Complement-mediated clearance of extracellular mitochondria

About This Grant

Summary Mitochondria are released by dying neutrophils in peripheral blood and tissue, contributing to inflammation and organ damage. Little is known about mechanisms to clear the extracellular mitochondria. Neutrophils can generate, and release, complement C3, an important opsonin in removal of cell debris. However, whether neutrophils can proteolytically activate C3, and the role of C3 in clearance of mitochondria is not known. The aim of the present study is to explore mechanisms of mitochondrial clearance, with a focus on the role of neutrophils in proteolytically opsonizing mitochondria with complement C3b, and consequences of C3b in downstream clearance of mitochondria by myeloid cells. The premise is that neutrophils, through proteolytical cleavage, opsonize mitochondria with C3b facilitating their clearance while limiting inflammation and tissue damage. To investigate this, we have two main aims. The first aim investigates mechanisms related to complement C3 release and proteolytic cleavage by neutrophils. Neutrophils from patients (n=40) and controls (n=20) will be analyzed for C3 content by qPCR and flow cytometry. The identify of the C3-cleaving protease(s) will be identified using select protease inhibitors in neutrophils, supernatants and isolated NETs. Binding of C3 to mitochondria will be characterized using immunoprecipitation, mass spectrometry, and fluorescence microscopy. The second aim investigates the role of complement C3 in mitochondrial clearance, both in vitro and in vivo. In vitro, the role of C3 in opsonizing mitochondria will be determined in myeloid cells assessing uptake, induction of inflammatory cytokines, as well as NET formation by neutrophils. In vivo, GFP- tagged mitochondria will be injected in WT or C3-/- mice and assessed for biodistribution over time using confocal microscopy. At the completion of this proposed research, our expected outcomes are to have identified novel mechanisms regulating complement C3 release and acitvation by neutrophils. Further, we anticipate to have identified key pathways involved in mitochondrial clearance, and its implication for downstream effector functions in recipient cells. We expect this work to have a positive impact because it will offer novel insight into basic neutrophil biology, as well as identify targetable pathways to limit mitochondrial- mediated inflammation and subsequent organ damage in SLE.