LC3-Associated Phagocytosis

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Abstract Two ancient processes, phagocytosis and macroautophagy, arose as ways to meet the energy demands of the cell. Both also evolved into mechanisms of host defense. During the previous support period for this grant, we discovered a process we term "LC3-Associated Phagocytosis" (LAP). In this process, signals that are generated upon engulfment of particles by phagocytic cells induce components of the autophagy machinery to associate with the phagosome, promoting its fusion to lysosomes (phagosome maturation). While engulfment of latex beads (for example) does not induce LAP, particles that engage TLR1/2, TLR2/6, TLR4, FcR, or receptors for engulfment of dying cells, cause recruitment of LC3 (ATG8) to the phagosome membrane. Like macroautophagy, this LC3 association depends on Beclin1, PI3P generation, ATG5, and ATG7, but unlike autophagy, LC3 associates with the single phagosome membrane (rather then the double membrane of autophagosomes). Further, unlike macroautophagy, LAP proceeds in the absence of elements of the autophagic pre-initiation complex, ULK1, ATG13, and FIP200. This raises an intriguing possibility: It is now well established that defects in some components of the autophagy machinery promote inflammatory disease and compromise host defense to intracellular infections. The existence of LAP as a discrete phenomenon suggests that at least some such effects may specifically relate to LAP. Here, we propose to characterize LAP, its relationship to phagosome maturation, and its roles in innate immune responses and normal homeostasis. Our central hypothesis, upon which this application is based, is that depending on signaling that accompany phagocytosis, LAP can be engaged to promote the sorting of the phagosome cargo to intracellular compartments for further signal detection, processing, or degradation. Specifically, we will ask: 1. What distinguishes the initiation of LAP versus macro-autophagy? Here we will explore the molecular events that initiate and propagate LAP and evaluate how these differ from those of conventional macroautophagy. 2. How does LAP promote phagosome maturation? Here we will investigate how the components of LAP greatly accelerate phagosome maturation and the points in each pathway where this enhancement occurs. We will further examine the consequences of LAP-induced phagosome maturation for macrophage-mediated host defense. 3. How does LAP impact on inflammation and homeostasis? Here we will use in vitro and in vivo systems to interrogate the roles of LAP in the inflammatory response to dying cells, in vitro and in vivo. While apoptotic cells are thought to be "immunologically silent" our evidence suggests that this may be, at least in part, due to suppression of the inflammatory cytokine response by LAP in phagocytes. We will test this exciting idea, and explore the long-term inflammatory consequences of defective LAP in macrophages and other compartments. Overall, our project seeks to characterize how LAP, as the conjunction of two ancient pathways, impacts innate immunity, offering new avenues for understanding inflammatory disease.