Rip Proteins in Innate Immune Signaling

项目总结/摘要 自身免疫性疾病和炎性疾病的发病率增加突出了改善免疫性疾病的需要。 治疗这些疾病的药物。我们的工作将RIPK 1激酶置于炎症通路的顶端 在那里它调节一种称为坏死性凋亡的炎性细胞死亡形式，这种坏死性凋亡由RIPK 1、RIPK 3和 MLKL。在这项工作中，我们将描述在自身免疫中重要的新的坏死性凋亡途径， 自身免疫性肝炎和病毒性肝炎中免疫介导肝损伤 我们证明了树突状细胞RIPK 1缺陷（Ripk 1DC KO）的小鼠会发生炎症， 自身免疫;出乎意料地揭示了RIPK 1抑制树突状细胞（DC）中的坏死性凋亡， 其作为坏死性凋亡的引发剂的作用已得到充分证实。在发育过程中，RIPK 1可防止坏死性凋亡 介导的核酸传感器Z-DNA结合蛋白（ZBP 1），导致我们假设ZBP 1- 介导的树突状细胞坏死性凋亡破坏耐受并诱导自身免疫。我们将检验这一假设 在目的1中，通过确定ZBP 1缺陷是否预防Ripk 1DC KO中的炎症和自身免疫， 小鼠我们将鉴定与ZBP 1结合的核酸种类，并将确定核酸结合是否 是诱导炎症和/或自身免疫所必需的。已知Z-型的核酸是高度同源的。 免疫原性和抗Z-DNA自身抗体发生在狼疮患者，因此，我们将测试遗传 抑制ZBP 1或坏死性凋亡是否能改善其他自身免疫易感小鼠的疾病。在目标2中， 将研究RIPK 1激酶依赖性坏死性凋亡是否有助于小鼠自身免疫性 由植物凝集素伴刀豆球蛋白A（ConA）诱导的肝炎（AIH）。根据我们的初步数据， 相互造血嵌合体我们假设AIH是由典型的坏死性凋亡介导的， 造血细胞和肝细胞中的非典型坏死性凋亡。与RIPK 1增加一致， MLKL在AIH患者肝脏中的表达，我们的初步数据表明RIPK 1和MLKL，但没有发现 RIPK 3在肝细胞死亡中的作用，使我们假设一种新的RIPK 3独立的坏死性凋亡 途径在肝细胞中被诱导。接下来，我们将通过培养小鼠来严格验证这一假设。 其在肝实质细胞中特异性表达激酶失活RIPK 1D 138 N或缺失Ripk 3或Mlk 1。一个 另一个目标是通过识别RIPK 1， 在肝细胞中负责MLKL活化的相互作用激酶，并评估其对AIH的贡献。 成功完成这些目标将阐明这些新的坏死性凋亡途径的作用 自身免疫和急性肝脏炎症的长期目标，以确定这些 可能受益于RIPK 1激酶靶向治疗的自身炎症/免疫性疾病

PROJECT SUMMARY/ABSTRACT The increased incidence of autoimmune and inflammatory disorders highlights the need for improved therapeutics to treat these diseases. Our work places RIPK1 kinase at the apex of inflammatory pathways where it regulates a form of inflammatory cell death called necroptosis mediated by RIPK1, RIPK3 and MLKL. In the work proposed, we will delineate novel necroptosis pathways important in autoimmunity and immune-mediated liver injury operative in autoimmune and viral hepatitis. We demonstrate that mice with a dendritic cell RIPK1-deficiency (Ripk1DC KO) develop inflammation and autoimmunity; unexpectedly revealing that RIPK1 restrains necroptosis in dendritic cells (DC) in contrast to it well established role as an initiator of necroptosis. During development, RIPK1 prevents necroptosis mediated by the nucleic acid sensor Z-DNA binding protein (ZBP1) leading us to hypothesize that ZBP1- mediated dendritic cell necroptosis breaks tolerance and induces autoimmunity. We will test this hypothesis in Aim 1 by determining whether a ZBP1-deficiency prevents inflammation and autoimmunity in Ripk1DC KO mice. We will identify the nucleic acid species bound to ZBP1 and will establish whether nucleic acid binding is required to induce inflammation and/or autoimmunity. Nucleic acids in the Z-form are known to be highly immunogenic and anti-Z-DNA autoantibodies occur in lupus patients; therefore, we will test genetically whether inhibiting ZBP1 or necroptosis ameliorates disease in other autoimmune prone mice. In Aim 2, we will investigate whether RIPK1-kinase-dependent necroptosis contributes to a mouse model of autoimmune hepatitis (AIH) induced by the plant lectin Concanavalin A (Con A). Based on our preliminary data in reciprocal hematopoietic chimeras we hypothesize that AIH is mediated by canonical necroptosis in hematopoietic cells and noncanonical necroptosis in hepatocytes. Consistent with increased RIPK1 and MLKL expression in the livers of AIH patients, our preliminary data implicate RIPK1 and MLKL but not RIPK3 in hepatocyte cell death, leading us to hypothesize that a novel RIPK3 independent necroptosis pathway is induced in hepatocytes. Going forward we will test this hypothesis rigorously by generating mice that express kinase inactive RIPK1D138N or delete Ripk3 or Mlkl specifically in liver parenchymal cells. An additional goal will be to delineate this non-canonical necroptosis pathway, by identifying the RIPK1- interacting kinase responsible for MLKL activation in hepatocytes and to assess its contribution to AIH. Successful completion of these Aims will elucidate the contribution(s) of these new necroptosis pathways to autoimmunity and acute liver inflammation with the long-term goal to identify those autoinflammatory/immune diseases that might benefit from RIPK1 kinase targeted therapy