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: 9175268
• 负责人: MARK J SHLOMCHIK
• 金额: $ 38.23万
• 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/9175268
• 关键词: Address; Animal Model; Autoantibodies; Autoimmune Diseases; Autoimmunity; B-Cell Activation; B-Lymphocytes; Backcrossings; Behavior; Binding; Biochemical; Biology; Birth; 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; Mouse Cell Line; Mus; Mutant Strains Mice; Nucleic Acids; Phenotype; Production; Reagent; Role; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Study models; Systemic Lupus Erythematosus; TLR7 gene; Tamoxifen; Testing; Therapeutic; Tissue Engineering; Tissues; Work; Yin-Yang; abstracting; base; cell type; design; genetic approach; genetic regulatory protein; improved; in vivo; inhibitor/antagonist; lupus prone mice; macrophage; mouse model; mutant; neutrophil; novel; overexpression; protein expression; research study; response; risk variant; targeted treatment; 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.

摘要 Shlomchik实验室的长期目标是了解TLR在SLE中的作用。在我们以前的工作中， 令人惊讶的发现是，在缺乏TLR 9的情况下，疾病明显加重，即使TLR 9是 抗DNA和抗染色质自身抗体产生所需的。这一点已经在多个国家得到证实。 小鼠狼疮的独立模型。这两个类似TLR的相反作用可能是一个基本的fea- TLR生物学的真实性以及SLE的真实性，但对其了解甚少。鉴于TLR信号传导在免疫调节中的重要作用， 人类狼疮和大量的SLE风险等位基因映射到TLR信号传导途径，它是免疫性的。 重要的是阐明这一基本生物学以及它如何应用于疾病机制和治疗。的 TLR在多种鼠狼疮模型中的关键作用的发现，加上遗传证据， 对于SLE和其他自身免疫性疾病临床中TLR抑制剂的开发和测试，高- 点亮重要性。本文提出的实验将直接解决TLR 7和TLR 9如何具有不同的 角色，它们如何相互交叉调节，以及这如何促进和调节疾病。我们的实验室有- 在动物模型中研究狼疮机制需要体内遗传学方法，部分导致了 在本PPG前期支持的研究中认识到TLR 7和TLR 9的作用。我们创造了- 艾德建立了新的模型，并使用遗传学方法来剖析细胞和分子信号通路 需要促进体内疾病。在这里，我们继续制作新的模型并在体内研究它们， 在细胞和生物化学水平上研究TLR 7和9的基本信号传导和生物学。 我们假设不同的组织特异性表达和信号传导可以解释不同的全球效应， TLR 7和TLR 9缺失的影响，这一想法得到了我们对新的TLR 9“floxed”小鼠的初步数据的支持。到 为了解决这一问题，我们将在体内操纵关键细胞类型中TLR的表达。在目标1中，我们将测试hy- 假设TLR 7和TLR 9的组织特异性表达解释了它们的部分或全部不同表型。 我们将把组织特异性TLR 9表达的工作扩展到巨噬细胞和中性粒细胞，并增加 狼疮模型。我们将制造新的floxed和rosa 26条件小鼠，以允许TLR 7组织特异性表达。 压力。这将让我们研究TLR 7和TLR 9的阴阳;到目前为止，我们只研究了TLR 9。在目标2 我们将阐述TLR 9以细胞内在方式调节TLR 7信号的假设。为了测试这一点，我们有 我们制造了几种工具，包括TLR 9和TLR 7的突变体，并将制造更多的突变体和突变小鼠。 我们的独特重点将是主要的B细胞，使目标1。这将使我们能够确定体内和 在体外，如果TLR 9通过竞争运输分子、信号分子或通过 诱导反调节蛋白的表达。此外，我们还将测试细胞质尾部是否 TLR 7和TLR 9介导不同的信号传导。最后，在目标3中，我们将测试TLR抑制肿瘤细胞增殖的治疗潜力。 使用可诱导的Cre来删除MyD 88并因此在疾病的不同阶段终止TLR 7/9信号传导。