Escherichia coli K88 activates NLRP3 inflammasome-mediated pyroptosis in vitro and in vivo

Pyroptosis induced by lipopolysaccharide (LPS) has an obvious impact on intestinal inflammation and immune regulation. Enterotoxigenic Escherichia coli (ETEC) K88 has been proved to induce inflammatory responses in several models, but whether E. coli K88 participates in the same process of pyroptotic cell death as LPS remains to be identified. We conducted a pilot experiment to confirm that E. coli K88, instead of Escherichia coli O157 and Salmonella typhimurium, promotes the secretion of interleukin-1 beta (IL-1β) and interleukin-18 (IL-18) in macrophages. Further experiments were carried out to dissect the molecular mechanism both in vitro and in vivo. The Enzyme-Linked Immunosorbent Assay (ELISA) results suggested that E. coli K88 treatment increased the expression of pro-inflammatory cytokines IL-18 and IL-1β in both C57BL/6 mice and the supernatant of J774A.1 cells. Intestinal morphology observations revealed that E. coli K88 treatment mainly induced inflammation in the colon. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of pyroptosis-related factors, such as NLRP3, ASC, and Caspase1, were significantly upregulated by E. coli K88 treatment. The RNA-seq results confirmed that the effect was associated with the activation of NLRP3, ASC, Caspase1, GSDMD, IL-18, and IL-1β, and might also be related to inflammatory bowel disease and the tumor necrosis factor pathway. The pyroptosis-activated effect of E. coli K88 was significantly blocked by NLRP3 siRNA. Our data suggested that E. coli K88 caused inflammation by triggering pyroptosis, which provides a theoretical basis for the prevention and treatment of ETEC in intestinal infection.