Necroptosis signaling adaptors in inflammatory diseases

 描述(由申请人提供)：炎症是一种关键的生物反应，在保护性免疫和自发性炎症性疾病中具有重要作用。坏死性下垂是一种非凋亡性的细胞死亡形式，由于细胞膜破裂释放细胞危险信号而具有促炎作用。受体相互作用蛋白激酶3(RIPK3)是肿瘤坏死因子样死亡受体和Toll样受体3(TLR3)和TLR4下游的重要受体。RIPK3基因缺陷的小鼠通过抑制caspase 8或细胞凋亡抑制蛋白(IAP)而免受许多炎症性疾病的影响。这些结果导致了被广泛接受的观点，即RIPK3通过坏死性下垂驱动炎症。与这些结果相反，我们发现RIPK3缺陷的小鼠患上了更严重的化学诱导的结肠炎。RIPK3基因缺陷小鼠肠道炎症的增加不是由于细胞死亡的变化。相反，RIPK3基因缺陷小鼠的IL-1β、IL-23和IL-22表达受损。与这些细胞因子在肠道组织修复中的已知功能一致，我们发现在RIPK3基因缺陷的小鼠中，损伤诱导的肠上皮细胞(IEC)增殖存在缺陷。这些结果表明，肠道固有层中的免疫哨兵需要RIPK3来启动细胞因子驱动的组织修复程序。我们发现，在骨髓来源的树突状细胞(BMDCs)中，IL-1β和IL-23的表达需要RIPK3来响应TLR4激动剂脂多糖的刺激。与使用结肠炎诱导剂治疗RIPK3缺陷小鼠类似，RIPK3缺陷BMDCs中细胞因子表达的减少并不是由于细胞死亡的减少。相反，它是由核因子-B激活缺陷和前-IL-1β的加工受损引起的。具体地说，RIPK3是RelB-p50异源二聚体最佳激活以促进IL-23表达所必需的。相反，RIPK3不是激活relA、c-rel或p52所必需的。此外，RIPK3还促进Caspase1和Caspase8介导的前IL-1的处理。这些结果表明，RIPK3关键控制依赖于RelB/p50的IL-23的表达和前IL-1的β处理，并且这些不依赖于坏死下垂的信号功能对于修复炎症是至关重要的。基于这些结果，我们提出了两个目的来研究RIPK3在组织修复反应中调节炎性细胞因子表达的分子机制。在目标1中，我们将研究控制RIPK3介导的RelB-p50在BMDCs和CX3CR1+巨噬细胞固有层巨噬细胞中核转位的生化机制。我们推测，RIPK3和TRIF通过“RIP同型相互作用基序”(RHIM)结合对RelB-p50激活和细胞因子基因转录至关重要。此外，我们假设，与坏死性下垂不同，这种由Rhim介导的相互作用不涉及淀粉样蛋白转化、RIPK1或RIPK3激酶活性。然而，它是由活性氧(ROS)控制的。我们进一步假设，在被TRIF激活后，RIPK3作为伴侣通过直接与RelB结合来促进RelB-p50核转位。除了RelB-p50，我们假设RIPK3还通过一个非典型的RIPK1-RIPK3-FADD-Caspase8复合体促进前IL-1的加工。此外，cFlIP-Long-form与该复合体的结合通过阻止caspase8介导的细胞凋亡来关键地促进IL-1的分泌。在目标2中，我们将验证这一假设，即肠道固有层中的CX3CR1+炎性巨噬细胞是以依赖于RIPK3的方式产生IL-1和IL-23的关键免疫效应细胞。为了验证我们的假设，我们将使用Cre介导的重组来产生在CD11c+或CX3CR1+细胞中具有RIPK3特异性失活的小鼠。此外，我们将使用表达RIPK1和RIPK3的激酶失活版本的小鼠来确定激酶活性在RIPK3依赖的细胞因子表达和结肠炎诱导中的作用。我们将进一步检验这一假设，即纠正细胞因子表达和组织修复的早期缺陷可以将慢性炎症和炎症诱导的结肠癌的易感性降至最低。这些研究的成功完成将极大地提高我们对RIPK3、树突状细胞/巨噬细胞、坏死性下垂、组织修复、炎症性疾病和癌症之间的免疫生物学及其相互关系的了解。

 DESCRIPTION (provided by applicant): Inflammation is a key biological response with essential roles in protective immunity and auto- inflammatory diseases. Necroptosis is a non-apoptotic form of cell death with pro-inflammatory effects due to the release of cellular danger signals from ruptured plasma membranes. Receptor interacting protein kinase 3 (RIPK3) is an essential adaptor for necroptosis downstream of TNF-like death receptors and toll-like receptor 3 (TLR3) and TLR4. Ripk3-deficient mice are protected from many inflammatory diseases driven by inhibition of caspase 8 or the cellular inhibitor of apoptosis (IAP) proteins. These results led to the widely accepted view that RIPK3 drives inflammation through necroptosis. In contrast to these results, we found that Ripk3-deficient mice developed more severe chemical-induced colitis. The increased intestinal inflammation in Ripk3-deficient mice was not due to changes in cell death. Rather, Ripk3-deficient mice were impaired for expression of IL-1β, IL-23 and IL-22. Consistent with the known function of these cytokines in intestinal tissue repair, we found that injury-induced intestinal epithelial cell (IEC) proliferation was defective in Ripk3-deficient mice. These results indicate that immune sentinels in the intestinal lamina propria require RIPK3 to initiate a cytokine-driven tissue repair program. We found that in bone