Unique immune regulation by alternatively spliced interleukin-4

描述（由申请人提供）： 白细胞介素（IL）-4在调节免疫稳态和各种免疫介导的疾病中发挥重要作用，包括但不限于哮喘、过敏、结核病和自身免疫相关的间质性肺病（硬皮病、类风湿性关节炎、多发性肌炎和皮肌炎）。我们最近描述了IL-4的剪接变体，即所谓的IL-442。IL-442的生物学似乎与IL-4的生物学非常不同，然而这种变体的功能仍然知之甚少。我们发现IL-442的表达水平在各种疾病中显著增加，包括哮喘，达到与IL-4相似或超过IL-4的水平。此外，我们最近发现IL-442改变了人T细胞中许多基因的表达水平，并且IL-442诱导的表达谱变化不同于IL-4诱导的表达谱变化。与IL- 4不同，IL-442在广泛的测试浓度范围内不刺激STAT 6的磷酸化。与IL- 4类似，IL-442以时间和剂量依赖性方式刺激T细胞中Jak 1、Jak 3和Tyk 2的磷酸化。将IL-442基因递送至小鼠肺引起肺环境中的促炎性变化，其中TNF-1、IL-11、IFN-3、IL-12 p40和MCP-1的诱导水平显著超过由IL-4基因递送诱导的水平。在哮喘患者支气管肺泡灌洗液和血T细胞中，IL-442的相对表达水平在许多情况下高于IL-4的相对表达水平。我们还发现IL-442有效地刺激人T细胞中MCP-1的产生，并且甚至更有效地（数百倍）以自分泌方式上调IL-442的产生。基于我们的初步数据，本研究的特异性假设是IL-442与T细胞上的特异性细胞表面受体结合;激活不同于IL-4诱导的信号传导的特征性细胞内信号传导;以不同于IL-4诱导的方式改变基因表达，特别是以自分泌方式刺激其自身的产生;并最终刺激大量促炎分子和Th 1分子的产生。此外，假设IL-442在体内引起肺中的显著促炎性变化，并且与人类患者中更严重的哮喘相关。在特定目的1中，将在细胞培养物中研究IL-442对原代人淋巴细胞的影响以及在分子水平上对其产生的调节。在特定目标2中，将在正常小鼠以及卵清蛋白致敏的哮喘小鼠模型中研究IL-442基因递送的体内效应。在具体目标3中，将研究IL-442与哮喘患者疾病严重程度的相关性。这项研究与退伍军人医疗保健直接相关。许多IL-4介导的疾病没有已知的治愈方法，并且在普通人群中广泛传播，特别是在退伍军人中。这些疾病是慢性的、严重的、使人衰弱的，甚至是致命的。这项研究的优势在于，它以机械的方式解决了一个新的主题;它结合了细胞培养、实验动物和人类患者的机械研究;它涉及了一种分子，这种分子参与了退伍军人中流行的许多疾病的机制。这项研究有望产生大量关于IL-442作为潜在诊断标志物以及未来治疗退伍军人多种疾病的重要靶点的新信息。

DESCRIPTION (provided by applicant): Project Summary Interleukin (IL)-4 plays a central role in the regulation of immune homeostasis and in various immune-mediated diseases including but not limited to asthma, allergies, tuberculosis, and autoimmunity-associated interstitial lung disease (scleroderma, rheumatoid arthritis, poly- and dermatomyositis). We recently described a splice variant of IL-4, so-called IL-442. The biology of IL-442 appears to be very different from that of IL-4, however the functions of this variant are still poorly understood. We found that the expression levels of IL-442 are dramatically increased in various diseases, including in asthma, to levels that are similar to or exceed those of IL-4. Furthermore, we recently found that IL-442 changes expression levels of numerous genes in human T cells, and that the IL-442-induced changes in the expression profile differ from those induced by IL-4. Unlike IL- 4, IL-442 does not stimulate phosphorylation of STAT6 in a broad range of tested concentrations. Similar to IL- 4, IL-442 stimulates phosphorylation of Jak1, Jak3, and Tyk2 in T cells in a time- and dose-dependent fashion. Gene delivery of IL-442 to mouse lungs causes proinflammatory changes in pulmonary milieu, with the induced levels of TNF-1, IL-11, IFN-3, IL-12p40, and MCP-1 significantly exceeding those induced by gene delivery of IL-4. In bronchoalveolar lavage and blood T cells of patients with asthma, relative expression levels of IL-442 were in many cases higher than those of IL-4. We also discovered that IL-442 potently stimulates production of MCP-1 in human T cells and even more potently (by several hundred fold) upregulates IL-442 production in autocrine fashion. Based on our preliminary data, the Specific Hypothesis of this study is that IL-442 binds to a specific cell surface receptor on T cells; activates characteristic intracellular signaling that is different from signaling induced by IL-4; changes gene expression in a fashion different from that induced by IL-4, particularly stimulating its own production in autocrine fashion; and ultimately stimulates production of numerous proinflammatory and Th1 molecules. Furthermore, the hypothesis is that IL-442 causes significant proinflammatory changes in the lungs in vivo, and is associated with more severe asthma in human patients. In Specific Aim 1, the effects of IL-442 on primary human lymphocytes, as well the regulation of its production at the molecular level, will be investigated in cell culture. In Specific Aim 2, effects of IL-442 gene delivery in vivo will be investigated in normal mice as well as in an ovalbumin-sensitized mouse model of asthma. In Specific Aim 3, the association of IL-442 with disease severity in asthma patients will be studied. This study is directly relevant to Veterans Healthcare. Many of the IL-4-mediated diseases have no known cures and are widely spread in the general population and particularly in veterans. These diseases are chronic, severe, debilitating, and even deadly. The strengths of this investigation are that it addresses a novel topic in a mechanistic way; that it combines mechanistic studies in cell culture, in experimental animals, and in human patients; and that it deals with a molecule that is involved in the mechanisms of numerous diseases that are prevalent in veterans. This study is expected to generate a wealth of novel information on IL-442 as a potential diagnostic marker as well as important target for future therapeutic modulation of numerous diseases in veterans.