A role for autophagy in increased cytosolic viral signaling in aging

描述(由申请人提供)：尽管对各种病原体的免疫反应随年龄的下降而伴随着循环中促炎症介质水平的增加，但这种联系的原因尚不清楚。对病毒感染的先天免疫是通过模式识别受体(PRRs)识别独特的病毒特征来启动的，模式识别受体(PRRs)介导了强大的抗病毒因子、I型干扰素(IFN)和其他促炎细胞因子的诱导。最近已经阐明，病毒在感染细胞内的识别是由胞浆中的维甲酸诱导的类基因受体(RLR)家族成员介导的，但到目前为止还没有研究RLR信号是否受年龄影响的报道。在这里，我展示了在几种情况下，病毒配体反应的促炎细胞因子的产生增加：(1)在没有自噬的情况下，(2)在氧化应激增加的情况下，以及(3)在老年人中。这些数据提示了在老年人中观察到的细胞因子产生增加和免疫功能受损的可能机制，我建议对此进行研究。自噬过程通过将细胞器和长寿蛋白运输到溶酶体进行降解来维持细胞器和长寿蛋白的完整性。我已经证明，没有自噬会以两种方式导致RLR信号的放大(和细胞因子产生的增加)。首先，在没有自噬的情况下，线粒体与线粒体相关蛋白IPS-1一起在细胞内积累，IPS-1是RLRs的关键信号蛋白。其次，在没有自噬的情况下，受损的线粒体不会被降解，这些线粒体提供了活性氧物种(ROS)的来源，ROS在ATG5基因敲除细胞中放大了RLR信号。有趣的是，我对老年志愿者的原代细胞的研究反映了这一模式。老年人的巨噬细胞表现出明显高于年轻人的巨噬细胞线粒体ROS和RLR信号水平。据推测，衰老过程中氧化应激水平的增加是许多衰老和迟发性疾病的潜在原因，从动物模型中可以推断，这种氧化应激的增加是由于自噬功能随着年龄的下降而导致的。我的目标是通过检测自噬减少或功能障碍的程度，以及这对受损线粒体的积累和由此导致的ROS水平增加的影响，来表征健康人类衰老中的自噬通量。正如我已经报道的，ROS增加了RLR信号，并发现两者在老年巨噬细胞中都显著升高，我的目标是检测氧化应激对胞浆抗病毒信号随年龄的影响，以了解在老年人中看到的炎症增加的情况，以及这对流感的促炎反应的影响。这项建议中概述的研究将检查衰老过程和抗病毒防御中基本的重要细胞过程之间的十字路口，目的是揭示老年人对病毒感染的先天免疫反应中受损线粒体积累和氧化应激的功能影响。 与公共健康相关：从身体上看，每个人都清楚地看到，我们正在不断衰老，然而，导致这一现象的生物学机制仍然不清楚。我提议调查细胞清理人员，即自噬，在多大程度上滞后于年龄，导致我们细胞内受损成分的积累。然后我将研究这如何影响衰老中的病理，特别是随着年龄的增长，细胞识别和应对病毒感染的方式。

DESCRIPTION (provided by applicant): While the age-dependent decline in the immune response to a variety of pathogens is accompanied by an increase in circulating levels of proinflammatory mediators, the reason for this association remains unclear. Innate immunity to viral infection is initiated through the recognition of unique viral signatures by pattern recognition receptors (PRRs) that mediate the induction of potent antiviral factors, type I interferon's (IFNs) and other pro-inflammatory cytokines. It has recently been elucidated that viral recognition within an infected cell is mediated by members of the retinoic acid inducible gene I -like receptor (RLR) family in the cytosol, yet no report to date has examined if RLR signaling is affected by age. Here, I show that pro-inflammatory cytokine production in response to viral ligands is increased in several situations: (1) in the absence of autophagy, (2) during increased oxidative stress, and (3) in the elderly. These data suggest a possible mechanism for the increased cytokine production and impaired immune function observed in the elderly which I propose to investigate. The process of autophagy maintains the integrity of cellular organelles and long-lived proteins by transporting them to the lysosomes for degradation. I have shown that the absence of autophagy leads to the amplification of RLR signaling (and increased cytokine production) in two ways. First, in the absence of autophagy, mitochondria accumulate within the cell along with the mitochondrial associated protein, IPS-1, a key signaling protein for RLRs. Second, damaged mitochondria that are not degraded in the absence of autophagy provide a source of reactive oxygen species (ROS), which amplified RLR signaling in Atg5 knockout cells. Interestingly, my studies on primary cells from elderly volunteers mirrored this pattern. Macrophages from elderly showed significantly higher levels of mitochondrial ROS and RLR signaling than macrophages from young adults. It has been postulated that the increased levels of oxidative stress seen in aging are the underlying cause for many of the pathologies seen in aging and in late onset diseases, and it has been inferred from animal models that this increased oxidative stress results from the decline of function of autophagy with age. I aim to characterize autophagic flux in healthy human aging by examining the extent to which autophagy is decreased or dysfunctional and the impact this bears on the accumulation of damaged mitochondria and the resultant increase in levels of ROS. As I have reported that ROS increases RLR signaling, and have found both to be strikingly elevated in elderly macrophages, I aim to examine the impact of oxidative stress on cytosolic antiviral signaling with age in order to understand the increased inflammatory profile seen in the elderly, and the effect that this has on the pro-inflammatory response to influenza. The study outlined in this proposal will examine the crossroads between important cellular processes fundamental in both the aging process and antiviral defense, with the goal of uncovering the functional implications for the accumulation of damaged mitochondria and oxidative stress on the innate immune response to viral infection in the elderly. PUBLIC HEALTH RELEVANCE: It is physically apparent to everyone observing from the outside that we are constantly aging, yet, the biological mechanisms responsible for this are still unclear. I am proposing to investigate to what extent the cellular clean-up crew, autophagy, lags with age, resulting in the accumulation of damaged components within our cells. I will then examine how this impacts the pathologies seen in aging, specifically the way in which a cell recognizes and responds to viral infection with age.