marrow derived dendritic cells (BMDCs), RIPK3 is required for IL-1β and IL-23 expression in response to stimulation with lipopolysaccharide (LPS), a TLR4 agonist. Similar to Ripk3-deficient mice treated with colitis-causing agent, reduced cytokine expression in Ripk3-deficient BMDCs was not due to reduced cell death. Rather, it was caused by defective activation of NF-B activation and impaired processing of pro-IL-1β. Specifically, RIPK3 is required for optimal activation of RelB-p50 heterodimer to promote IL-23 expression. In contrast, RIPK3 is not required for activation of RelA, c-Rel or p52. In addition, RIPK3 also promotes caspase 1 and caspase 8-mediated processing of pro-IL-1. These results indicate that RIPK3 critically controls RelB/p50-dependent IL-23 expression and pro-IL-1β processing and that these necroptosis-independent signaling functions are crucial for reparative inflammation. Based on these results, we propose two aims to investigate the molecular mechanisms by which RIPK3 regulates inflammatory cytokine expression in tissue repair responses. In Aim 1, we will study the biochemical mechanisms that controls RIPK3-mediated RelB-p50 nuclear translocation in BMDCs and CX3CR1+ macrophages from intestinal lamina propria. We hypothesize that RIPK3 and TRIF binding via the "RIP homotypic interaction motif" (RHIM) is critical for RelB-p50 activation and cytokine gene transcription. Moreover, we hypothesize that unlike necroptosis, this RHIM-mediated interaction does not involve amyloid conversion, RIPK1 or RIPK3 kinase activities. However, it is controlled by reactive oxygen species (ROS). We further hypothesize that upon activation by TRIF, RIPK3 functions as a chaperon to facilitate RelB-p50 nuclear translocation through direct binding to RelB. In addition to RelB-p50, we hypothesize that RIPK3 also promotes pro-IL-1 processing through an atypical RIPK1-RIPK3-FADD-caspase 8 complex. Moreover, cFLIP-long form binding to this complex critically promotes IL-1 secretion by preventing caspase 8-mediated apoptosis. In Aim 2, we will test the hypothesis that CX3CR1+ inflammatory macrophages in the intestinal lamina propria are the key immune effectors that produce IL-1 and IL-23 in a RIPK3-dependent manner. To test our hypothesis, we will use Cre-mediated recombination to generate mice with specific inactivation of RIPK3 in CD11c+ or CX3CR1+ cells. In addition, we will use mice expressing kinase inactive versions of RIPK1 and RIPK3 to determine the role of kinase activity in RIPK3-dependent cytokine expression and colitis induction. We will further test the hypothesis that correcting the early defect in cytokine expression and tissue repair can minimize chronic inflammation and susceptibility to inflammation-induced colon cancer. Successful completion of these studies will greatly enhance our understanding of the immunobiology of and relationship between RIPK3, dendritic cells/macrophages, necroptosis, tissue repair, inflammatory diseases and cancers.