TGF-beta2 and IL-4Ralpha Pathway Interactions in Asthma

描述（由申请方提供）：本提案的总体目标是更好地了解IL-4 R α相关通路与TGF-β 2在上皮细胞和成纤维细胞水平上的相互作用，重点关注这些相互作用对嗜酸性粒细胞增多和粘液产生的重要性。我们将具体解决的相互作用和相关的机制，影响粘液生产在上皮细胞水平和协同增加嗜酸性粒细胞趋化因子-1生产在成纤维细胞水平。我们将利用来自正常和患病受试者的原代人类细胞。由于我们的研究几乎完全是在“远系繁殖”的人类系统中进行的，因此将可能直接参与相互作用的遗传元件，特别是IL-4 R α的多态性纳入其中至关重要。在具体目标#1中，我们将扩展初步数据，其显示IL-4 R α刺激增加上皮细胞中的粘液、TGF-β 2和15 LO 1水平。然后，我们将进一步了解IL-4 R α和TGF-β 2通路之间的相互作用，以及15 S HETE或13 S HODE是否起中介作用。此外，由于我们计划使用人细胞，因此将对所有受试者进行IL-4 R α单核苷酸多态性（SNP）基因分型。这些数据将在体外水平上证实我们的体内和临床发现，这些发现表明I4 R或STAT-6结合位点的多态性影响功能结局。具体目标#2将利用原代人成纤维细胞（以及后来的上皮细胞）直接探索TGF-β 2与IL- 13/ IL-4 R α协同作用背后的信号传导和转录相互作用。由于人类原代细胞存在技术限制，但我们观察到的许多发现仅在人类细胞中发现，因此我们最初的机制研究将限于两种方法。第一项研究将评估TGF-β 2刺激对I4 R结合基序的影响及其对STAT-6活化和结合的后续影响。第二种方法将确定IL-13+ TGF-β 2刺激后产生的核蛋白的启动子结合模式。当确认一种或多种潜在的蛋白质结合模式时，将鉴定蛋白质并确认其复制协同作用的能力。最后，与目标#1相似，由于将评价原代人细胞，我们预计反应将发生变异性。因此，在目标#1中鉴定的IL-4 R α中相同多态性的影响将与信号转导和转录结合的变化相关。这些方法应该导致我们在组织和细胞水平上对这些先天性和适应性免疫应答元件之间的相互作用的理解的改善，以及它们对人类哮喘的潜在影响。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to better understand the interactions between IL-4Ralpha related pathways and TGF-beta2 at the level of the epithelial cell and fibroblast, focusing on the importance of these interactions to eosinophilia and mucus production. We will specifically address the interactions and associated mechanisms, which influence mucus production at the epithelial cell level and the synergistic increases in eotaxin-1 production at the fibroblast level. We will utilize primary human cells from normal and diseased subjects. As our studies are done almost entirely in the "out-bred" human system, it is of paramount importance to incorporate genetic elements that may be directly involved in the interactions, specifically polymorphisms in IL-4Ralpha. In Specific Aim #1, we will expand on preliminary data, which show IL-4Ralpha stimulation increases mucus, TGF-beta2 and 15 LO 1 levels in epithelial cells. We will then pursue an improved understanding of the interactions between IL-4Ralpha and TGF-beta2 pathways and whether 15S HETE or 13S HODE play intermediary roles. Additionally, as we propose to use human cells, all subjects will be genotyped for single nucleotide polymorphisms (SNPs) in IL-4Ralpha. These data will confirm at the in vitro level our in vivo and clinical findings which suggest polymorphisms in I4R or STAT-6 binding sites impact functional outcomes. Specific Aim #2 will directly explore the signaling and transcriptional interactions behind the synergy of TGF-beta2 with IL- 13/ IL-4Ralpha utilizing primary human fibroblasts (and later epithelial cells). As there are technical limits to human primary cells, yet many of the findings we observe are found only in human cells, our initial mechanistic studies will be limited to two approaches. The first will evaluate the impact of TGF-beta2 stimulation on the I4R binding motif and its subsequent effect on STAT-6 activation and binding. The second approach will determine the promoter-binding pattern of the nuclear proteins generated following IL-13+TGF-beta2 stimulation. When one or more potential protein binding patterns are confirmed, the proteins will be identified and their ability to replicate the synergy confirmed. Finally, similar to Aim #1, as primary human cells will be evaluated, we anticipate variability in the response will occur. Therefore, the impact of the same polymorphisms in IL-4Ralpha identified in Aim # 1 will be related to changes in signal transduction and transcriptional binding. These approaches should lead to improvement in our understanding of interactions between these innate and adaptive immune response elements at the tissue and cellular level, as well as their potential impact on human asthma.