RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection.

Pasteurella multocida is a gram-negative bacterium that causes serious diseases in a wide range of animal species. Inflammasomes are intracellular multimolecular protein complexes that play a critical role in host defence against microbial infection. Our previous study showed that bovine P. multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation. However, the exact mechanism underlying PmCQ2-induced NLRP3 inflammasome activation is not clear. Here, we show that NLRP3 inflammasome activation is positively regulated by a scaffold protein called receptor for activated C kinase 1 (RACK1). This study shows that RACK1 expression was downregulated by PmCQ2 infection in primary mouse peritoneal macrophages and mouse tissues, and overexpression of RACK1 prevented PmCQ2-induced cell death and reduced the numbers of adherent and invasive PmCQ2, indicating a modulatory role of RACK1 in the cell death that is induced by P. multocida infection. Next, RACK1 knockdown by siRNA significantly attenuated PmCQ2-induced NLRP3 inflammasome activation, which was accompanied by a reduction in the protein expression of interleukin (IL)-1ß, pro-IL-1ß, 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; these results showed that RACK1 is essential for NLRP3 inflammasome activation. Furthermore, RACK1 knockdown decreased PmCQ2-induced NF-κB activation, but RACK1 overexpression had the opposite effect. In addition, the immunofluorescence staining and immunoprecipitation results showed that RACK1 colocalized with NLRP3 and that NEK7 and interacted with these proteins. However, inhibition of potassium efflux significantly attenuated the RACK1-NLRP3-NEK7 interaction. Our study demonstrated that RACK1 plays an important role in promoting NLRP3 inflammasome activation by regulating NF-κB and promoting NLRP3 inflammasome assembly.

Chicken cathelicidin-2 promotes NLRP3 inflammasome activation in macrophages.

Chicken cathelicidin-2 (CATH-2) as a host defense peptide has been identified to have potent antimicrobial and immunomodulatory activities. Here, we reported the mechanism by which CATH-2 modulates NLRP3 inflammasome activation. Our results show that CATH-2 and ATP as a positive control induced secretion of IL-1ß and IL-1α in LPS-primed macrophages but did not affect secretion of IL-6, IL-12 and TNF-α. Furthermore, CATH-2 induced caspase-1 activation and oligomerization of apoptosis-associated speck-like protein containing a carboxy- terminal caspase recruitment domain (ASC), which is essential for NLRP3 inflammasome activation. However, CATH-2 failed to induce IL-1ß secretion in Nlrp3-/-, Asc-/- and Casp1-/- macrophages. Notably, IL-1ß and NLRP3 mRNA expression were not affected by CATH-2. In addition, CATH-2-induced NLRP3 inflammasome activation was mediated by K+ efflux but independent of the P2X7 receptor that is required for ATP-mediated K+ efflux. Gene interference of NEK7 kinase which has been identified to directly interact with NLRP3, significantly reduced IL-1ß secretion and caspase-1 activation induced by CATH-2. Furthermore, confocal microscopy shows that CATH-2 significantly induced lysosomal leakage with the diffusion of dextran fluorescent signal. Cathepsin B inhibitors completely abrogated IL-1ß secretion and caspase-1 activation as well as attenuating the formation of ASC specks induced by CATH-2. These results all indicate that CATH-2-induced activation of NLRP3 inflammasome is mediated by K+ efflux, and involves the NEK7 protein and cathepsin B. In conclusion, our study shows that CATH-2 acts as a second signal to activate NLRP3 inflammasome. Our study provides new insight into CATH-2 modulating immune response.

The Critical Role of Potassium Efflux and Nek7 in Pasteurella multocida-Induced NLRP3 Inflammasome Activation.

Pasteurella multocida is a zoonotic pathogen causing respiratory infection in different animal species such as cattle, sheep, pigs, chickens and humans. Inflammasome is a complex assembled by multiple proteins in the cytoplasm and plays an important role in the host defense against microbial infection. Bovine Pasteurella multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation and IL-1ß secretion, but the mechanism of PmCQ2-induced activation of NLRP3 inflammasome is still unknown. Therefore, the underlying mechanism was investigated in this study. The results showed that potassium efflux mediated PmCQ2-induced IL-1ß secretion and blocking potassium efflux attenuated PmCQ2-induced caspase-1 activation and ASC oligomerization. Furthermore, NIMA-related kinase 7 (Nek7) was also involved in PmCQ2-induced caspase-1 activation and IL-1ß secretion. In addition, PmCQ2 infection promoted Nek7-NLRP3 interaction, which is dependent on potassium efflux. In conclusion, our results indicate the critical role of potassium efflux and Nek7 in Pasteurella multocida-induced NLRP3 inflammasome activation, which provides useful information about Pasteurella multocida-induced host immune response.