The Role of TRIM6 and Ubiquitin in Influenza Virus-Induced Pathology

抽象的。甲型流感病毒 (IAV) 每年都会引发流行病和危险的大流行，涉及数百万人 全世界的患病和死亡病例。 IAV 病理学的主要原因是过度炎症， 因此，该提案的首要目标是了解如何研究炎症机制 操纵以提高对病毒感染的疾病耐受性。细胞因子产生的主要贡献者 炎症，在翻译后水平进行调节，以平衡有效的抗病毒反应和 破坏性炎症。主要的分子调控机制涉及信号传导的泛素化 组件。该提案的具体目标是确定炎症调节机制 IAV 体内感染过程中的泛素 (Ub) 系统。 我们最近报道了 E3-Ub 连接酶 TRIM6 催化非锚定多聚 Ub 链的合成， 促进抗病毒 IFN-I 反应。然而，TRIM6 在调节其他炎症中的作用 细胞因子尚不清楚。我们生成了 TRIM6 敲除小鼠 (Trim6-/-)，它提供了一种独特的工具 确定体内 TRIM6 和非锚定 Ub 调节的新免疫途径。我们的初步数据显示 尽管早期时间点 IAV 滴度增加，Trim6-/- 小鼠的病理迹象较少 感染后。我们还发现 CXCL1（一种众所周知的中性粒细胞化学吸引剂）的表达水平降低， 这与感染 IAV 的 Trim6-/- 小鼠肺部中性粒细胞浸润减少相关。我们发现 TRIM6 和未锚定的 Ub 与 PI3K/AKT 信号成分形成复合物，其磷酸化为 Trim6-/- 细胞受损。我们的数据还表明，受感染细胞产生的 TNFα 会诱导致病性 CXCL1 在旁观者细胞中招募中性粒细胞。已知中性粒细胞在 IAV 期间被募集至肺部 感染，可以发挥保护作用和有害作用。然而，哪些因素会导致中性粒细胞发生 感染期间的组织损伤尚不清楚。因此，在知识上存在着差距。 中性粒细胞募集的调节机制及其在保护性之间平衡中的作用 反应和致病性炎症。我们的假设是 TRIM6 被 TNFα 信号激活并且 促进早期 CXCL1 介导的致病性炎症，从而抑制疾病耐受性。在目标 1 中，我们 将确定 TRIM6 诱导的 CXCL1 的细胞来源及其在中性粒细胞募集到肺部中的作用， IAV 感染期间。我们将证明早期 CXCL1 产生在病理学中的作用以及 TNFα 是否 参与诱导 TRIM6 介导的 CXCL1。在目标 2 中，我们将确定 TRIM6 的机制 和 Ub 调节 PI3K-AKT 下游信号传导的激活以及 TRIM6 在 感染。结果包括致病性 CXCL1 细胞来源的鉴定，以及 TRIM6 被激活以进行信号传导的机制。这些信息将指导开发 通过靶向 TRIM6 和 CXCL1 产生细胞来减少炎症性疾病的治疗方法。

ABSTRACT. Influenza A virus (IAV) causes annual epidemics and dangerous pandemics involving millions of cases of illness and deaths worldwide. The main cause of pathology from IAV is excessive inflammation, therefore, the overarching goal of this proposal is to learn how mechanisms of inflammation can be manipulated to promote disease tolerance to virus infection. Cytokine production, a chief contributor of inflammation, is regulated at the post-translational level to balance between efficient antiviral responses and damaging inflammation. A major molecular regulatory mechanism involves ubiquitination of signaling components. The specific goal of this proposal is to identify mechanisms of regulation of inflammation by the ubiquitin (Ub) system during IAV infection in vivo. We recently reported that the E3-Ub ligase, TRIM6, catalyzes the synthesis of unanchored poly-Ub chains, which promote antiviral IFN-I responses. However, the role of TRIM6 in regulating other inflammatory cytokines is not known. We generated TRIM6 knockout mice (Trim6-/-), which provides a unique tool to identify novel immune pathways regulated by TRIM6 and unanchored Ub in vivo. Our preliminary data show that Trim6-/- mice have fewer signs of pathology even though there are increased IAV titers at early time points post-infection. We also found reduced expression levels of CXCL1, a well-known neutrophil chemo-attractant, which correlated with reduced neutrophil infiltration to the lungs of IAV-infected Trim6-/- mice. We found that TRIM6 and unanchored Ub form a complex with PI3K/AKT signaling components, and their phosphorylation is impaired in Trim6-/- cells. Our data also suggest that TNFα produced by infected cells induces pathogenic CXCL1 in bystander cells to recruit neutrophils. Neutrophils are known to be recruited to the lung during IAV infection and can play both protective and detrimental roles. However, what factors drive neutrophils to cause tissue damage during infection are not well-understood. Therefore, there is a gap in knowledge on the mechanisms of regulation of neutrophil recruitment and their roles in the balance between protective responses and pathogenic inflammation. Our hypothesis is that TRIM6 is activated by TNFα signaling and promotes early CXCL1-mediated pathogenic inflammation, thereby inhibiting disease tolerance. In Aim 1, we will determine the cellular source of TRIM6-induced CXCL1, and its role in neutrophil recruitment to the lungs, during IAV infection. We will demonstrate the role of early CXCL1 production in pathology and whether TNFα is involved in inducing TRIM6-mediated CXCL1. In Aim 2, we will determine the mechanism by which TRIM6 and Ub modulate the activation of PI3K-AKT for downstream signaling and how TRIM6 is activated during infection. The outcomes include the identification of the cellular source of pathogenic CXCL1, and the mechanism by which TRIM6 is activated for signaling. This information will guide the development of therapeutic approaches by targeting TRIM6 and CXCL1-producing cells to reduce inflammatory diseases.