Natural Killer Cell Responses of Aged Mice to Primary Influenza Infection

描述（由申请方提供）：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流感疫苗有限或无法获得，有可能导致30，000 - 90，000名65岁以下的人死亡。这些惊人的统计数据要求我们更好地了解急性流感感染的初级免疫反应中与年龄相关的变化。我们最近已经确定了一个关键作用，自然杀伤（NK）细胞在控制早期流感感染离体和体内。老年C57 BL/6小鼠在早期流感感染期间表现出肺病毒滴度增加、体重减轻、NK细胞毒性降低和NK细胞数量减少。重要的是，抗NK1.1 IgG抗体（PK 136）治疗的体内NK细胞耗竭诱导了年轻和老年小鼠流感感染后显著的体重减轻和肺病毒滴度增加。这些数据表明，NK细胞在流感感染的早期控制中至关重要。因此，我们的总体假设是，诱导有效的NK细胞应答对于控制脆弱人群（包括老年人）的早期流感感染至关重要。我们的研究将表征NK细胞功能的年龄相关变化，阐明NK细胞功能受损的潜在内在和外在机制，并建立NK细胞在体内控制流感病毒早期感染中的关键作用。在目标1中，将评估易感性的年龄相关差异和NK细胞功能在感染的第一个关键4天期间的作用。我们将进行流感剂量反应，并测量与年龄相关的差异，肺病毒滴度，病理学，组织学和肺细胞浸润，体重减轻和感染恢复。将在感染期间测量NK亚群、NK细胞的细胞溶解功能、树突状细胞（DC）功能以及局部和全身细胞因子产生的NK相关变化。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的年龄相关变化。将在纯化的NK细胞中测量裂解效率、活化受体功能/信号传导和细胞因子受体/功能的NK相关差异，以鉴定内在NK细胞缺陷。连续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效应。我们推测，在感染早期，树突状细胞是NK细胞功能的关键外部效应器。因此，我们将测量NK细胞的DC活化、DC细胞因子产生以及DC消融对NK细胞功能的影响。在目标3中，我们将验证NK细胞在控制体内对流感的应答中的重要作用。将沿着存活、体重减轻和肺病毒滴度评估年轻和老年小鼠的体内NK细胞溶解。NK细胞在控制流感易感性中的特定作用将在用PK 136体内NK耗竭后的小鼠中或在RAG-1-/-或NKD转基因小鼠中进行阐述。这三种小鼠模型将清楚地描述NK细胞在控制对流感的早期反应中的具体贡献。

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.