The role of adenosine in the pathogenesis of influenza

描述(申请人提供)：甲型流感病毒引起一种高度传染性的急性呼吸道疾病。季节性流感每年在全球造成30万人死亡，而大流行在20世纪造成了毁灭性的生命损失，并可能在21世纪造成这种情况。呼吸道合胞病毒(RSV)和流感病毒感染BALB/c小鼠均可诱导通道介导的核苷酸UTP和ATP释放到BALF中，在这两种感染中，BALF UTP升高导致肺水肿和低氧血症的发生。在BALB/c小鼠中，RSV感染只会引起轻微的疾病，而流感会引起更严重的疾病，具有许多急性肺损伤的病理生理特征，在人类的致死性大流行或流行性流感中也可以看到这种情况。重要的是，在流感而不是RSV感染之后，ATP释放到BALF中的增加伴随着ATP降解产物腺苷对A1亚型腺苷受体(ADAR)的激活。这一建议的中心假设是，流感刺激感染的ATII细胞从头合成和释放ATP，释放的ATP被加速代谢为腺苷(由于ECN CD73活性增加)，并且通过对A1-ADOR的影响，BALF中的腺苷在诱导流感肺损伤中发挥关键作用。该假说将被验证于3个特定目标：1)为了确定流感感染对肺核苷酸代谢的暂时影响，将测量流感感染后MLE-12细胞和纯化的肺泡II型(ATII)细胞以及FACS分选的C57BL/6或SP-C/GFP小鼠的ATII细胞的核苷酸代谢变化，以及流感感染对BALF核苷酸谱的影响，以及ERK图激酶在流感感染后诱导核苷酸合成中的作用。2)检测流感病毒感染对呼吸道上皮细胞CD73表达和活性的影响，药物阻断CD73对C57BL/6小鼠流感发病机制的影响，研究CD73基因敲除小鼠感染流感的转归，以确定CD73在流感发病机制中的作用。3)为确定腺苷/A1-ADAR受体信号在流感发病机制中的作用，将检测流感诱导的核因子-kB对ATII细胞和中性粒细胞A1-ADAR表达的影响；药理学上A1-ADOR阻断对C57BL/6小鼠流感发病的影响；以及在A1-ADOR/-基因敲除小鼠或A1-ADOR/骨髓嵌合小鼠中流感感染的结局。完成这些目标将使人们更好地了解腺苷在流感相关肺损伤的发病机制中的作用，并使人们能够确定其作为抗击流感所致肺损伤的治疗干预目标的潜力。 季节性流感每年在全球造成30万人死亡，而大流行在20世纪造成了毁灭性的生命损失，并可能在21世纪造成这种情况。建议的研究旨在提高我们对流感导致肺功能障碍的机制的理解，并确定流感治疗的新的潜在靶点。

DESCRIPTION (provided by applicant): Influenza A viruses cause a highly contagious acute respiratory disease. Seasonal influenza epidemics cause >300,000 deaths/yr worldwide, while pandemics elicited devastating loss of life in the 20th century, and may do so in the 21st. Currently, however, essential mechanisms underlying development of lung dysfunction and injury in influenza or any other pulmonary viral infection are poorly defined. Both respiratory syncytial virus (RSV) and influenza virus infection of BALB/c mice induce increased channel-mediated release of the nucleotides UTP and ATP into the bronchoalveolar lining fluid (BALF), and, in both infections, elevated BALF UTP contributes to development of lung edema and hypoxemia. RSV infection in BALB/c mice causes only mild disease while influenza causes more severe disease with many of the pathophysiologic features of acute lung injury, which is also seen in lethal pandemic or epidemic influenza in man. Importantly, following influenza, but not RSV, infection, elevated ATP release into BALF is accompanied by increased activation of A1-subtype adenosine receptors (AdoR) by the ATP degradation product adenosine. Adenosine stimulation of neutrophil A1-AdoR plays a significant role in the initiation and promotion of acute lung injury. The central hypothesis of this proposal is that influenza stimulates de novo ATP synthesis and release from infected ATII cells, that released ATP is metabolized to adenosine at an accelerated rate (due to increased ectonucleotidase CD73 activity), and that, by its effects on A1-AdoR, adenosine in the BALF plays a pivotal role in inducing lung injury in influenza. This hypothesis will be tested in 3 Specific Aims: 1) To determine temporal effects of influenza infection on pulmonary nucleotide metabolism, changes in nucleotide metabolism following influenza infection of MLE-12 cells and purified alveolar type II (ATII) cells, as well as FACS-sorted ATII cells from C57Bl/6 or SP- C/GFP mice will be measured, together with the impact of influenza infection on the BALF nucleotide profile, and the role of ERK MAP kinase in inducing nucleotide synthesis after influenza infection. 2) To determine the role of CD73 in influenza pathogenesis, effects of influenza infection on respiratory epithelial cell CD73 expression and activity will be measured; the impact of pharmacologic blockade of CD73 on influenza pathogenesis in C57BL/6 mice will be determined; and the outcome of influenza infection in CD73-knockout mice will be investigated. 3) To determine the role of adenosine/A1-AdoR receptor signaling in influenza pathogenesis, the effect of influenza-induced NF-kB activation on A1-AdoR expression in ATII cells and neutrophils will be measured; the impact of pharmacologic A1-AdoR blockade on influenza pathogenesis in C57BL/6 mice will be determined; and the outcome of influenza infection in A1-AdoR-/- knockout mice, or in A1- AdoR-/- bone marrow-chimeric mice will be investigated. Completion of these objectives will lead to increased understanding of the role of adenosine in the pathogenesis of influenza-associated lung injury, and permit determination of its potential as a target for therapeutic intervention to combat influenza-induced lung damage. PUBLIC HEALTH RELEVANCE: Influenza A virus causes a highly contagious acute respiratory disease. Seasonal influenza epidemics cause >300,000 deaths/yr worldwide, while pandemics elicited devastating loss of life in the 20th century, and may do so in the 21st. The proposed studies are aimed at improving our understanding of the mechanisms underlying lung dysfunction in influenza, and identifying a novel potential target for influenza therapy.