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

描述（由申请人提供）：流感是由流感病毒引起的一种传染性呼吸道疾病，是世界范围内主要疾病的罪魁祸首。治疗选择很少，大流行的威胁持续存在。我们已经鉴定出SERPINB1，一种内源性生物反应修饰蛋白，也被称为MNEI（单核中性粒细胞弹性酶抑制剂），它可以保护肺部免受细菌感染，我们假设它可能对预防流感有用。通过啮齿类动物肺部感染模型，我们发现SERPINB1/MNEI保护先天抗菌宿主防御并预防炎症和肺损伤。正如预期的那样，SERPINB1/MNEI在体外没有抗菌作用，因为它的生化功能是选择性和有效地抑制中性粒细胞丝氨酸蛋白酶（NSPs）。NSPs（弹力酶、组织蛋白酶G和蛋白酶-3）是储存在中性粒细胞颗粒中的活性蛋白酶，在炎症性肺病中大量释放，是一种强大的病理因子，可诱导炎症趋化因子过量产生，通过降解先天保护分子直接对肺组织造成蛋白水解损伤，丧失抗微生物防御能力。在NSPs靶向和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)，它可以中和许多流感病毒株，增强其清除能力，并通过对肺泡巨噬细胞的抗炎作用保护宿主防御。拟议的R21（试点）项目将测试中性粒细胞丝氨酸蛋白酶（NSPs）有助于流感致病性，而MNEI/SERPINB1可以预防流感致病性的假设。我们将使用最近在我们实验室通过删除SERPINB1/MNEI基因产生的NSP活性增加的小鼠模型，并将确定这些小鼠在感染甲型流感病毒（IAV）株时是否表现出增加的发病率（体重减轻）和死亡率。我们将通过测量病毒滴度、肺损伤、炎症细胞因子、肺泡巨噬细胞和中性粒细胞内流、中性粒细胞坏死以及完整SP-D和nsp -裂解灭活SP-D的水平来检测IAV感染mnei-/-小鼠的先天免疫反应（早期宿主反应）的异常情况。最后，我们将测试重组MNEI是否能拯救MNEI -/-小鼠的缺陷反应，并增强野生型小鼠对流感病毒的抗病毒宿主反应。成功鉴定NSPs作为流感致病性介质将是迈向新型治疗的一步。作为一种流感治疗药物，NSPs抑制剂，特别是MNEI（一种自然产生的生物反应调节剂）在机制上独立于现有药物，因此适合联合使用。由于MNEI通过保护先天宿主免疫发挥作用，预计对适应性免疫缺陷的个体（包括幼儿和老年人）有效。重要的是，MNEI不需要结合病毒决定因子，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，可导致大量疾病，并且存在出现更强毒性毒株的全球传播的实际威胁。我们已经确定了MNEI（单核细胞/中性粒细胞弹性酶抑制剂），一种内源性生物反应修饰蛋白，可以保护肺部免受细菌感染。在拟议的R21（探索性/发展性资助）项目中，我们将使用我们实验室开发的小鼠模型来测试MNEI将被证明对预防流感有用的假设。

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.