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

Abstract

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.

Keywords: C57BL/6 mouse; Escherichia coli K88; J774A.1 cells; LPS; Pyroptosis.

Fig. 1

Designs of (A) pilot experiment, (B) in Vivo experiment, and (C) in Vitro experiment.

Fig. 2

Pro-inflammatory cytokine levels in the supernatant of J774A.1 b y ELISA (n = 6). (A) IL-1β, and (B) IL-18.

Fig. 3

Pro-inflammatory cytokine levels in the serum and peritoneal lavage fluid of C57BL/6 mice by ELISA (n = 6). (A) IL-1β levels in the serum, (B) IL-1β levels in the peritoneal lavage fluid, (C) IL-18 levels in the serum, and (D) IL-18 levels in the peritoneal lavage fluid (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4

Effects of K88 and LPS on the intestinal morphology in the (A) duodenum, (B) jejunum, (C) ileum, and (D) colon by H&E at 200 × magnification.

Fig. 5

Pyroptosis-related gene and protein expressions in the colon of C57BL/6 mice. (A) relative mRNA expression of NLRP3 (n = 6), (B) relative mRNA expression of Caspase1 (n = 8), (C) relative mRNA expression of ASC (n = 8), (D) relative mRNA expression of GSDMD (n = 8), (E) relative mRNA expression of IL-1β (n = 8), (F) relative mRNA expression of IL-18 (n = 8), and (G) Western blot analysis of NLRP3, Caspase1 and ASC (n = 3) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 6

Pyroptosis-related gene and protein expressions in J774A.1 cells. (A) relative mRNA expression of NLRP3 (n = 9), (B) relative mRNA expression of GSDMD (n = 9), (C) relative mRNA expression of Caspase1 (n = 9), (D) relative mRNA expression of IL-1β, (E) relative mRNA expression of IL-18 (n = 9), and (F) Western blot analysis of NLRP3, Caspase1, IL-1β and ASC in the supernatant and cell lysate (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 7

RNA-seq analysis of differentially expressed genes in the CON, K88 and LPS group. (A) Heatmap of pyroptosis-related genes expression levels; (B) volcano map of differentially expressed genes between the K88 and CON groups; (C) volcano map of differentially expressed genes between the LPS and CON groups; (D) KEGG map of relative pathways between the K88 and CON groups; and (E) KEGG map of relative pathway between the LPS and CON groups.

Fig. 8

NLRP3 knockdown abolished the apoptosis-activated effects of E. coli K88. (A) qPCR result of NLRP3 mRNA expression level, (B) relative mRNA expression of IL-1β (n = 8), and (C) relative mRNA expression of IL-18. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with scramble siRNA-treated cells; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, compared with NLRP3 siRNA-treated cells).