The role of adenosine in the pathogenesis of influenza

描述（由申请人提供）：甲型流感病毒是一种高度传染性的急性呼吸道疾病。季节性流感流行每年在全世界造成150万人死亡，而大流行病在20世纪造成了毁灭性的生命损失，在21世纪也可能如此。然而，目前，流感或任何其他肺部病毒感染中肺功能障碍和损伤发展的基本机制尚不明确。呼吸道合胞病毒（RSV）和流感病毒感染BALB/c小鼠均诱导通道介导的核苷酸UTP和ATP向支气管肺泡衬液（BALF）释放增加，并且在这两种感染中，BALF - UTP升高有助于肺水肿和低氧血症的发展。在BALB/c小鼠中，RSV感染只引起轻微的疾病，而流感引起更严重的疾病，具有许多急性肺损伤的病理生理特征，这在致死性大流行或流行性流感中也可见到。重要的是，在流感（而非RSV）感染后，ATP释放到BALF的升高伴随着ATP降解产物腺苷对a1亚型腺苷受体（AdoR）激活的增加。腺苷刺激中性粒细胞A1-AdoR在急性肺损伤的发生和促进中起着重要作用。该建议的中心假设是，流感刺激受感染的ATII细胞重新合成和释放ATP，释放的ATP被加速代谢为腺苷（由于外核苷酶CD73活性增加），并且通过其对A1-AdoR的影响，BALF中的腺苷在流感诱导肺损伤中起关键作用。这一假设将在3个具体目标中进行检验：1)为了确定流感感染对肺核苷酸代谢的时间效应，我们将检测流感感染C57Bl/6或SP- C/GFP小鼠MLE-12细胞和纯化肺泡II型（ATII）细胞以及facs分选的ATII细胞后的核苷酸代谢变化，流感感染对BALF核苷酸谱的影响，以及流感感染后ERK MAP激酶在诱导核苷酸合成中的作用。2)为了确定CD73在流感发病中的作用，我们将检测流感感染对呼吸道上皮细胞CD73表达和活性的影响；确定药物阻断CD73对C57BL/6小鼠流感发病机制的影响；并对cd73敲除小鼠感染流感的结果进行研究。3)为确定腺苷/A1-AdoR受体信号在流感发病中的作用，将检测流感诱导的NF-kB活化对ATII细胞和中性粒细胞A1-AdoR表达的影响；确定药物阻断A1-AdoR对C57BL/6小鼠流感发病机制的影响；以及A1-AdoR-/-敲除小鼠或A1-AdoR-/-骨髓嵌合小鼠感染流感的结果将被研究。这些目标的完成将导致对腺苷在流感相关肺损伤发病机制中的作用的进一步了解，并允许确定其作为治疗干预目标的潜力，以对抗流感诱导的肺损伤。

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.