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: 9273442
• 负责人: MARK J SHLOMCHIK
• 金额: $ 38.54万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-16 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9273442
• 关键词: Address; Alpha Cell; Animal Model; Autoantibodies; Autoimmune Diseases; Autoimmunity; B-Cell Activation; B-Lymphocytes; Backcrossings; Behavior; Binding; Biochemical; Biology; Birth; Cell Line; Cells; Chromatin; Clinic; Complex; Coupled; Cytoplasmic Tail; DNA; Data; Development; Disease; Ear; Future; Genetic; Goals; Human; ITGAX gene; Immune system; In Vitro; Lighting; Limb structure; LoxP-flanked allele; Lupus; Maps; Mediating; Methods; Modeling; Molecular; Mus; Mutant Strains Mice; Nucleic Acids; Phenotype; Production; Reagent; Role; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Systemic Lupus Erythematosus; TLR7 gene; Tamoxifen; Testing; Tissue Engineering; Tissues; Work; Yin-Yang; base; cell type; design; experimental study; genetic approach; genetic regulatory protein; in vivo; inhibitor/antagonist; lupus prone mice; macrophage; mouse model; mutant; neutrophil; novel; overexpression; protein expression; response; risk variant; targeted treatment; therapeutic evaluation; tool; trafficking

Abstract The long-term goal of the Shlomchik lab is to understand the role of TLRs in SLE. In our previous work, a most surprising finding was that disease was markedly exacerbated in the absence of TLR9, even though TLR9 was required for anti-DNA and anti-chromatin autoantibody production. This has now been confirmed in multiple independent models of murine lupus. The opposite roles for these two similar TLRs is likely a fundamental fea- ture of TLR biology and thus of SLE, yet it is poorly understood. Given the prominent role of TLR signaling in human lupus and the large number of SLE risk alleles that map to the TLR signaling pathway, it is of im- portance to elucidate this fundamental biology and how it applies to disease mechanisms and therapy. The discovery of the key role of TLRs in multiple murine lupus models, coupled with the genetic evidence, has led to the development and testing of TLR inhibitors in the clinic for SLE and other autoimmune diseases, high- lighting the importance. Experiments proposed here will directly address how TLR7 and TLR9 have divergent roles, how they cross-regulated each other, and how this promotes and regulates disease. Our lab has pio- neered 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 creat- ed novel models and used genetic approaches in order to dissect the cells and molecular signaling pathways required to promote disease in vivo. Here we continue to make novel models and study them in vivo, coupled with studies of the basic signaling and biology of TLR7 and 9 at the cellular and biochemical levels. We hypothesize that differential tissue-specific expression and signaling can explain the disparate global ef- fects of TLR7 and TLR9 deletion, an idea supported by our preliminary data with new TLR9 “floxed” mice. To address this, we will manipulate the expression of TLRs in vivo in key cell types. In Aim 1 we will test the hy- pothesis that tissue-specific expression of TLR7 and TLR9 explains some or all of their divergent phenotypes. We will extend our work on tissue-specific TLR9 expression to both macrophages and neutrophils and to addi- tional lupus models. We will make new floxed and rosa26 conditional mice to allow TLR7 tissue-specific ex- pression. This will let us study the yin-yang of TLR7 and TLR9; to date we have only looked at TLR9. In Aim 2 we will address the hypothesis that TLR9 regulates TLR7 signals in a cell intrinsic way. To test this, we have made several tools, including mutants of TLR9 and TLR7, and will make additional mutants and mutant mice. Our unique focus will be on primary B cells, enabled by Aim 1. This will allow us to determine both in vivo and in vitro if TLR9 suppresses TLR7 signaling by competing for trafficking molecules, for signaling molecules or by inducing the expression of counter-regulatory proteins. In addition, we will test whether the cytoplasmic tails of TLR7 and TLR9 mediate different signaling. Finally, in Aim 3, we will test the therapeutic potential of TLR inhi- bition using an inducible Cre to delete MyD88 and thus end TLR7/9 signaling at different stages of disease.

抽象的 Shlomchik 实验室的长期目标是了解 TLR 在 SLE 中的作用。在我们之前的工作中，最 令人惊讶的发现是，尽管 TLR9 存在，但在缺乏 TLR9 的情况下，疾病明显恶化。 抗 DNA 和抗染色质自身抗体生产所需。目前这一消息已被多方证实 小鼠狼疮的独立模型。这两个相似的 TLR 的相反作用可能是一个基本特征 TLR 生物学的本质以及 SLE 的本质，但人们对其知之甚少。鉴于 TLR 信号传导在 人类狼疮和大量映射到 TLR 信号通路的 SLE 风险等位基因，它具有重要意义。 阐明这一基本生物学及其如何应用于疾病机制和治疗具有重要意义。这 TLRs 在多种小鼠狼疮模型中的关键作用的发现，加上遗传证据，导致 在临床上开发和测试针对 SLE 和其他自身免疫性疾病的 TLR 抑制剂，高 照明的重要性。这里提出的实验将直接解决 TLR7 和 TLR9 之间的差异 角色，它们如何相互交叉调节，以及如何促进和调节疾病。我们的实验室有 pio- 需要遗传体内方法来研究动物模型中的狼疮机制，部分导致 在本 PPG 前期支持的研究中认识到 TLR7 和 TLR9 的作用。我们有创造- 开发新模型并使用遗传方法来剖析细胞和分子信号传导途径 需要促进体内疾病。在这里，我们继续制作新颖的模型并在体内研究它们，结合 在细胞和生化水平上研究 TLR7 和 9 的基本信号传导和生物学。 我们假设差异组织特异性表达和信号传导可以解释不同的全局效应 TLR7 和 TLR9 缺失的影响，我们对新 TLR9“floxed”小鼠的初步数据支持了这一想法。到 为了解决这个问题，我们将操纵关键细胞类型体内 TLR 的表达。在目标 1 中，我们将测试 hy- TLR7 和 TLR9 的组织特异性表达解释了它们的部分或全部不同表型的假设。 我们将把组织特异性 TLR9 表达的工作扩展到巨噬细胞和中性粒细胞以及其他细胞。 狼疮模型。我们将制造新的 floxed 和 rosa26 条件小鼠，以允许 TLR7 组织特异性前 压力。这将使我们研究TLR7和TLR9的阴阳；到目前为止，我们只关注了 TLR9。目标 2 我们将提出这样一个假设：TLR9 以细胞固有的方式调节 TLR7 信号。为了测试这一点，我们有 制作了多种工具，包括 TLR9 和 TLR7 的突变体，并将制作更多的突变体和突变小鼠。 我们的独特重点将是通过 Aim 1 实现的原代 B 细胞。这将使我们能够确定体内和 在体外，如果 TLR9 通过竞争运输分子、信号分子或通过竞争抑制 TLR7 信号传导 诱导反调节蛋白的表达。此外，我们将测试细胞质尾部是否 TLR7 和 TLR9 介导不同的信号传导。最后，在目标 3 中，我们将测试 TLR 抑制的治疗潜力 使用诱导型 Cre 来删除 MyD88，从而在疾病的不同阶段终止 TLR7/9 信号传导。