The Roles and Regulation of TLR7 in Systemic Lupus Erythematosus

项目摘要：系统性红斑狼疮(SLE)是一种破坏性的自身免疫性疾病，可导致 严重的发病率和死亡率。尽管如此，目前只有一种FDA批准的治疗SLE的新疗法 在过去的60年里，这主要是由于对疾病机制的认识存在差距。在系统性红斑狼疮中，内体TLR7和 TLR9被认为是调节反自我反应的媒介。令人惊讶的是，我们的实验室发现TLR9在 尽管TLR9需要形成抗染色质，但SLE的Faslpr模型仍会加重疾病 抗体。这种恶化依赖于TLR7，这表明TLR9是保护性的，而TLR7是驱动器 病理学。我们实验室的一个主要目标是了解TLR7和TLR9的这些不同角色。我们发现了 TLR7和TLR9的下游接头(MyD88)是介导肾毒性T细胞活化所必需的 在B细胞和皮炎发生在树突状细胞。因此，我们假设TLR7在 不同免疫细胞亚群中SLE病理的不同方面。我们将定义这些细胞- 选择性缺失TLR7在两个MRL.Faslpr队列中的特定作用：一个在B细胞中，另一个在CD11c+中 树突状细胞。我们将通过肾脏病理、皮炎、抗自身抗体等综合评估疾病严重程度 自身免疫发作后的反应、淋巴结病和免疫细胞谱分析。考虑到 在缺乏TLR9的情况下，TLR7会加剧疾病，这将在TLR9缺陷小鼠中重复，以确定是否 这改变了TLR7导致疾病的方式。 由于其致病作用，了解TLR7的调控也很重要。我们的实验室发现 NOX2基因缺陷的小鼠有明显更严重的疾病，包括抗RNA自身抗体效价增加。这个 已知这些自身抗体的产生依赖于TLR7。最近的研究表明，NOX2- 依赖的活性氧物种(ROS)可以修饰TLR7特异性半胱氨酸残基以抑制抗病毒作用 发信号。这种半胱氨酸位于TLR7的胞外区，已被证明是RNA传感和 发信号。考虑到所有这些数据，我们假设NOX2的损失加剧了 通过TLR7调节失调引起的疾病。我们将从两个方面研究这一点：首先，我们将 NOX2缺陷到TLR7缺陷-小鼠确定由于缺乏NOX2而导致的疾病加重是否 依赖于TLR7。其次，我们将使用基于质谱学的方法(OxMRM)来解决NOX2缺陷 以确定NOX2信号如何影响SLE的TLR7氧化状态。这项研究将在 创新、协作和设备齐全的芝加哥大学M.Shlomchik实验室环境 匹兹堡。与这项重点研究相关的培训计划包括板凳工作的全面指导， 数据分析、科学交流和写作，以及职业发展。这将帮助我实现 我的长期目标是成为一名独立的科学家，为我们的自身免疫知识做出贡献。

Project Summary: Systemic lupus erythematosus (SLE) is a devastating autoimmune disease that causes significant morbidity and mortality. Despite this, there has been only one new FDA-approved therapy for SLE in the past 60 years, largely due to gaps in knowledge of disease mechanisms. In SLE, endosomal TLR7 and TLR9 are known to mediate the anti-self response. Surprisingly, our lab found that TLR9 deletion in the MRL.Faslpr model of SLE exacerbates disease, despite the requirement of TLR9 for formation of anti-chromatin antibodies. This exacerbation is dependent on TLR7, suggesting that TLR9 is protective while TLR7 drives pathology. A major goal of our lab is to understand these disparate roles of TLR7 and TLR9. We have found that the downstream adaptor of TLR7 and TLR9 (MyD88) is required to mediate nephrotoxic T cell activation in B cells and dermatitis development in dendritic cells. Thus, we hypothesize that TLR7 mediates different aspects of SLE pathology in different immune cell subsets. We will define these cell- specific roles by selectively deleting TLR7 in two MRL.Faslpr cohorts: one in B cells and the other in CD11c+ dendritic cells. We will comprehensively assess disease severity via kidney pathology, dermatitis, anti-self response, lymphadenopathy, and analysis of immune cell repertoire after onset of autoimmunity. Given that TLR7 exacerbates disease in the absence of TLR9, this will be repeated in TLR9-deficient mice to determine if this changes how TLR7 contributes to disease. Because of its pathogenic role, it also important to understand the regulation of TLR7. Our lab found that NOX2-deficient mice have remarkably worse disease, including increased anti-RNA autoantibody titers. The production of these autoantibodies is known to be TLR7-dependent. Recent work has shown that NOX2- dependent reactive oxygen species (ROS) can modify a TLR7-specific cysteine residue to inhibit anti-viral signaling. This cysteine, in the ectodomain of TLR7, has been shown to be required for RNA sensing and signaling. Taking all of this data into account, we hypothesize that loss of NOX2 exacerbates disease via dysregulation of TLR7. This will be studied using a twofold approach: First, we will cross NOX2-deficient to TLR7 deficient-mice to determine if exacerbated disease driven by the absence of NOX2 is dependent on TLR7. Second, we will use a mass spectrometry-based approach (OxMRM) in NOX2 deficient cells to determine how NOX2 signaling affects TLR7 oxidation state in SLE. This research will occur in the innovative, collaborative, and well-equipped environment of the M. Shlomchik laboratory at the University of Pittsburgh. The training plan related to this key research includes comprehensive instruction in bench work, data analysis, scientific communication and writing, and professional development. This will help me achieve my long-term goal of becoming an independent scientist that contributes to our knowledge of autoimmunity.