Regulation of Innate Immune Signaling by Lyn Kinase

 描述（由申请人提供）：越来越多的人认识到先天免疫细胞在引发和维持自身免疫性和炎症性疾病中起关键作用。林恩酪氨酸激酶是调节先天免疫细胞信号传导的主要抑制酶之一。林恩通过磷酸化抑制性受体发挥作用，该受体募集酪氨酸磷酸酶以下调细胞活化。在缺乏林恩激酶的情况下，小鼠产生类似于人类系统性红斑狼疮（SLE）的自发性自身免疫。在初步研究中，我们已经发现，与整体lyn-/-小鼠相比，树突状细胞（DC）中林恩激酶的单独损失足以引起严重的自身免疫性/炎性疾病，具有强烈的组织炎症和降低的存活率。这种疾病完全依赖于MyD 88信号传导，因为在DC中缺乏MyD 88和林恩的小鼠的产生缺乏炎症和自身免疫。这些观察结果提供了一个明确的例子，即导致DC中抑制性信号传导丧失的突变可导致淋巴细胞中免疫耐受的破坏，从而导致自身免疫。该计划包括四个具体目标。我们的第一个目标是确定抑制酪氨酸激酶通路影响树突状细胞中MyD 88信号传导的分子机制。我们假设这些通路之间的串扰通过CARD 9/Malt 1/Bcl 10复合物发生，我们将从生物化学和遗传学角度对其进行研究。我们将使用化学遗传学的方法来确定林恩在树突状细胞中的分子底物。目标#2将扩展我们与Lynch博士（UCSF）完成的初步观察结果，即DC中林恩的丢失导致肠道微生物群的生态失调，允许可能通过改变肠道屏障功能来驱动全身炎症的物种的生长。我们将在一系列微生物群分析实验中扩展这些观察结果，通过使用细菌补充方法，并直接检查各种Lyn-deficient菌株的肠道屏障功能。目标#3将 扩展初步数据，表明非造血细胞中未定义的Lyn-mediated信号通路可能对林恩-/-小鼠的疾病过程具有免疫调节作用。非造血细胞中的林恩相关抑制性信号传导的研究非常少。我们将使用生物化学和遗传学手段（包括在这些细胞中特异性地产生缺乏林恩的突变小鼠）专注于滤泡网状细胞。目的#4将扩展与Locksley博士（UCSF）进行的初步研究，该研究显示第2组先天淋巴细胞（ILC 2）在林恩-/-小鼠中扩增和激活。这一目标将解决单独ILC 2细胞中改变的信号传导是否可以导致自发性疾病表型（通过产生新的ILC 2特异性林恩突变体）以及这些细胞是否可能导致自身免疫性疾病。由于在人类SLE患者中观察到林恩激酶相关信号传导途径的变化，并且林恩现在是糖尿病治疗（治疗试验）中的治疗靶标，因此理解由该激酶在先天细胞中调节的抑制性信号传导途径将对人类自身免疫性和炎性病症具有直接影响。

 DESCRIPTION (provided by applicant): There is a growing recognition that innate immune cells play a critical role in initiating and sustaining autoimmune and inflammatory disorders. The Lyn tyrosine kinase is one of the primary inhibitory enzymes that regulate innate immune cell signaling. Lyn functions by phosphorylating inhibitory receptors that recruit tyrosine phosphatases to down modulate cellular activation. In the absence of Lyn kinase, mice develop spontaneous autoimmunity resembling human systemic lupus erythematous (SLE). In preliminary studies, we have found that loss of Lyn kinase in dendritic cells (DCs) alone is sufficient to cause a severe autoimmune/inflammatory disease, with strong tissue inflammation and reduced survival, compared to the global lyn-/- mice. This disease is completely dependent on MyD88-signaling, since generation of mice lacking MyD88 and Lyn in DCs alone lack the inflammation and autoimmunity. These observations provide a clear example that mutations resulting in loss of inhibitory signaling in DCs can result in breakdown of immune tolerance in lymphocytes, leading to autoimmunity. This proposal consists of 4 specific aims. Our first aim is to define the molecular mechanisms by which inhibition of tyrosine kinase pathways impacts MyD88 signaling in DCs. We hypothesize that the cross talk between these pathways occurs via the CARD9/Malt1/Bcl10 complex, which we will investigate biochemically and genetically. We will use a chemical genetic approach to define the molecular substrates of Lyn in DCs. Aim #2 will expand on our preliminary observations, done with Dr. Lynch (UCSF) that loss of Lyn in DCs results in dysbiosis of gut microbiota, allowing for outgrowth of species that may drive systemic inflammation by altering the intestinal barrier function. We will expand on these observations in a series of microbiota profiling experiments, by use of bacterial supplementation methods, and by directly examining intestinal barrier function in the various Lyn-deficient strains. Aim #3 will expand on preliminary data suggesting that an undefined Lyn-mediated signaling pathway in non- hematopoietic cells may have an immunomodulatory effect on the disease process in lyn-/- mice. Lyn related inhibitory signaling in non-hematopoietic cells is very poorly studied. We will focus on follicular retricular cells using biochemical and genetic means (including generating mutant mice lacking Lyn in these cells specifically). Aim #4 will expand on preliminary studies done with Dr. Locksley (UCSF) showing that group 2 innate lymphocytes (ILC2s) are expanded and activated in lyn-/- mice. This aim will address whether altered signaling in ILC2 cells alone can lead to spontaneous disease phenotypes (by generating novel ILC2-specific lyn mutants) and whether these cells may contribute to autoimmune disease. Since changes in Lyn kinase related signaling pathways have been observed in human SLE patients and Lyn is now a therapeutic target in diabetes treatments (therapeutic trials) understanding the inhibitory signaling pathways regulated by this kinase in innate cells will have direct impact on human autoimmune and inflammatory conditions.