Title of Document: Activation of NLRP3 inflammasomes during complement-mediated phagocytosis by macrophages

Title of Document: Activation of NLRP3 inflammasomes during complement-mediated phagocytosis by macrophages Rahul Suresh, Doctor of Philosophy, 2015 Directed by: Dr. David M. Mosser, Professor Department of Cell Biology & Molecular Genetics The Complement System is an important component of innate immunity, whose activation has been associated with acute inflammation. Activation of complement leads to the generation of a Membrane Attack Complex (MAC) composed of C5b-C9 proteins. The function of the MAC has been primarily associated with target cell death through the polymerization and insertion of C9 on the surface of complement activating particles. Here, I provide evidence that MAC assembled on the surface of complement activating particles, can be transferred to host macrophages during the process of phagocytosis. This “bystander activation” onto macrophages causes potassium efflux and ROS production, which induces the assembly of the NLRP3 inflammasome, which in turn results in the activation of caspase-1 and the processing and secretion of IL-1β and IL-18 to regulate both innate and adaptive immunity. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages is dependent on NLRP3, ASC, and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following complement-mediated phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promoted Th17 biasing. Leishmania major, an intracellular pathogen, follows a similar pattern, when infecting macrophages: Phagocytosis of the pathogen was accompanied by the activation of complement pathway resulting in “bystander activation” and NLRP3 inflammasome assembly. When macrophages are primed with IFN and IL-1β together prior infection, the killing of parasites is enhanced demonstrating that IL-1β can work in concert with IFNγ to activate macrophages and thus reduce the intracellular burden of the pathogen. This study demonstrates that the phagocytosis of complement-opsonized particles can induce inflammasome activation by a novel mechanism involving MAC-mediated “bystander activation” of host macrophages. This work provides another mechanism whereby complement activation can lead to acute inflammation and identifies a previously undescribed function of the MAC to activate inflammasomes on macrophages. Activation of NLRP3 inflammasomes during complement-mediated phagocytosis by macrophages