTest Elisa Kit】 Current Research on Autophagy and Autoimmune Disorder

FineTest Elisa kit contributes to the research on autophagy and autoimmune disorder. The immunoassay is designed to measure IL-8 and IL-1β concentration in THP-1 and RAW 264.7 cells.

Publication Details
Article Title: Vitamin D₃ and cArbamazepine Protect against Clostridioides Difficile Infection in Mice by Restoring Macrophage Lysosome Acidification
Journal Title: Autophagy
DOI: 10.1080/15548627.2021.2016004
IF: 16.016

Abstract:        Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D₃ and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/β-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1β (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D₃ or carbamazepine suppressed pro-inflammatory cytokine expression in vitro. In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D₃ and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D₃ and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice.
Keywords:      Autophagic Flux, Clostridium Difficile, MITF, Macrophages, Toxin B

Immunoassay

Validated Image

Figure Source: Autophagy, 2022 Jan 6;1-18. doi: 10.1080/15548627.2021.2016004.

Figure 1. Pro-inflammatory cytokine induction, autophagic flux impairment and NFKB activation by TcdB in human PMA-differentiated THP-1 macrophages: (E-F)Knockdown of SQSTM1 with siRNA abolished TcdB (10 ng/ml; 6 h)-induced (E) NFKB RELA/p65 phosphorylation at serine 536 and (F) mRNA expression of IL1B, IL8, and CXCL2.