Membrane trafficking in innate immunity to bacterial pathogens

总结 吞噬细胞中的抗菌炎症反应是由识别常见病原体启动的 相关的分子模式（PAMPs）的先天免疫受体。PAMP与膜相关 不同亚细胞部位的toll样受体（TLR）触发位点特异性反应， 受损的吞噬体膜另外触发细胞质受体，其中一些组装成 多亚基炎性小体，其加工和释放IL-1家族细胞因子。宿主体内膜动力学 吞噬细胞显著影响TLR和炎性小体的定位和信号传导，但宿主调节 这种动力学相对来说还没有被探索过。我们在遗传疾病模型中的研究确定了 内体适配器复合物AP-3作为调节TLR和TLR的细胞内运输途径的中心枢纽 树突状细胞（DCs）中吞噬细菌的炎性体反应-免疫细胞连接先天性 获得性免疫的反应。然而，AP-3间接影响这些反应，反映了AP-3的中心作用。 在内溶酶体蛋白分选中的作用。我们假设定义AP-3分选的直接靶点将阐明 控制先天信号传导和下游抗菌免疫应答的新膜途径。 AP-3将内体上的货物分类为与溶酶体、吞噬体或相关细胞器结合的囊泡。我们 假设AP-3缺陷DC中的先天性信号传导缺陷反映了来自这些DC的AP-3货物的耗尽， 细胞器初步数据表明，PI 4K 2 α是一种产生脂质的酶， 磷脂酰肌醇-4-磷酸通过其促炎衔接子将TLR募集到膜上。目标1将 测试吞噬体PI 4K 2 α是否募集TLR以启动促炎信号传导和抗原呈递。 其他推定的AP-3货物包括在溶酶体稳态中起作用的溶酶体转运蛋白。我们 假设这些货物的耗尽触发了溶酶体依赖性信号级联， 炎性小体通过自噬沉默。目标2将测试溶酶体破坏是否以及如何影响 Aim 3将测试特异性候选溶酶体膜是否耗尽， 通道类似地沉默炎性体并影响溶酶体信号传导和适应性免疫应答。 实现以下特定目的将阐明基因治疗潜在靶向的新途径。 疾病和/或病原体干扰以损害宿主对细菌病原体的免疫力。 1.为了测试来自吞噬体的TLR信号传导和下游反应是否受 AP-3相关PtdIns-4-激酶PI 4K 2 α。 2.为了测试受损的溶酶体功能是否通过营养- 依赖信号 3.检测溶酶体膜转运蛋白表达是否影响炎性小体激活。

SUMMARY 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 PI4K2α, an enzyme that generates the lipid phosphatidylinositol-4-phosphate to recruit TLRs via their proinflammatory adaptors to membranes. Aim 1 will test whether phagosomal PI4K2α 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 PI4K2α. 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.