Post Viral Bacterial Pneumonia: Role of MicroRNA and Autophagy

描述（由申请人提供）：流感感染是美国和世界范围内死亡的主要原因，仅在美国每年就有5万人死亡。尽管许多患者死于原发性病毒感染，但相当数量的流感相关死亡可归因于继发性细菌感染的发展。为什么宿主在流感后更容易受到细菌感染尚不清楚。为了开发改善结果的治疗方法，更好地了解宿主对连续流感+细菌感染的反应是必要的。使用小鼠序列性流感和耐甲氧西林金黄色葡萄球菌（MRSA）或肺炎链球菌感染模型，我们发现这些小鼠的存活率下降，抗菌反应受损。我们还发现，宿主对病毒后肺炎的反应以巨噬细胞自噬反应受损、巨噬细胞吞噬和肺部细菌杀伤受损为特征。与单独感染任一病原体的小鼠相比，这些发现与干扰素(IFN)γ的显著上调和miR 155的上调相关。因此，我们假设在流感后肺炎的情况下，对MRSA的先天免疫受损是由于ifn γ诱导miR 155的上调，miR 155反过来阻断保护性自噬反应，阻止细菌吞噬和杀伤。我们将通过以下具体目标来实现这些目标。目的1)确定IFNγ在流感单独感染、MRSA/肺炎链球菌单独感染或序列感染后调节miR 155表达、宿主防御、细胞因子产生和巨噬细胞自噬中的作用。该目标将在单次和连续感染中描述这些结果，并将测试使用野生型和IFNγ产生的嵌合小鼠的反应

DESCRIPTION (provided by applicant): Influenza infections are a leading cause of death in the United States and worldwide, with 50 000 of deaths occurring annually in the United states alone. Although many patients succumb to the primary viral infection, a significant number of influenza-related deaths are attributable to the development of secondary bacterial infections. Why the host is more susceptible to bacterial infections post-influenza is poorly understood. A better understanding of how the host responds to sequential influenza + bacterial infection is necessary in order to develop therapies to improve outcomes. Using a murine model of sequential pulmonary influenza and methicillin-resistant Staphylococcus aureus (MRSA) or Streptococcus pneumoniae infection, we have found that these mice have decreased survival and impaired anti-bacterial responses. We also showed that host responses to post-viral pneumonia are characterized by impaired macrophage autophagy responses, and impaired macrophage phagocytosis and killing of bacteria in the lungs. These findings correlate with significant upregulation of interferon (IFN)γ and upregulation of miR 155 when compared to mice infected with either pathogen alone. Thus, we hypothesize that impaired innate immunity against MRSA in the setting of post-influenza pneumonia is due to IFNγ-induced upregulation of miR 155 which in turn blocks protective autophagy responses and prevents bacterial phagocytosis and killing. We will address these goals with the following specific aims. Aim 1) To determine the role of IFNγ in regulating miR 155 expression, host defense, cytokine production, and macrophage autophagy following infection with influenza alone, MRSA/Streptococcus pneumoniae alone or sequential infection. This aim will characterize these outcomes in single and sequential infections, will test responses in chimeric mice generated using wild-type and IFNγ receptor-/- mice and will test a therapeutic approach to block IFNγ using a neutralizing mAb. Aim 2) To determine whether the impaired host defense noted during post-viral pneumonia is due to a failure of lung macrophages to upregulate protective autophagy responses This aim will determine whether autophagy is impaired in lung macrophages post- sequential infection compared to single infection, whether this is correlated with miR 155 inhibition of DAPK1 and whether enhancement of autophagy using rapamycin/Tat Beclin-1 can improve host defense by increasing bacterial phagocytosis and killing. Aim 3) To identify the mechanism that miR-155 uses to regulate macrophage recruitment, autophagy and to impair the anti-bacterial host-defense. This aim will characterize expression of miR 155 during post-viral pneumonia and explore inhibition of autophagy during post-viral pneumonia as a result of miR 155 inhibition of DAPK1. The ability of miR 155 to regulate bacterial phagocytosis and killing also will be explored. Additionally, transgenic mice will be generated using floxed miR155 and CD11b-Cre mice enabling cell type specific removal of miR155 from macrophages further providing mechanistic insight regarding regulation of autophagy by miR 155 expression on macrophages.