Natural Killer Cell Responses of Aged Mice to Primary Influenza Infection

描述（由申请人提供）：尽管接种了疫苗，流感和继发性肺炎仍是 65 岁及以上人群的第四大死因。最近出现的新型 2009-H1N1 流感，其疫苗有限或无法获得，有可能导致 65 岁以下人群死亡 30,000-90,000 人。这些令人震惊的统计数据表明，需要更好地了解对急性流感感染的初级免疫反应与年龄相关的变化。我们最近发现自然杀伤（NK）细胞在体外和体内控制早期流感感染中发挥着关键作用。老年 C57BL/6 小鼠在流感感染早期表现出肺部病毒滴度增加、体重减轻、NK 细胞毒性降低以及 NK 细胞数量减少。重要的是，在年轻和老年小鼠感染流感后，通过抗 NK1.1 IgG 抗体 (PK136) 治疗体内 NK 细胞耗竭可导致显着的体重减轻和肺部病毒滴度增加。这些数据表明 NK 细胞对于早期控制流感感染至关重要。因此，我们的总体假设是，诱导有效的 NK 细胞反应对于控制包括老年人在内的弱势群体的早期流感感染至关重要。我们的研究将描述 NK 细胞功能与年龄相关的变化，阐明 NK 细胞功能受损的潜在内在和外在机制，并确定 NK 细胞在控制体内流感病毒早期感染中的关键作用。在目标 1 中，将评估感染的前关键四天期间与年龄相关的易感性差异以及 NK 细胞功能的作用。我们将进行流感剂量反应，并测量与年龄相关的肺部病毒滴度、病理学、组织学和肺部细胞浸润、体重减轻和感染恢复的差异。感染期间将测量 NK 亚群与年龄相关的变化、NK 细胞的溶细胞功能、树突状细胞 (DC) 功能以及局部和全身细胞因子的产生。在目标 2 中，我们将阐明流感感染期间 NK 细胞功能受损的内在和外在机制中与年龄相关的变化。将在纯化的 NK 细胞中测量与年龄相关的裂解效率、激活受体功能/信号传导和细胞因子受体/功能的差异，以识别内在的 NK 细胞缺陷。过继转移研究将描述与流感易感性相关的 NK 细胞增殖、凋亡和归巢的内在和外在影响。我们假设 DC 是早期感染中 NK 细胞功能的关键外在效应器。因此，我们将测量 NK 细胞的 DC 激活、DC 细胞因子的产生以及 DC 消融对 NK 细胞功能的影响。在目标 3 中，我们将验证 NK 细胞在控制体内流感反应中的重要作用。将评估年轻和老年小鼠的体内 NK 细胞溶解情况以及存活率、体重减轻和肺部病毒滴度。 NK 细胞在控制流感易感性中的具体作用将在用 PK136 体内 NK 耗竭后的小鼠或 RAG-1-/- 或 NKD 转基因小鼠中进行研究。这三种小鼠模型将清楚地描述 NK 细胞在控制流感早期反应中的具体贡献。 公共卫生相关性：季节性流感的流行和最近猪 2009-H1N1 的出现要求有必要研究对流感感染的主要反应中与年龄相关的变化。老年小鼠在感染的前四天内表现出 NK 细胞功能下降和肺部病毒滴度升高，更重要的是，在适应性初级反应开始之前死亡。如果适用于人类，这表明年轻人和老年人对流感感染产生早期有效的 NK 细胞反应的能力至关重要，特别是在疫苗有限或无法获得的情况下。

DESCRIPTION (provided by applicant): Despite vaccination, influenza and secondary pneumonias are the fourth leading cause of death in individuals 65 years and older. The recent emergence of the novel 2009-H1N1 influenza, to which vaccines are limited or unavailable, has the potential to cause 30,000-90,000 deaths in people under 65 years of age. These staggering statistics mandate the need to better understand age-related changes in primary immune responses to acute influenza infection. We have recently identified a critical role for natural killer (NK) cells in controlling early influenza infection ex vivo and in vivo. Aged C57BL/6 mice exhibited increased lung virus titers, weight loss, decreased NK cytotoxicity, and a reduced number of NK cells during early influenza infection. Importantly, in vivo NK cell depletion by anti-NK1.1 IgG antibody (PK136) treatment induced marked weight loss and increased lung virus titers after influenza infection of both young and aged mice. These data suggest that NK cells are essential in early control of influenza infection. Thus, our overarching hypothesis is that the induction of an effective NK cell response is vital for controlling early influenza infection in vulnerable populations, including the elderly. Our studies will characterize age-related changes in NK cell function, elucidate potential intrinsic and extrinsic mechanisms for impaired NK cell function, and establish the critical role of NK cells in controlling early infection to influenza virus in vivo. In Aim 1, age-related differences in susceptibility and the role of NK cell function during the first critical four days of infection will be assessed. We will perform influenza dose responses and measure age-related differences lung virus titers, pathology, histology and cellular infiltrates in lung, weight loss, and recovery from infection. Age-related changes in NK subsets, cytolytic function of NK cells, dendritic cell (DCs) function, and local and systemic cytokine production will be measured during infection. In Aim 2, we will elucidate age-related changes in intrinsic and extrinsic mechanism(s) for impaired NK cell function during influenza infection. Age-related differences in lytic efficiency, activation receptor function/signaling, and cytokine receptor/function will be measured in purified NK cells to identify intrinsic NK cell defects. Adoptive transfer studies will delineate between intrinsic and extrinsic effects on NK cell proliferation, apoptosis, and homing as related to susceptibility to influenza. We hypothesize that DCs are critical extrinsic effectors on NK cell function in early infection. Thus, we will measure DC activation of NK cells, DC cytokine production, and the effects of DC ablation on NK cell function. In Aim 3, we will validate the essential role of NK cells in controlling the in vivo response to influenza. In vivo NK cytolysis in young and aged mice will be assessed along with survival, weight loss, and lung virus titers. The specific role of NK cells in controlling susceptibility to influenza will be addressed in mice after NK depletion in vivo with PK136 or in RAG-1-/- or NKD transgenic mice. These three mouse models will afford a clear delineation of the specific contribution of NK cells in controlling the early response to influenza. PUBLIC HEALTH RELEVANCE: The prevalence of seasonal influenza and the recent emergence of swine 2009-H1N1 mandate the need to study age-related changes in the primary response to influenza infection. Aged mice show decreased NK cell function and elevated lung virus titers during the first four days of infection, and more importantly, die prior the initiation of an adaptive primary response. If applicable to humans, this suggests that the ability of young and aged individuals to mount an early and effective NK cell response to influenza infection is vital, especially when vaccine is limited or unavailable.