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

项目摘要 细菌感染由进化上保守的模式识别受体感知，包括Toll样受体。 受体（TLR）。TLR信号传导诱导炎症介质的产生，其在炎症反应中起关键作用。 控制感染以及感染的病理后果，包括革兰氏阴性脓毒症。最近 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（Casp 8）作为一种蛋白酶的作用。 对细菌感染的转录反应的重要调节因子。Casp 8缺陷小鼠和人类 对粘膜细菌感染高度敏感，暗示Casp 8是抗菌免疫的关键调节剂 防御有趣的是，我们的初步研究表明，Casp 8在调节细胞凋亡中起着细胞内在的作用。 应答革兰氏阴性细菌感染的关键抗微生物炎症介质的转录， TLR刺激，并且这完全独立于Casp 8在调节细胞死亡中的公认作用。 Casp 8在关键天冬氨酸残基处的自动加工导致酶活性Casp 8的稳定化 同源二聚体，随后裂解其凋亡底物。相反，Casp 8作为一种 异源二聚体与cFLIP结合以限制细胞死亡并促进细胞存活。我们现在已经产生了一种新的Casp 8DA 突变小鼠，其中Casp 8同源二聚化的能力被消除，并且已经发现Casp 8DA 巨噬细胞在诱导炎症细胞因子产生的能力上有显著缺陷， TLR接合。这些研究提出了一个概念上新颖的假设，即Casp 8同源二聚体在细胞内起着重要作用。 在细菌感染或TLR接合期间诱导抗微生物应答中的非凋亡作用。 我们提出了两个具体目标，以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， 明确caspase-8介导的炎症细胞因子基因调控的分子机制 表情特别是，我们将测试caspase-8是否通过切割 和失活转录阻遏物或激活转录诱导物。二是要用好-- 确定的全身性细菌感染的小鼠模型，以询问caspase-8依赖的 先天性和适应性抗微生物免疫防御中的炎性细胞因子产生。

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.