The role of adenosine in the pathogenesis of influenza

描述（由申请方提供）：甲型流感病毒引起高度传染性急性呼吸道疾病。季节性流感流行每年在全世界造成> 300，000人死亡，而大流行在世纪引起毁灭性的生命损失，并且可能在21世纪这样做。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的基本机制尚不清楚。呼吸道合胞病毒（RSV）和流感病毒感染的BALB/c小鼠诱导增加的通道介导的核苷酸UTP和ATP释放到支气管肺泡衬里液（BALF），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。RSV感染BALB/c小鼠仅引起轻度疾病，而流感引起更严重的疾病，具有急性肺损伤的许多病理生理学特征，这也见于人类致命的大流行性流感或流行性流感。重要的是，流感（而非RSV）感染后，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（A1-subtype adenosine receptor，A1-subtype adenosine receptor）的激活增加。腺苷刺激中性粒细胞A1-AR在急性肺损伤的发生和发展中起重要作用。该提议的中心假设是，流感刺激从头ATP合成和从感染的ATII细胞释放，释放的ATP以加速的速率代谢为腺苷（由于增加的外核苷酸酶CD 73活性），并且通过其对A1-BXR的作用，BALF中的腺苷在诱导流感的肺损伤中起关键作用。将在3个特定目标中检验该假设：1）为了确定流感感染对肺核苷酸代谢的时间效应，将测量流感感染MLE-12细胞和纯化的肺泡II型（ATII）细胞以及来自C57 B1/6或SP-C/GFP小鼠的FACS分选的ATII细胞后核苷酸代谢的变化，以及流感感染对BALF核苷酸谱的影响，以及ERK MAP激酶在流感感染后诱导核苷酸合成中的作用。2)为了确定CD 73在流感发病机制中的作用，将测量流感感染对呼吸道上皮细胞CD 73表达和活性的影响;将确定CD 73的药理学阻断对C57 BL/6小鼠中流感发病机制的影响;并将研究CD 73敲除小鼠中流感感染的结果。3)为了确定腺苷/A1-A1 R受体信号传导在流感发病机制中的作用，将测量流感诱导的NF-κ B活化对ATII细胞和中性粒细胞中A1-A1 R表达的影响;将确定药理学A1-A1 R阻断对C57 BL/6小鼠中流感发病机制的影响;并且将研究A1-miR-/-敲除小鼠或A1-miR-/-骨髓嵌合小鼠中流感感染的结果。这些目标的完成将导致增加对腺苷在流感相关肺损伤的发病机制中的作用的理解，并允许确定其作为治疗干预对抗流感诱导的肺损伤的靶点的潜力。 公共卫生相关性：甲型流感病毒引起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> 300，000人死亡，而大流行在世纪引起毁灭性的生命损失，并且可能在21世纪这样做。拟议的研究旨在提高我们对流感肺功能障碍机制的理解，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.