Abstract: Objective To explore the mechanism of F. nucleatum-induced pyroptosis in macrophages and the regulatory role of inflammasomes. Methods Lactate dehydrogenase (LDH) cytotoxicity assay and Hoechst 33342/PI double fluorescence staining were used to analyze cytolysis in F. nucleatum- infected macrophage RAW264.7 cells. The expressions of pyroptosis-related proteins caspase-1, GSDMD and IL-1β were determined using Western blotting. Inflammasome activation in the cells was analyzed by detecting the mRNA expressions of NLRP3, NLRC4, AIM2, and NLRP1 with qRT-PCR. RNA interference technique was used to knock down the key molecules involved in pyroptosis regulation in the macrophages, and the pyroptosis and necrosis rates of the cells following F. nucleatum infection were examined. Results The results of LDH cytotoxicity assay and double-fluorescence staining showed that F. nucleatum infection caused swelling and lytic cell death in RAW264.7 cells. F. nucleatum infection resulted in the activation of caspase-1 and GSDMD and upregulated IL-1β expression in a multiplicity of infection (MOI)- and time-dependent manner (P<0.05). qRT-PCR revealed significantly increased expression of NLRC4 mRNA in the macrophages after F. nucleatum infection (P<0.05). NLRC4 silencing by siRNA strongly inhibited the activation of caspase-1/GSDMD pathway and reduced cell death (P<0.05) and IL-1β expression in F. nucleatum-infected cells. Conclusion NLRC4 inflammasome drives caspase-1/GSDMD-mediated pyroptosis and inflammatory signaling in F. nucleatuminfected macrophages, suggesting the potential of NLRC4 inflammasome as a therapeutic target for F. nucleatum infections.

Key words: Fusobacterium nucleatum; macrophages; pyroptosis; NLRC4 inflammasome