LPS-regulated SNAREs and control of cytokine secretion in macrophages.

TNF（肿瘤坏死因子α）是由免疫激活的巨噬细胞分泌的有效促炎细胞因子。TNF在宿主防御、肿瘤杀伤和能量代谢中具有重要作用。TNF在急性和慢性炎性病症（例如败血性休克、克罗恩病、类风湿性关节炎和癌症）中的过度分泌具有许多严重的、甚至致命的后果。在所有这些病症中，需要改进的抗TNF治疗剂用于临床管理。我们的研究主要集中在研究巨噬细胞如何合成和分泌TNF，最终目标是表征TNF分泌途径中的分子和囊泡。我们最近的研究结果表明，SNARE蛋白的表达，囊泡对接和融合机制的一部分，与细胞因子分泌和活化的巨噬细胞中的其他运输变化一致。我们鉴定了蛋白质Syntaxin 4、Munc-18 c和SNAP-23作为调节TNF递送至细胞表面的特异性t-SNARE复合物。在拟议的研究中，我们将通过研究它们的基因表达和蛋白质修饰来研究SNARE在巨噬细胞活化过程中是如何调节的。我们已经开发了一种单细胞测定法来测量巨噬细胞中的TNF运输;这允许鉴定在TNF分泌中起作用的分子，并且它将用于一系列实验以鉴定与t-SNARE合作用于TNF递送的特定v-SNARE蛋白。最后，我们将使用活细胞成像来研究TNF如何以及在何处被递送到巨噬细胞表面，并通过膜分离来研究膜微区在组织SNARE介导的TNF分泌中的作用。使用这些研究产生的数据操纵SNARE，具有开发新的抗TNF疗法的潜力。

TNF(tumour necrosis factor alpha) is a potent proinflammatory cytokine secreted by immune activated macrophages. TNF has essential roles in host defense, tumour killing and energy metabolism. Excessive secretion of TNF in acute and chronic inflammatory conditions, such as septic shock, Crohn s disease, rheumatoid arthritis and in cancer has many severe, even fatal, consequences. Improved anti-TNF therapeutics are needed for clinical management in all of these conditions. Our studies are focused on investigating how macrophages synthesize and secrete TNF, with the ultimate goal of characterizing the molecules and vesicles in the TNF secretory pathway. Our recent findings show the expression of SNARE proteins, part of the vesicle docking and fusion machinery, is regulated in concert with cytokine secretion and other trafficking changes in activated macrophages. We identified the proteins Syntaxin4, Munc-18c and SNAP-23 as the specific t-SNARE complex that regulates TNF delivery to the cell surface. In the proposed studies we will investigate how SNAREs are regulated during macrophage activation by studying their gene expression and protein modifications. We have developed a single-cell assay to measure TNF trafficking in macrophages; this allows the identification of molecules with roles in TNF secretion and it will be used in a series of experiments to identify the specific v-SNARE proteins that partner the t-SNARE for TNF delivery. Finally we will use live cell imaging to investigate how and where TNF is delivered to the macrophage cell surface and membrane fractionation to examine a role for membrane microdomains in organizing SNARE-mediated TNF secretion. Manipulation of SNAREs, using data generated by these studies, holds potential for the development of new anti-TNF therapies.