Membrane trafficking in innate immunity to bacterial pathogens

摘要 吞噬细胞中的抗菌炎性反应是通过识别常见病原体而启动的 先天免疫受体的相关分子模式(PAMP)。PAMP与膜相关的结合 位于不同亚细胞位置的Toll样受体(TLRs)可触发部位特异性反应，而细菌 受损的吞噬体膜还会触发细胞质受体，其中一些会组装成 多亚单位炎症体，处理和释放IL-1家族细胞因子。寄主体内的膜动力学 吞噬细胞显著影响TLR和炎症体的定位和信号转导，但宿主调节 这种动态相对来说还没有被探索过。我们在遗传病模型中的研究发现 内体适配器复合体AP-3作为调节两种TLR的细胞内转运途径的中心枢纽 树突状细胞(DC)--连接先天免疫细胞的免疫细胞--对吞噬细菌的炎症体反应 对适应性免疫的反应。然而，AP-3间接影响这些反应，反映了AP-3的S中枢 在内溶酶体蛋白分选中的作用。我们假设，定义AP-3分类的直接目标将阐明 控制先天信号和下游抗细菌免疫反应的新的膜途径。 AP-3将内体上的货物分类成小泡，运往溶酶体、吞噬体或相关细胞器。我们 假设AP-3缺陷树突状细胞中的先天信号缺陷反映了这些来源的AP-3货物的耗尽 细胞器。初步数据显示，其中一个这样的货物是PI4K2α，一种产生脂质的酶 磷脂酰肌醇-4-磷酸通过其促炎适配器将TLR募集到膜上。目标1将 测试吞噬小体PI4K2α是否招募TLR来启动促炎信号和抗原呈递。 其他推测的AP-3货物包括溶酶体转运体，功能在溶酶体动态平衡。我们 假设这些货物的耗尽触发了溶酶体依赖的信号级联反应，从而促进了 通过自噬使炎症体沉默。目标2将测试溶酶体破坏是否以及如何影响 炎症体活性，目标3将测试特定候选溶酶体膜是否耗尽 通道同样能抑制炎性小体，影响溶酶体信号和适应性免疫反应。 以下特定目标的实现将阐明潜在的基因靶向的新途径 疾病和/或病原体干扰，损害宿主对细菌病原体的免疫力。 1.测试吞噬小体的TLR信号和下游反应是否受 AP-3相关的PtdIns-4-激酶PI4K2α。 2.检测溶酶体功能受损是否通过营养抑制炎性小体活动。 依赖于信号。 3.检测溶酶体膜转运蛋白的表达是否影响炎性小体的激活。

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.