Neuroendocrine control of TLR4-dependent inflammation in influenza

摘要 流感是一种高度传染性的呼吸道疾病，疾控中心估计约有4750万人患有这种疾病 2019年10月1日至2020年4月4日期间死亡人数高达6.2万人。在没有“万能”的情况下 流感疫苗，“强烈建议每年接种疫苗；但每种疫苗的成分如下 基于对下一年哪些菌株将占主导地位的预测，这样的预测可能是 不正确。批准的抗病毒药物可以通过限制病毒复制来改善疾病，但必须给予 在感染早期要有效，并出现了耐药的流感毒株。而流感引发的疾病 是由病毒复制引发的，导致呼吸道上皮损伤，严重的炎症反应 作为代谢应激的结果，先天免疫细胞(如巨噬细胞)最终会产生一种“细胞因子” 这可能会导致急性呼吸窘迫综合征(ARDS)和死亡。因此，一种新的方法，即 靶向宿主的先天免疫反应将代表着流感治疗的重大进步 以及其他导致ARDS的呼吸道病毒，如SARS-CoV-2。我们已经鉴定出Toll样受体 4(TLR4)，一种模式识别受体，以感知革兰氏阴性脂多糖的能力而闻名 (LPs)，作为宿主对流感的炎症反应的关键。这最初是令人惊讶的，因为流感病毒 不表达任何通过TLR4引发炎症的“病原体相关分子模式”。 尽管如此，在流感小鼠模型中同时使用TLR4-/-小鼠和多个TLR4拮抗剂 感染，以及对非适应品系敏感的棉鼠(Sigmodon hispidus；CR) 人类流感，我们严格证明了TLR4信号是肺和 全身性炎症对感染的反应。我们鉴定了一种宿主来源的蛋白质，高迁移率族蛋白-1 (HMGB1)，在感染过程中从死亡细胞中释放出来，起到“危险相关的分子模式”的作用。 从而通过其辅助受体MD2触发TLR4。最近，我们发现了第二种宿主来源的蛋白质， 胃泌素释放肽(GRP)，作为流感介导性疾病的贡献者，使用三种不同的抑制物 GRP或GRP受体信号显著抑制细胞因子的产生、肺病理和致死性 以治疗性的方式给老鼠注射。我们已公布的和初步的数据支持中心假设 这两个介质相互关联，在宿主对流感感染的反应过程中汇聚在一起。分成两份 特定的目标，我们建议(1)描述流感介导的GRP受体(GRPR)- 和TLR4/MD2介导的信号转导，以及(2)GRP和HMGB1水平与疾病严重程度相关 流感引起的疾病和流感感染患者的不同实验模型。在这个探索性的 R21的应用，我们将寻求确定这两者之间关系的机制基础 具有确定治疗策略以改善疾病的潜力的信号通路。

SUMMARY Influenza is a highly contagious respiratory illness that the CDC has estimated to afflict ~47.5 million Americans with up to 62,00 deaths from October 1, 2019 to April 4, 2020. In the absence of a “universal influenza vaccine,” yearly vaccination is strongly recommended; however, the composition of each vaccine is based on predictions of which strains will predominate in the following year and such predictions may be incorrect. Approved antivirals can ameliorate disease by limiting viral replication, but they must be administered early in infection to be effective, and resistant influenza strains have emerged. While influenza-induced disease is initiated by viral replication resulting in airway epithelial damage, the severe inflammatory response that follows as a result of metabolic stress in innate immune cells (e.g., macrophages) ultimately elicits a “cytokine storm” that may lead to acute respiratory distress syndrome (ARDS) and death. Thus, a new approach that targets the host innate immune response would represent a highly significant therapeutic advance for influenza as well as other respiratory viruses that lead to ARDS, e.g., SARS-CoV-2. We have identified Toll-like receptor 4 (TLR4), a pattern recognition receptor best known for its ability to sense Gram-negative lipopolysaccharide (LPS), as key to the host inflammatory response to influenza. This was initially surprising since influenza virus does not express any “pathogen-associated molecular patterns” that trigger inflammation via TLR4. Nonetheless, using both TLR4-/- mice and multiple TLR4 antagonists in both a mouse model of influenza infection, as well as in cotton rats (Sigmodon hispidus; CR) that are susceptible to non-adapted strains of human influenza, we rigorously demonstrated that TLR4 signaling is central to the generation of lung and systemic inflammation in response to infection. We identified a host-derived protein, High Mobility Group Box-1 (HMGB1), released from dying cells during infection, that acts as a “danger-associated molecular pattern,” thereby triggering TLR4 through its co-receptor, MD2. Recently, we identified a second host-derived protein, gastrin-releasing peptide (GRP), as contributory to influenza-mediated disease, using three distinct inhibitors of GRP or GRP receptor signaling to significantly blunt cytokine production, lung pathology, and lethality when administered to mice therapeutically. Our published and preliminary data support the central hypothesis that these two mediators are interrelated and converge during the host response to influenza infection. In two Specific Aims, we propose to (1) delineate influenza-mediated interactions between GRP receptor (GRPR)- and TLR4/MD2-mediated signaling, and (2) correlate GRP and HMGB1 levels with disease severity in two distinct experimental models of influenza-induced disease and in influenza-infected patients. In this exploratory R21 application, we shall seek to identify the mechanistic underpinnings of the relationship between these two signaling pathways with the potential of identifying therapeutic strategies to ameliorate disease.