Role Of AIM2Inflammasome In Mycobacterium Bovis-mediated IL-1β Secrection And Cell Apoptosis

Mycobacterium bovis (M.bovis) is the causative agent of tuberculosis in a range of animal species and humen, with wordwide annual losses to agriculture and serious implications for human health. The host immune response to M. bovis infection is similar to Mycobacterium tuberculosis infection in humans. M. bovis infects and primarily replicates within macrophages, which serve as key effector cells in activating the innate and adaptive immune responses required to determine the outcome of in fection. IL-1β is one of the key proinflammatory cytokines that are essential for the recruitment of immune cells from neighboring blood vessels to the site of mycobacterial infection and regulation of granuloma formation. The mechanism by which macrophages become activated and secrete IL-1β in tuberculosis has not yet been elucidated. In this study, we investigated the role of AIM2inflammasome in the response of mouse macrophages to infection with pathogenic strain of M. bovis, and elucidated the location of M. bovis in infected macrophages, the results povided new theory for the study of innate immunity in M. bovis-infected animals.1. Firstly, J774a.1macrophages and bone-marrow derived macrophages (BMDMs) were infected with M. bovis at a multiplicity of infection (MOI) of10:1. Activated caspase-1p10and IL-1β p17in the supernatant were assesed using western blot, and the results showed that M. bovis activated caspase-1activation and lead to IL-1β production. In order to study the relationship between M. bovis and AIM2inflammasome, the inflammasome components AIM2and ASC were knocked down by gene silencing, after that, we detected activation of caspase-1and IL-1β activation, the rate of cell death, and mRNA expression of TNF-a and CCL3. The results showed that M. bovis-induced caspase-1activation and IL-1P release depended on AIM2inflammasome activation, and inflammasome activatoin was required for the synthesis of proinflammatory and chemotactic factors by M. bovzs-activated macrophage.2. Then, we examined the relevance of high extracellular K+, ROS scavenger NAC, phagocytosis to the activation of the AIM2inflammasome in M. bovis-infected macrophages. IL-1β production was assessed using western blot, the mRNA expression of AIM2and ASC were measured with qPCR. Our results showed that high extracellular K+and the inhibition of phagosytosis by cytochalasin D significantly reduced IL-1β production, and downregulated AIM2and ASC expression. Then, we investigated the relationship between AIM2and IFN, and our results indicated that knock down expression of AIM2significantly up regulated IFN-β expression, and IFN-β could reduced IL-iβ release by targeting pro-IL-1β, but IFN-β had no effect on caspase-1activation; besides, IFN-y had no impact on IL-1β production. Lastly, we examined the intracellular location of M. bovis in macrophage, and our data showed that at24h postinfection, M. bovis escaped from the phagosome to the cytosol.3. In order to investigate the relationship between AIM2inflammasome and apoptosis, AIM2was knocked down by gene silencing, after that, macrophages were infected with M. bovis. Caspase-3, caspase-8and caspase-9were detected using western blot, and mitochondrial transmembrane potential was assessed using JC-1. Our results indicated that knock down AIM2suppressed apoptosis and the change of mitochondrial transmembrane potential.