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

肿瘤坏死因子是由免疫激活的巨噬细胞分泌的一种强有力的促炎细胞因子。肿瘤坏死因子在宿主防御、肿瘤杀伤和能量代谢中具有重要作用。肿瘤坏死因子的过度分泌在急慢性炎症条件下，如感染性休克、克隆氏病、类风湿性关节炎和癌症中有许多严重的，甚至是致命的后果。在所有这些情况下，临床治疗都需要改进的抗肿瘤坏死因子疗法。我们的研究集中在研究巨噬细胞如何合成和分泌肿瘤坏死因子，最终目的是表征肿瘤坏死因子分泌途径中的分子和小泡。我们最近的发现表明，作为囊泡停靠和融合机制的一部分，SNARE蛋白的表达与激活的巨噬细胞中细胞因子的分泌和其他运输变化是一致的。我们发现Synaxin4、MUNC-18c和SNAP-23蛋白是调节肿瘤坏死因子转运到细胞表面的特异性t-SNARE复合体。在拟议的研究中，我们将通过研究SNARs的基因表达和蛋白质修饰来研究SNARs在巨噬细胞激活过程中是如何调节的。我们已经开发了一种单细胞实验来测量巨噬细胞中的肿瘤坏死因子的转运；这使得能够识别在肿瘤坏死因子分泌中起作用的分子，并将在一系列实验中使用它来识别与t-SNARE配对的特定的v-SNARE蛋白来传递肿瘤坏死因子。最后，我们将使用活细胞成像来研究肿瘤坏死因子如何和在哪里被运送到巨噬细胞表面，并通过膜分级来研究膜微区在组织圈套介导的肿瘤坏死因子分泌中的作用。利用这些研究产生的数据操纵陷阱，具有开发新的抗肿瘤坏死因子疗法的潜力。

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.