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，先天性免疫细胞在启动和维持自身免疫和炎症性疾病方面发挥着关键作用。Lyn酪氨酸激酶是调节天然免疫细胞信号转导的主要抑制酶之一。LYN的功能是通过磷酸化抑制受体招募酪氨酸磷酸酶来下调细胞激活。在没有Lyn激酶的情况下，小鼠会发展成类似于人类系统性红斑狼疮(SLE)的自发自身免疫。在初步研究中，我们发现，与全球Lyn-/-小鼠相比，单是树突状细胞(DC)中Lyn激酶的丢失就足以导致严重的自身免疫性/炎症性疾病，伴随着强烈的组织炎症和生存减少。这种疾病完全依赖于MyD88信号，因为在DC中缺乏MyD88和Lyn的一代小鼠缺乏炎症和自身免疫。这些观察结果提供了一个明显的例子，即导致DC抑制信号丢失的突变可以导致淋巴细胞免疫耐受性的崩溃，从而导致自身免疫。这项建议包括4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MyD88信号的分子机制。我们假设这些通路之间的串扰是通过CARD9/MALT1/Bcl10复合体发生的，我们将对其进行生化和遗传学研究。我们将使用化学遗传学方法来确定DC中LYN的分子底物。目的#2将扩展我们与Lynch博士(加州大学旧金山分校)共同完成的初步观察，即树突状细胞中LYN的丢失会导致肠道微生物区系的失调，从而允许通过改变肠道屏障功能而导致全身炎症的物种的生长。我们将在一系列微生物区系图谱实验中，通过使用细菌补充方法，并通过直接检查各种Lyn缺陷菌株的肠道屏障功能，来扩展这些观察结果。目标#3将 对初步数据的扩展表明，非造血细胞中未明确的Lyn介导的信号通路可能对Lyn-/-小鼠的疾病过程具有免疫调节作用。非造血细胞中的LYN相关抑制信号研究很少。我们将利用生物化学和遗传学手段重点研究卵泡回缩细胞(包括产生在这些细胞中特异性缺失Lyn的突变小鼠)。目的#4将扩展与Locksley博士(加州大学旧金山分校)所做的初步研究，表明在Lyn-/-小鼠中，第二组固有淋巴细胞(ILC2s)被扩增和激活。这一目标将解决ILC2细胞中信号变化是否单独导致自发性疾病表型(通过产生新的ILC2特异性LYN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患者中已经观察到Lyn激酶相关信号通路的变化，并且Lyn现在是糖尿病治疗(治疗试验)的治疗靶点，了解由该激酶调节的先天细胞中的抑制信号通路将直接影响人类自身免疫和炎症状况。

 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.