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

描述(申请人提供)：这项建议的总体目标是在上皮细胞和成纤维细胞水平上更好地了解IL-4Rα相关通路和转化生长因子-β2之间的相互作用，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。我们将具体讨论在上皮细胞水平影响粘液产生的相互作用和相关机制，以及在成纤维细胞水平协同增加嗜酸性粒细胞趋化因子-1的产生。我们将利用来自正常和疾病受试者的原代人类细胞。由于我们的研究几乎完全是在“外交”的人类系统中完成的，因此纳入可能直接参与相互作用的遗传因素是至关重要的，特别是IL-4Rpha的多态。在特定的目标#1中，我们将扩展初步数据，这些数据表明IL-4Rα刺激会增加上皮细胞中粘液、转化生长因子-β2和15LO 1的水平。然后，我们将进一步了解IL-4Rα和转化生长因子-β2途径之间的相互作用，以及15S HETE或13S HODE是否起中介作用。此外，由于我们建议使用人类细胞，所有受试者都将接受IL-4Rpha单核苷酸多态(SNPs)的基因分型。这些数据将在体外水平上证实我们的体内和临床研究结果，表明I4R或STAT-6结合位点的多态影响功能结果。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)直接探索转化生长因子-β2与IL-13/IL-4Rα协同作用背后的信号和转录相互作用。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，我们最初的机制研究将仅限于两种方法。首先将评估转化生长因子-β2刺激对I4R结合基序的影响以及随后对STAT-6激活和结合的影响。第二种方法将确定IL-13转化生长因子-β2刺激后产生的核蛋白的启动子结合模式。当一个或多个潜在的蛋白质结合模式得到确认时，这些蛋白质将被鉴定，它们复制协同作用的能力也将得到确认。最后，与目标1类似，由于将对原代人类细胞进行评估，我们预计反应将发生变化。因此，在AIM#1中发现的相同的IL-4Rα基因多态性的影响将与信号转导和转录结合的变化有关。这些方法应该有助于我们更好地了解这些先天和获得性免疫反应元件在组织和细胞水平上的相互作用，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.