A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4–ROS–NLRP3 pathway

BACKGROUND: Giant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases. METHODS: We analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory–secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed. RESULTS: The activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4–ROS–NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome. CONCLUSIONS: rBsCPI-1 activates the inflammasome through the TLR4–ROS–NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1β and IL-18, thus promoting the occurrence and development of the inflammatory response in the host. GRAPHICAL ABSTRACT: [Image: see text]