Role of mitochondrial anti-viral signaling protein and dynamin-related protein 1 for the NLRP3 inflammasome activation

Abstract

NLRP3 inflammasome is an intracellular protein complex composed of NLRP3, adaptor protein ASC and procaspase-1, and activates caspase-1 in response to various stimuli such as pathogens, microbial toxin, crystalline materials, and endogenous danger signals. Many studies have tried to figure out how NLRP3 inflammasome is activated, but molecular mechanisms are still unclear. Recent studies suggest that mitochondria play an important role in NLRP3 inflammasome activation by releasing of mitochondrial product from damaged mitochondria such as mitochondrial ROS (mtROS) or mitochondrial DNA (mtDNA). However, despite diverse studies, how mitochondria regulate NLRP3 inflammasome remains to be explored. Therefore, I attempt to demonstrate the mitochondrial regulation of NLRP3 inflammasome activation by focusing mitochondrial protein MAVS and abnormal mitochondrial dynamics induced by decrease of mitochondrial fission-associated protein Drp1 protein expression. In PART1, I demonstrated that mitochondrial anti-viral signaling protein MAVS interacts with NLRP3, and mitochondrial MAVS promoted NLRP3 inflammasome activation by facilitating ROS-dependent NLRP3 oligomerization on mitochondrial outer membrane in response to Sendai virus infection. Supporting this, knockdown of MAVS expression in macrophages showed a significant decrease in NLRP3-mediated caspase-1 activation in response to Sendai virus infection. Moreover, both mouse macrophages and human THP-1 cells exhibited MAVS-mediated NLRP3 inflammasome assembly and activation in response to Sendai-virus infection. Thus, mitochondrial MAVS facilitates ROS-dependent NLRP3 oligomerization, and promotes NLRP3 inflammasome activation in response to Sendai virus infection. In PART 2, I demonstrated that abnormal mitochondrial dynamics caused by knockdown of Drp1 protein expression was associated with NLRP3 inflammasome activation. Drp1 knockdown macrophages exhibited elongated mitochondria and increase NLRP3 inflammasome activation in response to LPS followed by ATP. Furthermore, Drp1 knockdown-mediated mitochondrial elongation enhanced the formation of NLRP3 inflammasome assembly. On the other hand, mitochondrial fragmentation induced by mitochondrial fission inducer CCCP treatment show highly attenuated NLRP3 inflammasome assembly and activation. At molecular levels, Drp1 knockdown macrophages show increase of the release of mitochondrial product such as mtROS or cytosolic mtDNA. However, caspase-1-selective inhibitor YVAD treatment prevented the release of mitochondrial products. Thus, this phenomenon might be the consequences of caspase-1 activation. Interestingly, Drp1 knockdown macrophages show constitutively phosphorylated extracellular signal-regulated kinase (ERK) without any stimulation, suggesting that ERK has a critical role in NLRP3 inflammasome assembly by localizing NLRP3 into mitochondria. Supporting this, ERK phosphorylation inhibitor U0126 treatment induced markedly impaired NLRP3 inflammasome assembly and activation. Therefore, these data show that Drp1 knockdown-mediated abnormal mitochondrial elongation enhances NLRP3 inflammasome activation in response to classical NLRP3 activating stimuli such as LPS plus ATP in an ERK signaling dependent manner. Collectively, I demonstrated MAVS- and Drp1-dependent mitochondrial regulation to the NLRP3 inflammasome activation.