Project Summary/Abstract: Macrophages are critical innate immune cells which are necessary for anti-pathogen immunity, cancer immunosurveillance and wound healing. Powering these diverse responses in macrophages is the dynamically regulated electron transport chain (ETC) within the mitochondria. An important regulator of ETC function is complex IV (CIV), which is critical for oxygen consumption, ATP generation, and cell survival. Humans with loss- of-function mutations in CIV subunits present with severe immunodeficiencies characterized by recurrent bacterial and viral infection, highlighting the importance of CIV in immune responses. Intriguingly, CIV is the only respiratory complex which remodels its protein subunit composition in response to environmental stimuli, such as hypoxia, to modulate its function. This is achieved through the upregulation of subunit isoforms which replace highly homologous core CIV subunits that function to improve cellular fitness. However, whether immune cells remodel CIV subunit composition during immune responses and whether this remodeling is important for myeloid cell function is unknown. Since inflammatory responses are powerful external stimuli that result in mitochondrial reprogramming, I hypothesized that CIV subunit composition is remodeled in macrophages and functions to regulate inflammatory responses. To this end, I identified a single transcript (AA467197) induced in pro- inflammatory macrophages which encodes a miRNA (miR-147) and a highly conserved peptide (NMES1) with striking homology to the CIV subunit NDUFA4. Intriguingly, my preliminary data and recent studies indicate that NMES1 and miR-147 work in concert to replace the core CIV subunit NDUFA4 with NMES1 in CIV. Strikingly, while remodeling of CIV subunit composition by NMES1 and miR-147 was dispensable for ATP production, I showed that this subunit switch played a critical role in regulating inflammatory cell death induced by NLRP3 inflammasome activation, termed pyroptosis. While mitochondrial function is modulated in macrophages during priming by pro-inflammatory signals such as LPS, it also can be critical in enhancing activation of the NLRP3 inflammasome through the production of ROS, release of mtDNA, externalization of cardiolipin, and regulation of ion flux in the mitochondria. Thus, our overall hypothesis is that NMES1 and miR-147 remodel CIV subunit composition in macrophages to increase NLRP3 inflammasome activation in vitro and in vivo. To explore this hypothesis, I will use macrophages sufficient or deficient in AA467197 to uncover how remodeling of CIV by NMES1 and miR-147 impacts CIV function to induce pyroptotic cell death. Additionally, I will conditionally delete AA467197 in relevant cell types and employ bacterial infection models to pinpoint how NMES1 and miR-147 regulate NLRP3 inflammasome activation and anti-bacterial immunity in vivo. Altogether, this proposal will elucidate the cellular and molecular mechanisms that und...