Regulation of TLR signaling, Inflammation and Antigen Presentation by VPS33B

项目摘要 先天性免疫应答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）。识别后，受体启动 下游信号转导途径的激活，通常涉及下游衔接子的募集 和激酶。Toll样受体家族的PRR，这是目前研究的主题，表达 在质膜和内体上都有。最近的几项研究表明， 质膜TLR（尤其是TLR 4）的内吞作用在调节细胞质和细胞周期中起关键作用。 反应性巨噬细胞中炎症反应的程度。其他研究表明TLR 信号转导增强了微生物货物的吞噬作用，但不增强凋亡细胞货物的吞噬作用，这表明在一定程度上， 不被理解的特殊性。此外，尽管TLR 4的内吞作用和内吞作用后的事件与TLR 4的内吞作用有关，但TLR 4的内吞作用可能与TLR 4的内吞作用有关。 影响信号转导的TLR 4的内吞作用已经被很好地研究，但内吞作用是否以及如何发生尚不完全清楚。 影响其他质膜和内体TLR下游的信号传导。我们的研究发现， 一种名为Vps 33 B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，Vps 33 B直接影响TLR下游信号传导的结果 以及果蝇的Toll和IMD通路。基因VPS 33 B和VPS 16 B的突变与一个基因的突变有关。 一种罕见的人类疾病，称为ARC（Arthrogryposis-renal dysfunction-cholestasis）综合征。这两个ARC 基因编码HOPS复合物亚单位的旁系同源物，表明在膜融合中的作用，但如何干扰 这些蛋白质的功能导致ARC患者疾病症状的多样性并不完全是 清楚然而，有文献记载，ARC患者患有败血症和复发性细菌感染 因此我们研究了这些蛋白质在影响免疫反应中的作用。我们发现在 在缺乏VPS 33 B情况下，果蝇对微生物刺激反应强烈。夸大的免疫反应是 响应活的或死的细菌而产生，并且Toll和IMD途径的纯化配体导致死亡 Vps 33 B突变体，而不是野生型苍蝇。Vps 33 B的这种功能在脊椎动物中是保守的，我们发现， 当受到刺激时，缺乏Vps 33 B的小鼠巨噬细胞分泌非常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， 识别受体，特别是TLR，导致依赖于Vps 33 B的特化内体的形成 用于溶酶体融合。Vps 33 B的缺乏可能影响先天性和适应性免疫的几个方面， 为了验证这个假设，我们提出1。定义在核内体中调节Vps 33 B功能的分子事件 成熟，2.定义Vps 33 B调节的TLR运输和信号传导的作用3.研究Vps 33 B的作用 规管区议会的货物处理;及研究Vps 33 B在调节抗原呈递和免疫应答中的作用。 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.