Detection & Use of Novel Therapeutics to Stimulate NLRP3 Activity in the Elderly

描述(由申请人提供)：流感病毒感染和继发性肺炎球菌感染是全世界约500万人的重大发病率和死亡率的罪魁祸首，其中老年人的感染率最高(65岁)。众所周知，随着年龄的增长，对流感和肺炎链球菌的先天和获得性免疫反应会受到损害；然而，增强这些反应从而降低老年宿主高死亡率和发病率的治疗方法还没有得到很好的研究。随着人口老龄化和肺部感染日益成为发病率和死亡率的重要原因，迫切需要研究新的治疗方法来刺激这一人群的先天免疫反应。NLRP3炎症体是一个关键的信号复合体，负责处理和激活IL-1和IL-18，在流感病毒感染时通过M2离子通道激活，在肺炎链球菌细菌感染时激活。我们的初步结果表明，在体外和体内感染HKx31(小鼠适应株流感病毒)和原发和继发性肺炎链霉菌时，老年宿主损害了炎症体的激活，降低了NLRP3炎症体信号通路几个关键组成部分的基因表达，降低了caspase-1活性和IL-1β的产生。在流感和肺炎链球菌的体外感染过程中，经三磷酸腺苷或黑素刺激后，老年树突状细胞(DC)的IL-1β表达增加，NLRP3炎症体成分的基因表达增加，caspase-1活性增强。尽管有这些发现，由于三磷酸腺苷或黑素的化学性质，这些化合物的使用可能会在肺部感染期间对老年宿主产生有害的副作用，因此迫切需要研究其他治疗方法。这项拟议工作的目标是研究类似于ATP或黑素的新化合物，这些化合物具有刺激老年宿主NLRP3炎症体激活的潜力，并具有救援功能和改善老年宿主的临床结果。为此，在目标1中，我们将检测NLRP3炎症体功能对一种被证明可改变氧化应激的药物吡非尼酮的反应，以及寻找可用于在流感感染期间增强老年宿主NLRP3活性的其他化合物(目标2)。具体地说，我们将使用一种化学物质 用于鉴定NLRP3炎性小体激活剂的文库，我们将在体外使用这些文库进行研究 以及活体小鼠模型。我们将测试这一假设，即在流感或肺炎链球菌感染早期对老年宿主进行早期干预，如刺激NLRP3炎症小体，将改善先天免疫反应，从而降低这一高度易感人群的发病率和死亡率。摘要和影响：由于流感感染和肺炎球菌病仍然是老年人发病率和死亡率的主要原因，即使在成人常规接种疫苗的时代，也迫切需要研究新的治疗方法和治疗策略，以减少严重疾病并改善临床结果，为应对当前和新出现的流感或抗生素耐药细菌株做准备。R21中提出的目标的完成将进一步定义NLRP3炎症体作为流感和肺炎球菌感染期间重要的固有信号通路的作用，并产生可以在原代人类细胞中容易测试并在其他模型系统中进行评估的新疗法。

DESCRIPTION (provided by applicant): Influenza viral infections and secondary pneumococcal infections are responsible for significant morbidity and mortality involving approximately five million people worldwide, with the highest infection rates found in the elderly (>65 years). It has been well established that innate and adaptive immune responses to influenza as well as Streptococcus pneumoniae are impaired with aging; however therapeutic treatments to augment these responses and thereby reduce high levels of mortality and morbidity in elderly hosts have not been well investigated. With an aging population and pulmonary infections becoming an increasingly significant cause of morbidity and mortality, there is an urgent need to investigate new therapeutics that can stimulate innate immune responses within this population. The NLRP3 inflammasome, a key signaling complex responsible for the processing and activation of IL-1¿ and IL-18, is activated in the lung during influenza viral infection by the M2 ion channel as well as during S. pneumoniae bacterial infection by pneumolysin. Our preliminary results demonstrate that elderly hosts have impaired inflammasome activation, decreased gene expression of several key components of the NLRP3 inflammasome signaling pathway, decreased caspase-1 activity and decreased IL-1¿ production in response to in vitro and in vivo infection with HKx31, a mouse adapted strain of influenza, and primary and secondary S. pneumoniae. Stimulation with ATP or nigericin, which have been previously shown to promote K+ efflux, augmented IL-1¿ expression by elderly dendritic cells (DC) as well as increased gene expression of NLRP3 inflammasome components and augmented caspase-1 activity during in vitro influenza as well as S. pneumoniae infection. Despite these findings, due to the chemical properties of ATP or nigericin, usage of these compounds may result in deleterious side effects in elderly hosts during pulmonary infections and hence there is a pressing need to investigate additional therapeutics. The goal of the proposed work is to investigate new compounds that, similar to ATP or nigericin, have the potential to stimulate NLRP3 inflammasome activation in elderly hosts and rescue function and improve clinical outcomes in elderly hosts. To this extent, in Aim 1, we will examine NLRP3 inflammasome function in response to an agent shown to alter oxidative stress, pirfenidone, as well as to search for additional compounds that can be used to heighten NLRP3 activity in elderly hosts during influenza infection (Aim 2). Specifically, we will employ a screen of chemical libraries to identify NLRP3 inflammasome activators that we will readily investigate using in vitro and in vivo murine models. We will test the hypothesis that an early intervention, such as stimulation of the NLRP3 inflammasome, in elderly hosts early during influenza or S. pneumoniae infection will improve innate immune responses and thereby decrease morbidity and mortality in this highly susceptible population. Summary and impact: As influenza infections and pneumococcal disease remain a substantial cause of morbidity and mortality in the elderly, even in an era of routine adult vaccination, there is a pressing need to investigate novel therapeutics and treatment strategies that reduce serious disease and improve clinical outcomes, in preparation for current and emergent strains of influenza or antibiotic resistant bacteria. Completion of the aims proposed in this R21 will further define the role of the NLRP3 inflammasome as an important innate signaling pathway during influenza and pneumococcal infections as well as yield new therapeutics that can be readily tested in primary human cells and evaluated in additional model systems.