Role of the antiviral ribonuclease, RNase-L, in the host antibacterial response

描述(申请人提供)：1型干扰素(IFN)被发现是宿主抗病毒功能的媒介，最近被证明在对非病毒病原体的先天免疫反应中发挥重要作用。因此，已确定的干扰素抗病毒作用效应物可能在抗菌免疫中发挥以前未被认识的作用。为了了解干扰素是如何发挥其抗菌活性的，必须确定特定的抗菌效应物的特性和作用机制。为了实现这一目标，我们确定了干扰素调节的抗病毒核糖核酸酶-L在宿主抗菌反应中的一个新角色。核糖核酸酶-L-/-小鼠在细菌攻击后死亡率显著增加。核糖核酸酶-L-/-小鼠对细菌攻击的敏感性增加是由于免疫反应受损导致细菌负荷增加。基因芯片分析确定可能介导其抗菌活性的核糖核酸酶-L调节的宿主转录本，确定了两类依赖核糖核酸酶的基因调控，影响编码已建立的免疫介质的转录本。具体地说，在RNase-L-/-巨噬细胞中：1.介导内溶酶体相关免疫功能的组织蛋白酶-E(cathepsin-E，CATE)的基础表达增加，表明该基因是RNase-L的底物；2.细菌诱导的促炎细胞因子和趋化因子的表达减少，表明RNase-L通过调节上游信号通路间接影响它们的表达(S)。与核糖核酸酶-L-/-巨噬细胞中Cate表达的变化一致，细菌感染后内溶酶体介导的吞噬空泡清除被破坏。细菌诱导的信号转导在核糖核酸酶-L-/-巨噬细胞中受损，这为细胞因子和趋化因子的表达减少以及相应的中性粒细胞募集的调节提供了机制基础。根据这些发现，我们推测核糖核酸酶-L通过多种机制发挥其抗菌活性，包括调节CATE介导的内溶酶体功能，以及诱导宿主细胞因子和趋化因子。这项建议的目的是剖析核糖核酸酶-L调节宿主免疫反应基因表达的机制(目标1和2)，并确定这种调节在其抗菌活性中的作用(目标3)。还将检测核糖核酸酶-L激活剂的抗菌活性。这项拟议的研究将提供有关核糖核酸酶-L在抗菌活性中作用的第一个信息，并将确定核糖核酸酶-L作用的特定成分作为治疗调节天然免疫反应的靶点。公共卫生相关性：细菌感染是一个主要的公共卫生问题，免疫系统对抗细菌感染的机制尚不完全清楚。识别细菌防御的关键介质对于开发增强抗菌免疫反应的治疗方法至关重要。这些研究将首次为核糖核酸酶-L作为一种新的抗菌活性介体的作用提供信息，并将评估核糖核酸酶-L激活剂作为一类新的治疗剂的抗菌活性。

DESCRIPTION (provided by applicant): Type 1 interferons (IFNs) were discovered as mediators of host antiviral functions, and were recently shown to play essential roles in the innate immune response to nonviral pathogens. Established effectors of IFN antiviral action may thus serve previously unrecognized roles in antibacterial immunity. To understand how IFN exerts its antibacterial activities, the identities and mechanisms of action of specific antibacterial effectors must be determined. Towards this goal, we identified a novel role for RNase-L, an IFN-regulated antiviral ribonuclease, in the host antibacterial response. RNase-L-/- mice exhibited a significant increase in mortality following bacterial challenge. The increased susceptibility of RNase-L-/- mice to bacterial challenge was due to a compromised immune response resulting in an increased bacterial load. Microarray analysis to identify RNase- L-regulated host transcripts that may mediate its antibacterial activity identified two classes of RNase-L- dependent gene regulation that affected transcripts encoding established immune mediators. Specifically, in RNase-L-/- macrophages: 1. basal expression of cathepsin-E (CatE), that mediates endolysosome-associated immune functions, was increased suggesting that this mRNA is an RNase-L substrate; and 2. bacteria-induced expression of proinflammatory cytokines and chemokines was diminished, suggesting that RNase-L indirectly impacts their expression via the modulation of upstream signaling pathway(s). Consistent with the altered CatE expression in RNase-L-/- macrophages, the endolysosome-mediated clearance of phagocytic vacuoles was disrupted following bacterial infection. Bacteria-induced signal transduction was impaired in RNase-L-/- macrophages, providing a mechanistic basis for the diminished cytokine and chemokine expression, and corresponding modulation of neutrophil recruitment. Based on these findings, we hypothesize that RNase-L exerts its antibacterial activity through multiple mechanisms including the regulation of CatE- mediated endolysosome functions, and the induction of host cytokines and chemokines. The goals of this proposal are to dissect the mechanisms by which RNase-L regulates the expression of host immune response genes (aims 1 and 2), and to determine the roles of this regulation in its antibacterial activity (aim 3). The antibacterial activity of RNase-L activators will also be examined. The proposed studies will provide the first information on the role of RNase-L in antibacterial activity, and will identify specific components of RNase-L action as targets for therapeutic modulation of the innate immune response. Public Health Relevance: Bacterial infections are a major public health problem, and the mechanisms by which the immune system combats bacterial infection are incompletely understood. The identification of key mediators of bacterial defense is essential for the development of therapies to enhance the antibacterial immune response. The proposed studies will provide the first information on the role of RNase-L as a novel mediator of antibacterial activity, and will evaluate the antibacterial activity of RNase-L activators as a new class of therapeutic agent.