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

抽象的。甲型流感病毒(IAV)导致每年流行和危险的大流行，涉及数百万人 世界各地的疾病和死亡病例。IAV的主要病理原因是过度炎症， 因此，这项建议的首要目标是了解炎症的机制如何 被操纵以提高对病毒感染的疾病耐受性。细胞因子的产生，一个主要贡献者 炎症，在翻译后水平上进行调节，以平衡有效的抗病毒反应和 破坏性炎症。一种主要的分子调控机制涉及信号的泛素化。 组件。这项建议的具体目标是确定炎症的调节机制，通过 泛素(Ub)系统在体内感染IAV中的作用 我们最近报道了E3-Ub连接酶TRIM6催化合成未锚定的多Ub链， 促进抗病毒的干扰素-I反应。然而，TRIM6在调节其他炎症反应中的作用 细胞因子尚不清楚。我们产生了TRIM6基因敲除小鼠(Trim6-/-)，这提供了一个独特的工具来 在体内发现受TRIM6和非锚定Ub调节的新的免疫通路。我们的初步数据显示 Trim6-/-小鼠的病理迹象较少，即使在早期时间点有IAV滴度增加 感染后。我们还发现，众所周知的中性粒细胞化学诱导剂CXCL1的表达水平降低， 这与IAV感染的Trim6-/-小鼠肺内中性粒细胞渗入减少有关。我们发现 TRIM6和未锚定的Ub与PI3K/AKT信号元件形成复合体，它们的磷酸化是 Trim6-/-细胞受损。我们的数据还表明，感染细胞产生的肿瘤坏死因子α诱导致病 CXCL1在旁观者细胞中募集中性粒细胞。已知中性粒细胞在IAV期间被招募到肺中。 感染，既可以起到保护作用，也可以起到有害作用。然而，是什么因素驱使中性粒细胞导致 感染过程中的组织损伤还不是很清楚。因此，在认识上存在差距。 中性粒细胞募集的调节机制及其在保护之间平衡中的作用 反应和致病性炎症。我们的假设是，TRIM6被肿瘤坏死因子α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，以及它在中性粒细胞重新聚集到肺中的作用。 在IAV感染期间。我们将展示早期CXCL1的产生在病理中的作用以及肿瘤坏死因子α 参与诱导TRIM6介导的CXCL1。在目标2中，我们将确定TRIM6 和Ub调节PI3K-AKT的激活以进行下行信号传递，以及TRIM6是如何在 感染。结果包括确定致病CXCL1的细胞来源，以及 激活TRIM6进行信号传递的机制。这些信息将指导 通过靶向TRIM6和CXCL1产生细胞的治疗方法来减少炎症性疾病。

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.