Investigating How TLR7 Activates and TLR9 Regulates Systemic Autoimmunity

研究 TLR7 如何激活和 TLR9 如何调节系统性自身免疫

• 批准号: 10598477
• 负责人: MARK J SHLOMCHIK
• 金额: $ 45.56万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598477
• 关键词: Address; Age Months; Agonist; Animal Model; Autoantibodies; B-Lymphocytes; Biochemical; Biological; Biology; Cells; Chromatin; DNA; Data; Disease; Genetic; Goals; Image; Instruction; LoxP-flanked allele; Lupus; Macrophage; Maps; Methods; Modeling; Molecular; Mus; Mutant Strains Mice; Phenotype; Production; Property; Role; Series; Signal Pathway; Signal Transduction; Systemic Lupus Erythematosus; TLR7 gene; TLR8 gene; Testing; Therapeutic Uses; Tissues; Work; age related; cell type; experimental study; genetic approach; in vivo; inducible Cre; insight; mutant; neutrophil; novel; overexpression; risk variant; systemic autoimmunity; therapeutic evaluation

The long-term goal of the Shlomchik lab is to understand the role of TLRs in SLE. Prior work by us and others revealed that, surprisingly, disease was markedly exacerbated in the absence of TLR9, even though TLR9 was required for anti-DNA and anti-chromatin autoantibody production. The opposite roles for these two similar TLRs is likely a fundamental feature of TLR biology and of SLE, yet it is poorly understood. Given the large number of SLE risk alleles that map to the TLR signaling pathway, we will elucidate this fundamental biology and how it applies to disease mechanisms and therapy. Experiments proposed in the original application and which will be continued during the MERIT extension will directly address how TLR7 and TLR9 have divergent roles, how they cross-regulate each other, and how this promotes and regulates disease. Our lab has pioneered genetic in vivo methods for the study of lupus mechanism in animal models, in part leading to the recognition of the roles of TLR7 and TLR9, in studies supported in the prior period of this PPG. We have created novel models and used genetic approaches in order to dissect the cells and molecular signaling pathways required to promote disease in vivo. In the first three years we have made and analyzed multiple novel models, which have already greatly informed our insights into how TLR7 and 9 function differently and control lupus. Additional models are needed, and the mice we have recently made will require substantial in depth analysis; we must wait 6 months for age-dependent lupus to evolve in these mice. In addition to in vivo studies, we are now on the verge of determining biochemical and signaling properties of the mutants, which will provide very novel insights into TLR7 and 9 biology. Our mutant mice allow us to study this in primary B cells, which our data from Aim 1 shows us are the key cell type. In Aim 1 we will test the hypothesis that tissue-specific expression of TLR7 and TLR9 explains some or all of their divergent phenotypes. We will use our new TLR7 floxed mice to examine tissue-specific TLR7 expression to both macrophages and neutrophils given our recent results that TLR7 expression in B cells is not the whole story. We will also now extend our studies to TLR8. In Aim 2 we will address the hypothesis that TLR9 regulates TLR7 signals in a cell intrinsic way. To test this, we are making a series of mutant mice, whose phenotypes need to be determined together in order to determine how TLR7 and 9 differ and how they cross-regulate. We propose new imaging, biochemical and cell biological experiments that will illuminate how TR7 vs TLR9 function in primary B cells. Finally, in Aim 3, we will test the therapeutic potential of TLR inhibition using an inducible Cre to overexpress TLR9. Last, we will test TLR9 agonists to determine if TLR9 can be used therapeutically to treat disease. RELEVANCE (See instructions):

Shlomchik实验室的长期目标是了解TLRs在SLE中的作用。我们和其他人之前的工作 研究表明，令人惊讶的是，在没有TLR9的情况下，疾病明显恶化，即使TLR9 是产生抗DNA和抗染色质自身抗体所必需的。这两个角色的对立面 类似的TLRs可能是TLR生物学和SLE的基本特征，但人们对此知之甚少。给定 大量的SLE风险等位基因映射到TLR信号通路，我们将阐明这一基本原理 生物学以及它如何应用于疾病机制和治疗。原文中提出的实验 将直接解决TLR7和TLR9的问题 有不同的角色，它们如何相互交叉调节，以及这如何促进和调节疾病。我们的 实验室开创了在动物模型中研究狼疮发病机制的体内遗传方法，在一定程度上领先于 认识到TLR7和TLR9在本项目小组前期支持的研究中的作用。我们 创造了新的模型并使用了遗传方法来剖析细胞和分子信号 在体内促进疾病所需的途径。在最初的三年里，我们做出并分析了多个 新颖的模型，这些模型已经极大地启发了我们对TLR7和TLR9如何不同的功能和 控制狼疮。我们需要更多的模型，我们最近制造的老鼠将需要大量的 深度分析：我们必须等待6个月才能在这些小鼠中进化出年龄依赖型狼疮。除了在体内 研究，我们现在即将确定突变体的生化和信号特性，这是 将为TLR7和9生物学提供非常新颖的见解。我们的突变小鼠允许我们在B小学研究这一点 我们来自Aim 1的数据告诉我们，细胞是关键的细胞类型。在目标1中，我们将检验假设 TLR7和TLR9的组织特异性表达解释了它们部分或全部不同的表型。我们将使用 我们的新TLR7小鼠检测组织特异性TLR7对巨噬细胞和中性粒细胞的表达 鉴于我们最近的结果，TLR7在B细胞中的表达并不是全部。我们现在还将延长我们的 TLR8的研究。在目标2中，我们将解决TLR9调节细胞内固有的TLR7信号的假设 道路。为了测试这一点，我们正在制造一系列突变小鼠，它们的表型需要一起确定 以确定TLR7和TLR9的不同之处以及它们如何交叉调节。我们提出了新的成像方法， 生物化学和细胞生物学实验将阐明TR7和TLR9如何在原代B细胞中发挥作用。 最后，在目标3中，我们将使用可诱导的Cre过度表达来测试TLR抑制的治疗潜力 TLR9.最后，我们将测试TLR9激动剂，以确定TLR9是否可以用于治疗疾病。 相关性(请参阅说明)：