Post Viral Bacterial Pneumonia: Role of MicroRNA and Autophagy

描述（由申请人提供）：流感感染是美国和全球的主要死因，仅在美国每年就有50，000例死亡。虽然许多患者死于原发性病毒感染，但大量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还知之甚少。更好地了解宿主如何应对流感+细菌感染是必要的，以便开发治疗方法来改善结果。使用连续肺流感和耐甲氧西林金黄色葡萄球菌（MRSA）或肺炎链球菌感染的小鼠模型，我们发现这些小鼠的存活率降低，抗菌反应受损。我们还表明，宿主对病毒感染后肺炎的反应的特征是巨噬细胞自噬反应受损，巨噬细胞吞噬作用受损，并杀死肺部的细菌。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，针对MRSA的先天免疫受损是由于IFNγ诱导的miR 155上调，其进而阻断保护性自噬应答并阻止细菌吞噬和杀伤。我们将通过以下具体目标实现这些目标。目的1）研究IFNγ对流感病毒、MRSA/肺炎链球菌感染及流感病毒感染后miR 155表达、宿主防御、细胞因子产生及巨噬细胞自噬的影响。这一目标将描述这些结果在单一和连续感染，将测试反应的嵌合小鼠产生的野生型和IFNγ 受体-/-小鼠，并将测试使用中和mAb阻断IFNγ的治疗方法。目的2）确定在病毒后肺炎期间注意到的宿主防御受损是否是由于肺巨噬细胞未能上调保护性自噬应答。该目的将确定与单一感染相比，在连续感染后肺巨噬细胞中自噬是否受损，这是否与miR 155对DAPK 1的抑制相关，以及使用雷帕霉素/达特Beclin-1增强自噬是否与miR 155对DAPK 1的抑制相关。1可以通过增加细菌的吞噬和杀伤作用来提高宿主的防御能力。目的3）探讨miR-155对巨噬细胞募集、自噬及宿主防御功能的影响机制。该目的将表征病毒后肺炎期间miR 155的表达，并探索病毒后肺炎期间由于miR 155抑制DAPK 1而导致的自噬抑制。还将探索miR 155调节细菌吞噬和杀伤的能力。此外，将使用floxed miR 155和CD 11b-Cre小鼠产生转基因小鼠，使得能够从巨噬细胞中特异性去除miR 155，进一步提供关于巨噬细胞上miR 155表达调节自噬的机制见解。

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.