Defining the non-apoptotic role of Caspase-8 activity in anti-bacterial immune defense

项目摘要 细菌感染是通过进化上保守的模式识别受体来感知的，包括Toll样受体 受体(TLR)。TLR信号诱导炎症介质的产生，而炎症介质在 控制感染以及感染的病理后遗症，包括革兰氏阴性败血症。近期 我们实验室和其他实验室的研究揭示了半胱氨酸蛋白酶Caspase-8(Caspase-8)作为一种 细菌感染时转录反应的重要调节因子。Casp8缺陷小鼠和人类 对粘膜细菌感染高度敏感，暗示Casp8是抗细菌免疫的关键调节因子 防守。有趣的是，我们的初步研究表明，Casp8在调节 革兰氏阴性杆菌感染后关键抗菌炎性介质转录水平的变化 TLR刺激，这完全独立于Casp8在调节细胞死亡中公认的作用。 Casp8在关键天冬氨酸残基上的自动处理导致具有酶活性的Casp8的稳定 同源二聚体，以及随后对其凋亡底物的切割。相反，Casp8充当 异源二聚体与cFLIP一起限制细胞死亡并促进细胞存活。我们现在已经生成了一个新的Casp8DA Casp8同源二聚化能力被消除的突变小鼠，并发现Casp8DA 巨噬细胞在诱导炎性细胞因子产生方面存在明显缺陷 TLR接洽。这些研究引发了一种概念上的新假设，即Casp8同源二聚体在 非凋亡性作用在细菌感染或TLR参与的抗微生物反应诱导中的作用。 我们提出了两个具体目标来解决我们知识中的这一重要差距。首先，我们将利用新的- 产生的突变小鼠区分caspase-8同源和异源二聚体的功能 确定caspase-8调控炎症细胞因子基因的分子机制 表情。特别是，我们将测试caspase-8是否通过切割来调节炎症基因的表达。 以及使转录抑制物失活或激活转录诱导物。二是用好-- 确定的全身性细菌感染小鼠模型以探讨依赖于caspase-8的体内作用 先天和获得性抗微生物免疫防御中炎性细胞因子的产生。

Project Summary Bacterial infection is sensed by evolutionarily conserved pattern recognition receptors, including Toll Like Receptors (TLRs). TLR signaling induces production of inflammatory mediators that play a key role in controlling infection as well as in the pathologic sequellae of infection, including gram-negative sepsis. Recent studies in our laboratory and others revealed a role for the cysteine protease, caspase-8 (Casp8) as an important regulator of the transcriptional response to bacterial infection. Casp8-deficient mice and humans are highly susceptible to mucosal bacterial infection, implicating Casp8 as a key regulator of anti-bacterial immune defense. Intriguingly, our preliminary studies demonstrate that Casp8 plays a cell-intrinsic role in regulating transcription of key anti-microbial inflammatory mediators in response to gram-negative bacterial infection and TLR stimulation, and this is entirely independent of the well-established role of Casp8 in regulating cell death. Autoprocessing of Casp8 at a key aspartate residue leads to stabilization of the enzymatically active Casp8 homodimer, and subsequent cleavage of its apoptotic substrates. Conversely, Casp8 functions as a heterodimer with cFLIP to limit cell death and promote cell survival. We have now generated a novel Casp8DA mutant mouse in which the ability of Casp8 to homodimerize is eliminated, and have found that Casp8DA macrophages have a significant defect in their ability to induce inflammatory cytokine production in response to TLR engagement. These studies provoke the conceptually novel hypothesis that the Casp8 homodimer plays a non-apoptotic role in the induction of antimicrobial responses during bacterial infection or TLR engagement. We propose two Specific Aims to address this important gap in our knowledge. First we will utilize newly- generated mutant mice that distinguish between the function of the caspase-8 homo- and hetero-dimers to define the define molecular mechanism of caspase-8-mediated control of inflammatory cytokine gene expression. In particular we will test whether caspase-8 regulates inflammatory gene expression by cleaving and inactivating transcriptional repressors or activating a transcriptional inducer. Second, we will use well- defined murine models of systemic bacterial infection to interrogate the in vivo role of caspase-8-dependent inflammatory cytokine production in innate and adaptive antimicrobial immune defense.