Anti-bacterial inflammatory responses in phagocytes are initiated by recognition of common pathogen associated molecular patterns (PAMPs) by innate immune receptors. PAMP binding to membrane-associated toll-like receptors (TLRs) at distinct subcellular sites triggers site-specific responses, and bacteria that compromise phagosomal membranes additionally trigger cytoplasmic receptors, some of which assemble into multisubunit inflammasomes that process and release IL-1 family cytokines. Membrane dynamics within host phagocytes dramatically influence TLR and inflammasome localization and signaling, but host regulation of such dynamics has been relatively unexplored. Our studies in a genetic disease model identified the endosomal adaptor complex AP-3 as a central hub for intracellular trafficking pathways that regulate both TLR and inflammasome responses to phagocytosed bacteria in dendritic cells (DCs) - immune cells that link innate responses to adaptive immunity. However, AP-3 impacts these responses indirectly, reflecting AP-3's central role in endolysosomal protein sorting. We hypothesize that defining direct targets of AP-3 sorting will elucidate new membrane pathways controlling innate signaling and downstream anti-bacterial immune responses. AP-3 sorts cargoes on endosomes into vesicles bound for lysosomes, phagosomes or related organelles. We hypothesize that innate signaling defects in AP-3-deficient DCs reflect depletion of AP-3 cargoes from these organelles. Preliminary data suggest that one such cargo is PI4K2?, an enzyme that generates the lipid phosphatidylinositol-4-phosphate to recruit TLRs via their proinflammatory adaptors to membranes.
Aim 1 will test whether phagosomal PI4K2? recruits TLRs to initiate pro-inflammatory signaling and antigen presentation. Other putative AP-3 cargoes include lysosomal transporters that function in lysosome homeostasis. We hypothesize that depletion of such cargoes triggers a lysosome-dependent signaling cascade that promotes inflammasome silencing by autophagy.
Aim 2 will test whether and how lysosomal disruption impacts inflammasome activity, and Aim 3 will test whether depletion of specific candidate lysosomal membrane channels similarly silence inflammasomes and impact lysosome signaling and adaptive immune responses. Fulfillment of the following Specific Aims will elucidate novel pathways that are potentially targeted by genetic disease and/or by pathogen interference to compromise host immunity to bacterial pathogens. 1. To test whether TLR signaling from phagosomes and downstream responses are regulated by the AP-3-associated PtdIns-4-kinase PI4K2?. 2. To test whether impaired lysosomal function dampens inflammasome activity through nutrient- dependent signaling. 3. To test whether lysosomal membrane transporter expression influences inflammasome activation.
Dendritic cells are immune sentinels that capture bacterial pathogens and stimulate protective inflammatory and adaptive immune responses against them. The nature and strength of these responses are controlled by membrane dynamics within dendritic cells, and genetic diseases that alter these dynamics impact the ability to fight bacterial infections. In this proposal, we exploit our observations in a mouse model of a human immunodeficiency disorder to discover new dendritic cell membrane trafficking pathways that regulate inflammatory anti-bacterial responses and that may be targeted in as yet undiscovered or poorly characterized immune disorders.
