Defining the mechanism and functions of RIPK1-induced cell death in anti-bacterial immune defense

项目摘要 针对细菌感染的免疫防御需要信号传导途径，其激活 炎症介质来控制和清除感染。许多病原体抑制这些信号通路， 以逃避宿主的免疫防御特别是，耶尔森氏菌注入了一种毒力因子YopJ， 阻断NF-ΚB B和MAPK信号通路的关键方面。免疫防御是如何介导 阻断免疫信号通路的病原体仍然知之甚少。巨噬细胞NF-κB阻断 暴露于细菌PAMP导致细胞死亡。重要的是，我们最近公布的数据表明， RIPK 1激酶活性是耶尔森氏菌诱导的细胞死亡所必需的，并且RIPK 1激酶活性对于 对耶尔森氏菌感染和体内先天性炎性细胞因子产生的抗性。这表明RIPK 1- 由耶尔森氏菌引发的诱导死亡是关键的免疫保护机制。我们的新研究表明IKK 磷酸化RIPK 1以限制炎症刺激诱导的细胞死亡，这表明耶尔森氏菌阻断作用 IKK释放RIPK 1的抑制作用。RIPK 1激酶活性和细胞死亡如何促进宿主 对细菌感染的防御是未知的。我们的中心假设是RIPK 1介导的细胞死亡 响应于病原体介导的NF-κB和MAPK阻断而触发释放促炎信号， 例如IL-1细胞因子和细胞内警报蛋白，其使未感染的旁观者细胞能够产生关键的 炎症介质如TNF，其控制干扰先天信号传导的病原体的感染。 这是一个重要的问题，因为这一途径可能会响应许多病原体，阻断关键的先天性 免疫信号通路和病理刺激的背景下，导致RIPK 1诱导的细胞死亡。 我们提出了三个具体目标，以解决我们知识中的这一重要差距。首先，我们将定义 RIPK 1激酶激活如何响应YopJ阻断IKK信号传导而发生的分子基础，以及 测试这一途径在抗细菌免疫防御中的作用。其次，我们将定义免疫反应， 感染和旁观者细胞，并将定义RIPK 1依赖性细胞死亡途径对抗- 体内细菌宿主防御。最后，我们将确定旁观者细胞的TNF信号传导对 抗菌免疫防御，并将确定RIPK 1和TNFR的细胞类型特异性要求， 依赖性细胞死亡的宿主免疫防御对耶尔森氏菌。

Project Summary Immune defense against bacterial infection requires signaling pathways that activate expression of inflammatory mediators to control and clear infection. Many pathogens inhibit these signaling pathways in order to evade host immune defenses. In particular, Yersinia injects a virulence factor, YopJ, which potently blocks key aspects of NF-ΚB and MAPK signaling pathways. How immune defense is mediated against pathogens that block immune signaling pathways remains poorly understood. NF-κB blockade in macrophages exposed to bacterial PAMPs leads to cell death. Importantly, our recently published data demonstrate that RIPK1 kinase activity is required for Yersinia-induced cell death, and that RIPK1 kinase activity is critical for resistance to Yersinia infection and innate inflammatory cytokine production in vivo. This suggests that RIPK1- induced death triggered by Yersinia is a key immune protective mechanism. Our new studies indicate that IKK phosphorylates RIPK1 to limit induction of cell death by inflammatory stimuli, suggesting that Yersinia blockade of IKK releases RIPK1 from this inhibitory effect. How RIPK1 kinase activity and cell death promote host defense against bacterial infection is not known. Our central hypothesis is that RIPK1-mediated cell death triggered in response to pathogen-mediated NF-κB and MAPK blockade releases pro-inflammatory signals, such as IL-1 cytokines and intracellular alarmins that enable uninfected bystander cells to produce key inflammatory mediators such as TNF, which control infection by pathogens that interfere with innate signaling. This is an important problem as this pathway likely responds to many pathogens that block critical innate immune signaling pathways and in the context of pathological stimuli that lead to RIPK1-induced cell death. We propose three Specific Aims to address this important gap in our knowledge. First we will define the molecular basis for how RIPK1 kinase activation occurs in response to YopJ blockade of IKK signaling, and test the role of this pathway in anti-bacterial immune defense. Second, we will define immune responses of infected and bystander cells, and will define the contribution of RIPK1-dependent cell death pathways to anti- bacterial host defense in vivo. Finally, we will determine the contribution of TNF signaling by bystander cells to antibacterial immune defense and will define the cell-type specific requirement for RIPK1- and TNFR- dependent cell death in host immune defense against Yersinia.