Interplay between Type I IFN and TGF-beta signaling in systemic sclerosis

描述(申请人提供)：尽管人们普遍认为转化生长因子-β(TGF？)在系统性硬化症(SSC)的纤维化维持中起核心作用，但其触发、激活和细胞内信号转导仍不清楚。这笔赠款的目标是了解SSC中转化生长因子信号的激活是如何上调的，重点是先天性免疫、炎症和纤维化之间的联系。新的证据表明先天免疫和干扰素(IFN)在包括SSc在内的许多自身免疫性疾病中的重要性，我们发现除了SSC皮肤和外周血单个核细胞(PBMC)中上调的TGFβ基因表达外，SSC皮肤和外周血单个核细胞(PBMC)也显示出上调的干扰素基因表达。有趣的是，最近我们发现，Toll样受体3(TLR3)配体PolyIC可以激活人成纤维细胞(FB)，不仅诱导已知受IFN调控的基因，还诱导由转化生长因子β诱导的基因，这表明转化生长因子β的激活和随后的纤维化受到先天免疫反应的调节。因此，这项应用建议研究先天免疫激活如何通过干扰素/转化生长因子之间的串扰来调节炎症和瘢痕形成。我们最令人兴奋的初步结果之一是PolyIC和干扰素都能诱导Smad1的磷酸化，表明干扰素能交叉激活转化生长因子信号。此外，我们出人意料地发现，在干扰素受体1(IFNAR1-/-)缺陷的小鼠中，干扰素α受体2(IFNAR2)激活了一条“非经典”的干扰素途径，该途径可能参与了全新的生物学功能，如控制转化生长因子信号成分的诱导。在第一个目标中，拟议的实验将通过野生型(WT)小鼠FB中干扰素和转化生长因子β之间的相互作用，研究Smad1/2/3的表达和磷酸化，IRF7和Smad7基因的表达，来研究控制促炎程序诱导和抑制之间的生理平衡的机制。然后，我们将研究异常的干扰素信号促进了干扰素和转化生长因子之间的新的串扰，并导致了TLR激活的修改平衡促进了IFNAR1-/-小鼠FB的促纤维化表型。目的2将探讨干扰素/干扰素信号障碍是否像我们在IFNAR1-/-FB中观察到的那样，是SSC FB的促纤维化表型的基础。为此目的的实验将测试干扰素和PolyIC对SSC中转化生长因子信号成分的影响以及与正常Fb相比INF/信号的功能。最后，我们将检测选定的干扰素和转化生长因子介体IRF7和Smad7在SSC和对照皮肤中的体内表达。综上所述，这些结果将极大地扩展我们对天然免疫激活、干扰素和转化生长因子在真皮纤维化中的作用的理解，为皮肤干细胞发病机制提供了一个新的观点，即干扰素/转化生长因子的损伤可能是转化生长因子失控激活的主要原因。此外，IFNAR2在控制转化生长因子信号的激活方面的新发现，为干扰素介导的信号的复杂性增加了一种新的生物学反应，可能影响针对IFN的治疗的临床应用，不仅在SSC，而且在其他疾病中也是如此。 公共卫生相关性：纤维化是疤痕形成的一种形式，是弥漫性皮肤系统性硬化症(SSC)患者临床问题的主要原因，SSC是一种涉及皮肤、肺、肾脏和胃肠道的疾病。人们对此知之甚少。这个项目的主要目标是确定干扰素(干扰素)之间的相互作用，干扰素是炎症的关键信号， 转化生长因子-β(转化生长因子-β)是瘢痕形成/纤维化的关键信号，在炎症期间，转化生长因子-β在皮肤细胞中相互作用，导致SSC中的纤维化。这些研究可能会 提供有关这些介质如何导致SSc瘢痕形成/纤维化的重要信息，并为在SSc患者中使用旨在干扰这些介质的药物的临床试验计划提供重要信息。

DESCRIPTION (provided by applicant): Although it is generally agreed that transforming growth factor-beta (TGF¿) plays a central role in sustaining fibrosis in systemic sclerosis (SSc), the triggers of TGF¿, TGF¿ activation and TGF¿ intracellular signaling are still unknown. The goal of this grant is to understand how TGF¿ signal activation is up-regulated in SSc focusing on the link between innate immunity, inflammation and fibrosis. New emerging evidence points to the importance of innate immunity and interferons (IFNs) in many autoimmune diseases including SSc, and we found that in addition to upregulated TGF¿ gene expression SSc skin and peripheral blood mononuclear cells (PBMC) also show upregulated IFN gene expression. Interestingly more recently we found that polyIC, a toll like receptor 3 (TLR3) ligand, activates human fibroblasts (fb), inducing not only genes known to be regulated by IFNs but also genes that are induced by TGF¿, suggesting that TGF¿ activation and then fibrosis is regulated by the innate immune response. Thus this application proposes to investigate how innate immune activation regulates inflammation and scarring through cross talk between IFN¿/¿and TGF¿. One of the most exciting of our preliminary results is that polyIC and IFN¿ both induce Smad1 phosphorylation, indicating crossactivation of TGF¿ signaling by IFN¿/¿. In addition we unexpectedly discovered that IFN¿ a receptor 2 (IFNAR2) in mice deficient of the IFN¿ receptor 1 (IFNAR1 -/-) activates a "non classical" IFN¿/¿ pathway, and that this pathway may be involved in entirely new biological functions such as controlling the induction of TGF¿ signaling components. In the first aim, proposed experiments will examine the mechanism controlling the physiologic balance between the induction and suppression of the pro-inflammatory program through cross talk between IFN and TGF¿ in fb from wild type (WT) mice, studying Smad1/2/3 expression and phosphorylation, IRF7 and Smad7 gene expression. Then we will investigate the consequences of aberrant IFN signaling that promote novel cross talk between IFN and TGF¿, and causing the modified equilibrium in TLR activation promoting the profibrotic phenotype in fb from IFNAR1-/- mice. Aim 2 will explore whether impairment of IFN¿/¿ signaling might underlie the pro-fibrotic phenotype that characterizes SSc fb, as we observed in IFNAR1 -/- fb. Experiments in this aim will test the effect of IFN¿ and polyIC on TGF¿ signaling components and the functioning of INF¿/¿ signaling in SSc compared to normal fb. Finally we will examine the in vivo expression of selected IFN¿/¿ and TGF¿ mediators IRF7 and Smad7, in SSc and control skin. Together, these results should significantly extend our understanding of the role of innate immune activation, IFNs and TGF¿ in dermal fibrosis, enlightening a new standpoint of SSc pathogenesis that IFN¿/¿ impairment might be the primary cause of uncontrolled TGF¿ activation. In addition the new finding of IFNAR2 function in controlling activation of TGF¿ signaling, add a novel biological response to the complexity of IFN-mediated signaling potentially influencing the clinical use of therapeutics targeting IFNs not only in SSc but also in other diseases. PUBLIC HEALTH RELEVANCE: Fibrosis, a form of scarring, is responsible for the majority of clinical problems in patients with diffuse cutaneous systemic sclerosis (SSc), a disease that involves the skin, lung, kidney and gastrointestinal tract. Little is known about. The major goal o this project is to identify the interaction between interferon (IFN), a key signal of inflammation, and transforming growth factor-beta (TGF¿), a key signal for scarring/fibrosis interact to cause fibrosis in SSc, in skin cell known as fibroblasts during inflammation. These studies are likely to provide important information about how these mediators cause scarring/fibrosis in SSc and also toward planning of clinical trials using medicines designed to interfere with these mediators in patients with SSc.