The Roles and Regulation of TLR7 in Systemic Lupus Erythematosus

系统性红斑狼疮（SLE）是一种毁灭性的自身免疫性疾病， 严重的发病率和死亡率。尽管如此，只有一种新的FDA批准的SLE治疗方法 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SLE中，内体TLR 7和 已知TLR 9介导抗自身应答。令人惊讶的是，我们的实验室发现，TLR 9缺失， SLE的Faslpr模型加重疾病，尽管TLR 9需要形成抗染色质 抗体的这种恶化依赖于TLR 7，表明TLR 9是保护性的，而TLR 7驱动 病理我们实验室的一个主要目标是了解TLR 7和TLR 9的不同作用。我们发现 TLR 7和TLR 9的下游衔接子（MyD 88）是介导肾毒性T细胞活化所必需的 在B细胞和皮炎发展的树突状细胞。因此，我们假设TLR 7介导 不同免疫细胞亚群中SLE病理学的不同方面。我们将定义这些细胞- 在两个MRL.Faslpr群组中通过选择性地删除TLR 7来确定特异性作用：一个在B细胞中，另一个在CD 11 c + 树突状细胞我们将通过肾脏病理学、皮炎、抗自身抗体、 反应、淋巴结病和自身免疫发作后免疫细胞库的分析。鉴于 如果TLR 7在缺乏TLR 9的情况下加重疾病，则将在TLR 9缺陷小鼠中重复这一过程以确定是否存在TLR 9缺陷。 这改变了TLR 7对疾病的贡献。 由于其致病作用，了解TLR 7的调节也很重要。我们的实验室发现 NOX 2缺陷小鼠的疾病明显更严重，包括抗RNA自身抗体滴度增加。的 已知这些自身抗体的产生是TLR 7依赖性的。最近的研究表明，NOX 2- 依赖性活性氧（ROS）可以修饰TLR 7特异性半胱氨酸残基以抑制抗病毒 信号在TLR 7的胞外域中的这种半胱氨酸已被证明是RNA传感所需的， 发信号。考虑到所有这些数据，我们假设NOX 2的损失加剧了 疾病通过TLR 7的失调。这将使用双重方法进行研究：首先，我们将交叉 将NOX 2缺陷小鼠与TLR 7缺陷小鼠进行比较，以确定由NOX 2缺乏引起的疾病恶化是否是 依赖于TLR 7其次，我们将使用基于质谱的方法（OxMRM）在NOX 2缺乏的 细胞来确定NOX 2信号传导如何影响SLE中的TLR 7氧化状态。这项研究将在 创新，协作，和装备精良的环境的M。莫斯科大学的Shlomchik实验室 匹兹堡与本重点研究相关的培训计划包括长凳工作的综合指导， 数据分析，科学交流和写作，以及专业发展。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.