Gender and Host Defense Against Pneumonia

描述（由申请人提供）：问题：性别差异存在于适应性和先天免疫，但仍然知之甚少。其中一个例子是肺炎这一常见而严重的问题，女性比男性表现出更大的抵抗力。虽然雌激素介导了许多对免疫系统的影响，但在确定雌激素改善肺宿主防御的特定细胞和分子机制方面几乎没有取得进展。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，我们观察到女性或雌激素治疗的男性对感染的抵抗力更强，表现为体内细菌清除率更高，肺部炎症减少，存活率更高。我们还确定了组成型表达的一氧化氮合酶-3（aka内皮NOS或NOS 3）产生的一氧化氮的关键作用，最有可能是在雌激素介导的信号传导事件引发NO释放后。假设：雌激素介导的通过PI 3 K-Akt磷酸化的NOS 3活化增强AM的细菌杀伤，并且是女性对肺炎的更大抗性的基础。具体目标：目的1将测试的假设，即NOS 3-生产NO的吞噬体内的行为，它与NADPH氧化酶衍生的超氧化物相互作用，形成有效的杀微生物剂，过氧亚硝酸盐。体内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（NOS 3、NOS 2）遗传缺陷小鼠的清除率、肺炎和存活率。来自这些小鼠的AM的体外分析将测量吞噬作用和杀伤作用。目的2将测试雌激素通过PI 3 K-Akt磷酸化途径激活NOS 3增强AM对肺炎球菌的杀伤的假设。将使用蛋白质印迹分析、药理学抑制剂和敲除（KO）小鼠检测雌激素对NOS 3和Akt磷酸化状态和功能的影响。将通过活体显微镜评价吞噬触发的钙流在激活雌激素引发的NOS 3中的作用。目标3将测试预测，宿主对肺炎的防御将在更年期小鼠模型中下降，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。重要性：这项研究将确定宿主防御肺炎的新机制，并将告知公共卫生政策激素替代疗法的潜在益处。

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.