Regulation of Proinflammatory Cytokine Responses by a Caspase-8-N4BP1 Axis

项目总结/摘要 罕见的单基因免疫疾病已经阐明了炎症的关键方面，但许多潜在的 其机制仍然知之甚少。例如，自身免疫性淋巴增生综合征（ALPS）， 一种T细胞不能进行凋亡的疾病，通常是由死亡受体的遗传缺陷引起的 FAS或其配体FASL。然而，介导细胞死亡的caspase-8或其衔接子FADD -的突变， FAS下游-导致ALPS和严重免疫缺陷的组合。由于免疫缺陷是 在FAS或FASL突变的患者中通常观察不到，我假设FADD-caspase-8必须具有 FAS以外的免疫受体下游的非骨化症依赖性功能。事实上，我最近 发现多种免疫受体的激活可激活caspase-8介导的Nedd 4- 结合蛋白1（N4 BP 1），一种新的细胞因子抑制因子。这代表了监管的关键点， 炎症值得注意的是，N4 BP 1的缺失通常不影响Toll样细胞的TRIF依赖性亚群。 激活半胱天冬酶-8的受体（TLR）（例如，TLR 3和TLR 4）。然而，受损的细胞因子的产生， 用TLR 4激动剂刺激的半胱天冬酶-8缺陷型巨噬细胞通过共缺失N4 BP 1而恢复正常。 相反，N4 BP 1缺失导致TRIF非依赖性TLR的过度细胞因子应答（例如，TLR1/2， TLR 7和TLR 9），不直接激活caspase-8。因此，N4 BP 1被胱天蛋白酶-8切割使N4 BP 1失活。 完整的、未切割的N4 BP 1的抗炎活性。这些发现提供了一个新的机械解释， 由FADD-半胱天冬酶-8突变引起的免疫缺陷，由此不能切割N4 BP 1导致其免疫缺陷。 细胞因子反应的异常持续和收缩。与TLR 3和TLR 4激动剂一样， (TNF)还导致N4 BP 1的半胱天冬酶-8裂解，赋予TNF使N4 BP 1活化的能力， 从而允许不依赖TRIF的TLR产生细胞因子。后一项发现强调了一个关键点， TNF和TLR系统之间的分子串扰，其会聚于N4 BP 1的半胱天冬酶-8裂解。在 根据目前的建议，我已经将N4 BP 1抑制细胞因子产生的机制与一系列 这些蛋白质在炎症中既有以前认识到的作用，也有迄今未知的作用。在目标1中，我将尝试 为了破译N4 BP 1控制这种新型激酶依赖性途径活性的机制， 抑制炎症。在目的2中，我将剖析N4 BP 1如何抑制晚期炎症基因， 使用基因组规模的技术进行表达。在目标3中，我将探索机制和体内结果 TNF-半胱天冬酶-8-N4 BP 1轴的信号整合。总之，这些目标将提供新的机械 解释炎症基础的关键调节回路的见解。他们也将为我的 独立的研究生涯。

Project Summary/Abstract Rare monogenic immune disorders have illuminated key aspects of inflammation, but many of the underlying mechanisms remain poorly understood. For example, autoimmune lymphoproliferative syndrome (ALPS), a disorder in which T cells fail to undergo apoptosis, is most often caused by genetic defects in the death receptor FAS or its ligand FASL. However, mutations in caspase-8 or its adaptor FADD – which mediate cell death downstream of FAS – cause a combination of ALPS plus severe immunodeficiency. Since immunodeficiency is not generally observed in patients with FAS or FASL mutations, I hypothesized that FADD-caspase-8 must have an apoptosis-independent function downstream of an immune receptor other than FAS. Indeed, I recently discovered that activation of multiple immune receptors elicits the caspase-8-mediated cleavage of Nedd4- binding protein 1 (N4BP1), a novel cytokine suppressor. This represents a critical point of regulation during inflammation. Notably, deletion of N4BP1 does not ordinarily affect the TRIF-dependent subset of toll-like receptors (TLRs) that activate caspase-8 (e.g., TLR3 and TLR4). However, the impaired cytokine production of caspase-8-deficient macrophages stimulated with a TLR4 agonist is restored to normal by co-deletion of N4BP1. In contrast, N4BP1 deletion leads to exorbitant cytokine responses by the TRIF-independent TLRs (e.g., TLR1/2, TLR7 and TLR9) that do not directly activate caspase-8. Thus, N4BP1 cleavage by caspase-8 inactivates the anti-inflammatory activity of intact, un-cleaved N4BP1. These findings offer a novel mechanistic explanation for immunodeficiency caused by FADD-caspase-8 mutations, whereby the inability to cleave N4BP1 results in its aberrant persistence and constriction of cytokine responses. Like TLR3 and TLR4 agonists, tumor necrosis factor (TNF) also leads to caspase-8 cleavage of N4BP1, endowing TNF with the ability to inactivate N4BP1 and thereby license cytokine production by the TRIF-independent TLRs. This latter finding highlights a key point of molecular crosstalk between the TNF and TLR systems that converges on caspase-8 cleavage of N4BP1. In the current proposal, I have linked the mechanism by which N4BP1 suppresses cytokine production to a series of proteins with both previously recognized and heretofore unknown roles in inflammation. In Aim 1, I will attempt to decipher the mechanism by which N4BP1 controls the activity of this novel kinase-dependent pathway that suppresses inflammation. In Aim 2, I will dissect how N4BP1 suppresses late phase inflammatory gene expression using genome-scale technologies. In Aim 3, I will explore the mechanisms and in vivo consequences of signal integration by the TNF-caspase-8-N4BP1 axis. Together, these aims will provide novel mechanistic insights explaining a key regulatory circuit underlying inflammation. They also will serve to launch my independent research career.