Gender and Host Defense Against Pneumonia

描述(申请人提供)：问题：适应性免疫和先天免疫方面存在性别差异，但仍知之甚少。一个例子是常见而严重的肺炎问题，女性表现出比男性更强的抵抗力。虽然雌激素对免疫系统有多种调节作用，但在确定雌激素改善肺宿主防御的具体细胞和分子机制方面进展甚微。试点数据：使用最常见的肺炎形式肺炎球菌肺炎的小鼠模型，我们观察到女性或接受雌激素治疗的男性对感染的抵抗力更强，表现为更好地清除体内细菌，减少肺部炎症和更好的存活率。我们还确定了由结构性表达的一氧化氮合酶-3(又名内皮型一氧化氮合酶或NOS3)产生的一氧化氮的关键作用，很可能是在雌激素介导的信号事件启动NO释放之后。假设：雌激素通过PI3K-Akt磷酸化激活NOS3，增强AM对细菌的杀灭作用，这是女性对肺炎更具抵抗力的基础。具体目标：目标1将测试假设，即NOS3-产生的NO在吞噬体内作用，在那里它与NADPH氧化酶衍生的超氧化物相互作用，形成有效的杀菌剂过氧亚硝酸盐。活体研究将测量正常小鼠或NADPH氧化酶、内皮或诱导型一氧化氮合酶(NOS3，NOS2)基因缺陷小鼠的清除率、肺炎和存活率。这些小鼠的AM的体外分析将测量吞噬和杀伤能力。目的2将验证雌激素通过PI3K-Akt磷酸化途径激活NOS3来增强AM对肺炎球菌的杀伤力的假设。雌激素对NOS3和Akt的磷酸化状态和功能的影响将使用Western印迹分析、药物抑制剂和基因敲除(KO)小鼠来测试。吞噬触发的钙流在激活雌激素诱导的NOS3中的作用将通过活显微镜进行评估。目的3将在更年期小鼠模型中测试宿主对肺炎的抵抗力将下降的预测，以及雌激素替代疗法(HRT)将逆转这种下降并恢复正常增加的女性对肺炎球菌肺炎的抵抗力。意义：这项研究将确定宿主抵抗肺炎的新机制，并将向公共卫生政策通报激素替代疗法的潜在好处。

DESCRIPTION (provided by applicant): The Problem: Gender differences exist in adaptive and innate immunity, but remain poorly understood. One example is the common and serious problem of pneumonia, where females show greater resistance than males. Although estrogen mediates numerous effects on the immune system, little progress has been made in identifying the specific cellular and molecular mechanisms by which estrogen improves lung host defense. Pilot Data: Using a mouse model of the most common form of pneumonia, pneumococcal pneumonia, we observe greater resistance to infection in females or estrogen-treated males, demonstrated by better clearance of bacteria in vivo, diminished lung inflammation and better survival. We also identify a critical role for nitric oxide generated by constitutively expressed nitric oxide synthase-3 (aka endothelial NOS or NOS3), most likely after priming for NO release by estrogen-mediated signaling events. Hypothesis: Estrogen- mediated activation of NOS3 via PI3K-Akt phosphorylation enhances bacterial killing by AMs and is the basis for greater resistance to pneumonia in females. Specific Aims: Aim 1 will test the postulate that NOS3-production of NO acts within phagosomes where it interacts with NADPH oxidase-derived superoxide to form the potent microbicide, peroxynitrite. In vivo studies will measure clearance, pneumonia and survival in normal mice or mice genetically deficient in NADPH oxidase, endothelial or inducible nitric oxide synthase (NOS3, NOS 2). In vitro analysis of AMs from these mice will measure phagocytosis and killing. Aim 2 will test the postulate that estrogen augments AM killing of pneumococci by activating NOS3 through a PI3K-Akt phosphorylation pathway. Estrogen effects on phosphorylation status and function of NOS3 and Akt will be tested using Western blot analysis, pharmacologic inhibitors, and knockout (KO) mice. The role of a phagocytosis-triggered calcium flux in activating estrogen-primed NOS3 will be evaluated by live microscopy. Aim 3 will test the prediction that host defense against pneumonia will decline in mouse models of menopause, and that estrogen hormone replacement therapy (HRT) will reverse this decline and restore the normal increased female resistance to pneumococcal pneumonia. Significance: This research will identify novel mechanisms in host defense against pneumonia and will inform public health policy about a potential benefit of hormone replacement therapy.