SERPINB1/MNEI: Role in innate immune defense against influenza virus infection

描述（由申请人提供）：流感是一种由流感病毒引起的传染性呼吸道疾病，在全球范围内引起大量疾病。治疗选择很少，流行病的威胁持续存在。我们已经确定了SERPINB1，一种内源性生物反应调节蛋白，也称为MNEI（单核细胞中性粒细胞弹性蛋白酶抑制剂），可保护肺部免受细菌感染，我们假设，可能证明可用于预防流感。使用肺部感染的啮齿动物模型，我们表明SERPINB1/MNEI保护先天性抗细菌宿主防御并防止炎症和肺损伤。正如预期的那样，SERPINB1/MNEI在体外没有抗菌作用，因为其生物化学功能是选择性和有效地抑制嗜中性粒细胞丝氨酸蛋白酶（NSP）。NSP（弹性蛋白酶、组织蛋白酶G和蛋白酶-3）是储存在嗜中性粒细胞颗粒中的活性蛋白酶，其在炎性肺病中大量释放，并且是诱导炎性趋化因子过度产生、对肺组织的直接蛋白水解损伤和通过降解先天保护性分子而丧失抗微生物防御的强大病理剂。在被NSP靶向并被MNEI保护的宿主防御分子中突出的是肺表面活性蛋白-D（SP-D），其中和许多流感病毒株并增强它们的清除并通过对肺泡巨噬细胞的抗炎作用来保护宿主防御。拟议的R21（试点）项目将测试中性粒细胞丝氨酸蛋白酶（NSP）有助于流感致病性和MNEI/SERPINB1保护流感致病性的假设。我们将使用我们实验室最近通过缺失SERPINB1/MNEI基因产生的NSP活性增加的小鼠模型，并将确定这些小鼠感染甲型流感病毒株（IAV）后是否显示发病率（体重减轻）和死亡率增加。我们将通过测量病毒滴度、肺损伤、炎性细胞因子、肺泡巨噬细胞和嗜中性粒细胞的流入、嗜中性粒细胞的坏死以及完整SP-D和NSP裂解的失活SP-D的水平来测试IAV感染的mnei-/-小鼠的先天免疫应答（早期宿主应答）的异常。最后，我们将测试重组MNEI是否挽救了mnei-/-小鼠的缺陷反应，并增强了野生型小鼠对流感病毒的抗病毒宿主反应。成功鉴定NSP作为流感致病性介质将是迈向新型治疗的一步。作为流感的治疗剂，NSP的抑制剂，特别是天然存在的生物反应调节剂MNEI的抑制剂，在机制上与现有药物无关，因此适合联合使用。由于MNEI通过保护先天宿主免疫而发挥作用，因此预期其对适应性免疫缺陷的个体（包括非常年轻和非常年老的个体）有效。重要的是，MNEI不需要结合病毒决定簇，因此预计不会产生耐药性。流感是由流感病毒引起的一种传染性呼吸道疾病，是造成大量疾病的原因，并且存在着新出现的更具毒性的毒株在世界范围内传播的真实的威胁。我们已经确定了MNEI（单核细胞/中性粒细胞弹性蛋白酶抑制剂），一种内源性生物反应调节蛋白，可保护肺部免受细菌感染。在拟议的R21（探索/发展补助金）项目中，我们将使用我们实验室开发的小鼠模型来测试MNEI将被证明有助于预防流感的假设。

DESCRIPTION (provided by applicant): Influenza, a contagious respiratory illness caused by influenza virus, is responsible for substantial illness worldwide. Treatment options are few and the threat of a pandemic is constant. We have identified SERPINB1, an endogenous biological response modifier protein, also called MNEI (monocyte neutrophil elastase inhibitor) that protects the lung against bacterial infection and that, we hypothesize, may prove useful in protecting against influenza. Using rodent models of pulmonary infection, we showed that SERPINB1/MNEI protects innate anti-bacterial host defense and prevents inflammation and lung injury. SERPINB1/MNEI had no anti-bacterial effect in vitro, as anticipated, since its biochemical function is to selectively and efficiently inhibit neutrophil serine proteases (NSPs). The NSPs (elastase, cathepsin G and proteinase-3) are active proteases stored in neutrophil granules, which are released in large amounts in inflammatory lung disease and are powerful pathological agents that induce overproduction of inflammatory chemokines, direct proteolytic injury to lung tissue and loss of anti-microbial defense by degrading innate protective molecules. Prominent among the host defense molecules targeted by NSPs and protected by MNEI is pulmonary surfactant protein-D (SP-D), which neutralizes many influenza virus strains and enhances their clearance and protects host defense by anti- inflammatory action on alveolar macrophages. The proposed R21 (pilot) project will test the hypothesis that neutrophil serine proteases (NSPs) contribute to, and MNEI/SERPINB1 protects against, pathogenicity of influenza. We will use a mouse model of increased NSP activity recently generated in our laboratory by deletion of the SERPINB1/MNEI gene and will determine whether these mice show increased morbidity (loss of body weight) and mortality on infection with strains of influenza A virus (IAV). We will test for abnormalities of the innate immune response (early host response) of IAV infected mnei-/- mice by measuring viral titer, lung injury, inflammatory cytokines, influx of alveolar macrophages and neutrophils, necrosis of neutrophils, and levels of intact SP-D and NSP-cleaved inactivated SP-D. Finally, we will test whether recombinant MNEI rescues the defective response of mnei-/- mice and enhances the anti-viral host response of wild-type mice to influenza virus. Successful identification of NSPs as mediators of pathogenicity in influenza would be a step toward a novel therapeutic. As a therapeutic for influenza, inhibitors of NSPs and especially MNEI, a naturally occurring biological response modifier, would be mechanistically independent of current drugs and therefore suitable for combination use. Because MNEI functions by protecting innate host immunity, efficacy is anticipated for individuals with deficient adaptive immunity including the very young and very old. Importantly, MNEI has no need for binding viral determinants, and thus development of resistance is not anticipated. Influenza, a contagious respiratory illness caused by influenza virus, is responsible for substantial illness, and there is real threat of worldwide spread of emerging more virulent strains. We have identified MNEI (monocyte/neutrophil elastase inhibitor), an endogenous biological response modifier protein, that protects the lung against bacterial infection. In the proposed R21 (Exploratory/- Developmental Grant) project, we will use a mouse model developed in our laboratory to test the hypothesis that MNEI will prove useful in protecting against influenza.