Regulation of TLR signaling, Inflammation and Antigen Presentation by VPS33B

项目摘要 先天免疫反应的启动依赖于种系编码模式之间的同源相互作用 识别受体及其配体(由微生物表达)。在识别之后，受体启动 激活下游信号转导通路，通常涉及下游适配器的招募 和激活酶。表达了目前研究的PRRs的Toll样受体家族 无论是在质膜上，还是在内吞体内。最近的几项研究表明， 质膜TLRs(尤其是TLR4)的内吞作用在调节TLR4的质量和 反应的巨噬细胞的炎症反应的大小。其他研究表明，TLR 信号增强微生物货物的吞噬作用，但不能吞噬凋亡细胞货物，这表明在一定程度上 不被理解的专一性。此外，尽管TLR4的内吞作用和内吞作用后的事件 对影响信号转导的TLR4进行了很好的研究，目前还不完全清楚是否以及如何内吞作用 影响其他质膜和内体TLR下游的信号传导。在研究中，我们发现 一种名为Vps33B的蛋白质在模式识别后调节吞噬和内吞货物的处理 受体激活。更重要的是，Vps33B直接影响TLR下游信号传递的结果 在小鼠和果蝇的Toll-和IMD通路中。基因VPS33B和VPS16B的突变与 罕见的人类疾病称为ARC(关节紊乱-肾功能障碍-胆汁淤积症)综合征。这两个ARC 基因编码啤酒花复合亚单位的并列，暗示在膜融合中发挥作用，但如何扰动 这些蛋白的功能改变导致ARC患者的各种疾病症状并不完全 安全。然而，已有文献证明，ARC患者患有败血症和反复的细菌感染。 因此，我们研究了这些蛋白质在影响免疫反应中的作用。我们发现这一点 缺少VPS33B，果蝇对微生物的侮辱反应强烈。夸大的免疫反应是 对活的或死亡的细菌以及Toll和IMD途径的纯化配体产生的反应会导致死亡 Vps33B突变型，而不是野生型。Vps33B的这一功能在脊椎动物中是保守的，我们发现 缺乏Vps33B的小鼠巨噬细胞在受到刺激时会分泌大量的炎性细胞因子 通过质膜或内体TLR配体。因此，我们假设模式的激活 识别受体，特别是TLRs，导致依赖Vps33B的特化内体的形成 用于溶酶体融合。缺乏Vps33B可能会影响先天免疫和获得性免疫的几个方面，并 验证这一假说，我们建议1.定义调节内体Vps33B功能的分子事件 成熟，2.确定Vps33B调节的TLR运输和信号转导的作用3.研究Vps33B的作用 4.研究Vps33B在调节抗原提呈和调节抗原提呈中的作用 适应性免疫。

Project Summary Initiation of innate immune responses depends on cognate interaction between germline-encoded pattern recognition receptors and their ligands (expressed by microbes). Following recognition, the receptors initiate activation of downstream signal transduction pathways that often involve recruitment of downstream adapters and kinases. The Toll-like receptor family of PRRs, which are the subject of current investigation, are expressed both on the plasma membrane and in the endosomes. Several recent studies have demonstrated that endocytosis of the plasma membrane TLRs (especially TLR4) plays a critical role in regulating both quality and magnitude of inflammatory responses in a responding macrophage. Other studies have demonstrated that TLR signaling enhances phagocytosis of microbial cargo but not of apoptotic cell cargo suggesting a degree of specificity that is not understood. In addition, although endocytosis of TLR4 and the events following endocytosis of TLR4 that influence signal transduction are very well studied, it is not entirely clear if and how endocytosis influences signaling downstream of other plasma membrane and endosomal TLRs. In our studies, we find that a protein called Vps33B regulates handling of the phagocytic and endocytic cargo following pattern recognition receptor activation. More importantly, Vps33B directly influences the outcome of signaling downstream of TLRs in mice and Toll- and IMD pathways in Drosophila. Mutations in the genes VPS33B and VPS16B are linked to a rare human disease called ARC (Arthrogryposis-renal dysfunction-cholestasis) syndrome. Both of these ARC genes encode paralogs of HOPS complex subunits suggesting a role in membrane fusions but how perturbation of function of these proteins results in a diverse spectrum of disease symptoms in ARC patients is not entirely clear. It has however been documented that ARC patients suffer from sepsis and recurrent bacterial infections and we were therefore investigated the role of these proteins in influencing immune responses. We find that in the absence of VPS33B, Drosophila respond vigorously to microbial insult. Exaggerated immune responses are generated in response to live or dead bacteria and purified ligands of the Toll and IMD pathway results in death of Vps33B mutant, but not wild-type flies. This function of Vps33B is conserved in vertebrates and we find that mouse macrophages lacking Vps33B secrete very high quantities of inflammatory cytokines, when stimulated by either plasma membrane or endosomal TLR ligands. We therefore hypothesize that activation of pattern recognition receptors and specifically TLRs leads to formation of specialized endosomes that depend on Vps33B for lysosomal fusion. Lack of Vps33B is likely to affect several aspects of innate and adaptive immunity and to test this hypothesis, we propose to 1. Define the molecular events that regulate Vps33B function in endosomal maturation, 2. Define the role of Vps33B-regulated TLR trafficking and signaling 3. Investigate the role of Vps33B in regulating cargo handling by DCs and 4. Investigate the role of Vps33B in regulating antigen presentation and adaptive immunity.