Investigating Dead Cell-Phagocyte Interaction at the Phagosomal Interface to Decode Death-Associated Responses - PROJECT SUMMARY/ABSTRACT Cell death and the effective clearance of dead or dying cells are essential processes for maintaining homeostasis. Cells die in various physiological or pathological contexts, including tissue injury, infection, anti- tumor chemotherapy, or radiation therapy. Dead or dying cell corpses, along with the released intracellular contents, are potent activators of cellular responses such as wound healing and inflammation. These processes must be tightly regulated to prevent triggering autoimmune, fibrotic, or other diseases. Moreover, cell death can occur through distinct pathways, such as non-lytic apoptosis, or lytic necroptosis and pyroptosis, each having different effects on the surrounding milieu. Therefore, manipulating the mode of cell death or its subsequent biological effects holds great potential for developing more effective therapies against diseases like cancer. Efferocytosis, the process by which phagocytes internalize dead cells, removes and processes dead cell remnants to prevent harmful accumulation. Dead cell-containing phagosomes formed during efferocytosis are filled with entities that bind and recognize dead cells, serving as a platform for decoding death-associated information via processes such as antigen presentation. Recently, we have established a proximity-labeling- based approach, dubbed PhagoPL, to profile the protein compositions of microbe-containing phagosomes, systematically exploring microbe-sensing mechanisms in macrophages (Li et al., Nature 2024). This proof-of- concept application of the PhagoPL technique demonstrates that the composition of the phagosome is adaptive to the biochemical nature of its cargo and proves that PhagoPL holds the potential to uncover novel players in cell recognition. In this proposal, the cutting-edge PhagoPL approach will be used to investigate the protein compositions of various dead cell-containing phagosomes. By doing this, we seek to address two significant gaps in the literature related to how phagocytes interact with dead cells: (1) What are the different sets of proteins used by phagocytes to uptake, process and respond to distinct types of dead cells, specifically apoptotic, necroptotic, and pyroptotic cells? (2) How does phagosome composition dictate the ability of phagocytes, especially different types of dendritic cells, to perform antigen cross-presentation? By answering these unexplored but fundamentally important questions, this research program will provide guidance and potentially novel targets for the development of new therapeutics for autoimmune diseases, cancer, and infections.
