RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

Abstract

Abstract Pasteurella multocida is gram-negative bacteria that causes serious diseases in a wide range of animal species. Inflammasome as an intracellular multimolecular protein complex plays a critical role in host defense against microbial infection. Our previous study showed that bovine Pasteurella multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation. However, the exact mechanism of PmCQ2-induced NLRP3 inflammasome activation is less studied. Here, we show that NLRP3 inflammasome activation is positive regulated by a scaffold protein called receptor for activated C kinase 1 (RACK1). This study showed that RACK1 expression was downregulated by PmCQ2 infection in mice primary peritoneal macrophages and mice tissues while overexpression of RACK1 rescued PmCQ2-induced cell death, indicating the positive role of RACK1 in the host. Next, RACK1 knockdown by si-RNA significantly attenuated PmCQ2-induced NLRP3 inflammasome activation with the reduction of protein expression of interleukin (IL)-1β, pro-IL-1β, caspase-1 and NLRP3 as well as the formation of ASC specks while RACK1 overexpression by pcDNA3.1-RACK1 plasmid transfection significantly promoted PmCQ2-induced NLRP3 inflammasome activation, indicating RACK1 is essential for NLRP3 inflammasome activation. Furthermore, RACK1 knockdown decreased PmCQ2-induced NF-κB activation but RACK1 overexpression increased its activation. In addition, results of immunofluorescence staining and immunoprecipitation showed that RACK1 colocalized with NLRP3 and NEK7 and interacted with them. However, blockage of potassium efflux significantly attenuated RACK1-NLRP3-NEK7 interaction. Our study demonstrated that RACK1 plays an important role in promoting NLRP3 inflammasome activation by regulating NF-κB and promoting the NLRP3 inflammasome assembly.