Role of Cul4A in Governing Th2-Type Tolerance

项目总结/摘要 过敏性疾病的标志是Th 2细胞在炎症部位的浸润/积聚; 然而，控制不需要的Th 2驱动的气道疾病的发展的机制很差， 明白因此，现在越来越清楚的是，了解Th 2型免疫应答和途径 控制它们对于治疗过敏性炎症可能是非常宝贵的。 最近，我们和其他人一样，已经证明了E3泛素连接酶在T细胞耐受性中的作用。 Cullin 4A（Cul 4A）属于进化上保守的E3连接酶家族，并且已经被认为是 参与造血细胞活化和染色质调节;然而，Cul 4A在T细胞中的作用 淋巴细胞尚未得到解决。有趣的是，Cul 4A的表达在耐受T细胞中上调， 细胞此外，Cul 4A敲除（KO）CD 4 + T细胞在致耐受性条件下在体外和体内活化， 与野生型细胞相比，显示出增殖沿着IL-2和IL-4产生水平的提高， 提示Cul 4A在调节T细胞耐受性中的重要作用。引人注目的是，我们发现Cul 4A KO 效应T细胞和Treg细胞表达更高水平的Th 2细胞因子，但不表达Th 1和Th 17细胞因子，这与 Cul 4A KO小鼠血清中IgG 1和IgE水平升高，表明Cul 4A在 控制炎症性Th 2型反应。重要的是，我们在CD 4 + T细胞中检测到Cul 4A表达减少， 与来自健康供体的CD 4 + T细胞相比，来自哮喘患者的Cul 4A细胞进一步表明， 可能有助于气道免疫耐受诱导。此外，在细胞中Cul 4A的shRNA敲低， 人CD 4 + T细胞导致Th 2特异性细胞因子的上调表达。基于这些发现，我们 假设Cul 4A对Th 2型免疫应答调节可能是一个重要的检查点 在气道耐受诱导中，这对于预防变应性疾病的发展至关重要。 在目的1中，我们提出确定Cul 4A通过利用Cul 4A调控T细胞活化的机制。 基因敲低方法和体外和体内Th 2耐受诱导模型。在目标2中，我们 确定Cul 4A在Th 2细胞编程中的作用及其潜在机制。我们会委聘 尖端技术，包括双杂交筛选和基因表达的微阵列分析， 鉴定Cul 4A的确切靶标以确定其在Th 2发育中的功能。此外，我们将评估 Cul 4A控制Th 2型过敏性哮喘的细胞机制。在目标3中，我们将确定 Cul 4A调节Treg功能的机制，特别是在控制Th 2炎性细胞中， 应答将在不同的体内模型中分析这一发现的生理意义。 拟议的研究将提供新的重要洞察表征的机制 潜在的Cul 4A介导的Th 2型耐受性可能具有治疗意义， 过敏性疾病

PROJECT SUMMARY/ABSTRACT The hallmark of allergic diseases is the infiltration/accumulation of Th2 cells at the sites of inflammation; however the mechanisms governing the development of unwanted Th2-driven airway diseases are poorly understood. Thus, today it is becoming clear that understanding the Th2-type immune responses and ways to control them may prove to be invaluable for the treatment of allergic inflammation. Recently, we as well as others, have demonstrated the role of E3 ubiquitin ligases in T cell tolerance. Cullin4A (Cul4A) belongs to the evolutionally conserved E3 ligase family and has been suggested to be involved in hematopoietic cell activation and in chromatin regulation; however, the role of Cul4A in T lymphocytes has not been addressed yet. Interestingly, expression of Cul4A was upregulated in tolerant T cells. Moreover, Cul4A knockout (KO) CD4+ T cells activated in vitro and in vivo under tolerogenic conditions exhibited an enhanced level of proliferation along with IL-2 and IL-4 production compared to wild-type cells, suggesting an important role of Cul4A in regulation of T cell tolerance. Strikingly, we found that Cul4A KO effector T cells and Treg cells expressed higher levels of Th2 but not Th1 and Th17 cytokines, which correlates with increased levels of IgG1 and IgE in the sera of Cul4A KO mice, indicating the selective role of Cul4A in controlling inflammatory Th2-type responses. Importantly, we detected reduced Cul4A expression in CD4+ T cells from asthmatic patients compared to CD4+ T cells from healthy donors, further indicating that Cul4A potentially contributes to airway immune tolerance induction. In addition, shRNA knockdown of Cul4A in human CD4+ T cells resulted in upregulated expression of Th2-specific cytokines. Based on these findings, we hypothesize that regulation of Th2-type immune responses by Cul4A may be an important checkpoint in airway tolerance induction, which is crucial to prevent development of allergic diseases. In Aim 1, we propose to determine the mechanisms whereby Cul4A controls T cell activation by utilizing gene knockdown approaches and in vitro and in vivo Th2 tolerance induction models. In Aim 2, we will determine the role of Cul4A in Th2 cell programming and its underlying mechanisms as well. We will employ cutting edge technologies, including two-hybrid screening and microarray analysis of gene expression, to identify the exact target(s) of Cul4A to determine its function in Th2 development. In addition, we will assess the cellular mechanisms whereby Cul4A controls Th2 type-driven allergic asthma. In Aim 3, we will determine the mechanism by which Cul4A regulates Treg function, particularly in controlling Th2 inflammatory responses. The physiological significance of this finding will be analyzed in different in vivo models. The proposed research will provide new significant insight into characterization of mechanisms underlying Cul4A-mediated Th2-type tolerance that may potentially have therapeutic implications for allergic diseases.