Exploring the Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

项目摘要 过度溶血与单核细胞、巨噬细胞和内皮细胞的炎性激活有关。 上皮细胞，并经常导致不受控制的无菌炎症，可增加感染的易感性， 血管闭塞溶血产物，血红素，激活半胱天冬酶-1，一种酶，需要确保正确的重组， 通过促炎细胞因子白细胞介素（IL）-1 β和 IL-18。我们的初步数据显示，血红素促进了原代人巨噬细胞中IL-1 β的释放， SCD巨噬细胞对血红素刺激的反应更灵敏我们的数据还表明，caspase-5被激活， 单核细胞系中caspase-5缺失导致IL-1 β释放显著增加 在血红素处理后。这些结果表明，caspase-5调节caspase-1的活性。此外，我们有 确定了SCD患者中炎性半胱天冬酶基因的五种变体。caspase-1变异与 降低了中风的风险，而caspase-4的变体与风险增加有关。是否 这些变体影响对促炎刺激的反应仍然是难以捉摸的。我的目标是描述 炎症性半胱天冬酶对血红素的激活机制，并确定其在临床中的作用， SCD患者的结局。我的主要假设是血红素激活了炎症性半胱天冬酶， 积极和消极地调节SCD中的炎症。具体目标是：（1）确定后果 血红素诱导的炎症性半胱天冬酶激活的分子要求; 2）确定血红素诱导的炎症性半胱天冬酶激活的分子要求。 炎症性半胱天冬酶的激活;以及3）确定炎症性半胱天冬酶中的自然遗传变异如何影响炎症性半胱天冬酶的活性。 ses影响SCD中的炎症。我将使用双分子荧光互补（BiFC），一种成像- 基于我们实验室开发的技术，以揭示炎症性半胱天冬酶的上游需求 activation.为了完成目标1，我将对比IL-1 β、炎性caspase和底物裂解的水平， a）来自健康供体和SCD患者的免疫细胞中响应于血红素的炎性细胞死亡;和 B）野生型或半胱天冬酶-1、-4或-5缺陷的免疫细胞。对于目标2，我将使用BiFC来表征血红素- 诱导半胱天冬酶活化，并确定单细胞中BiFC发作的动力学和定位。通信- 血红素诱导的半胱天冬酶-1、-4和-5的活化的可能性将通过BiFC实验来鉴定， 寡聚化破坏突变体和炎性体基因沉默。炎性体相互作用将是一致的- 通过免疫沉淀或血红素的直接结合而确定，将使用无细胞测定来探索。最后，为了COM- 完成目标3，每个变体对半胱天冬酶激活，蛋白质结构和血红素结合的影响将被确定。 使用生物化学和细胞分析以及计算机研究进行开采。将评估SCD患者的数据 以确定这些结果如何与携带这些变体的SCD患者的生理症状相关。 这些研究将提供深入了解半胱天冬酶激活，血红素诱导的 炎症和细胞死亡，以阐明这些机制如何在SCD中改变和放大。

PROJECT SUMMARY Excessive hemolysis has been implicated in the inflammatory activation of monocytes, macrophages and endo- thelial cells and often results in uncontrolled sterile inflammation that can augment susceptibility to infections and vaso-occlusion. The hemolysis product, heme, activates caspase-1, an enzyme required to ensure correct reg- ulation of inflammatory signaling through the maturation of the proinflammatory cytokines, interleukin (IL)-1β and IL-18. Our preliminary data shows that heme promotes the release of IL-1β in primary human macrophages and that SCD macrophages are more responsive to heme stimulation. Our data also show that caspase-5 is activated by heme and that deletion of caspase-5 in a monocytic cell line results in a striking increase of IL-1β release upon heme treatment. These results suggest that caspase-5 regulates caspase-1 activity. Furthermore, we have identified five variants of the inflammatory caspases genes in SCD patients. A caspase-1 variant was associated with a decreased risk stroke, while a variant of caspase-4 was associated with increased risk. Whether or not these variants affect the response to pro-inflammatory stimuli remains elusive. My objective is to characterize the activation mechanism of the inflammatory caspases in response to heme and to identify their role in clinical outcomes in SCD patients. My central hypothesis is that heme activates the inflammatory caspases, which both positively and negatively regulate inflammation in SCD. The specific aims are to: 1) determine the consequences of heme-induced inflammatory caspases activation; 2) identify the molecular requirements for heme-induced activation of the inflammatory caspases; and 3) determine how natural genetic variation in inflammatory caspa- ses impacts inflammation in SCD. I will use Bimolecular Fluorescence Complementation (BiFC), an imaging- based technique developed by our laboratory, to reveal the upstream requirements for inflammatory caspases activation. To complete Aim 1, I will contrast levels of IL-1β, inflammatory caspase and substrate cleavage, and inflammatory cell death in response to heme in a) immune cells from healthy donors and from SCD patients; and b) immune cells wild-type or deficient in caspase-1, -4, or -5. For Aim 2, I will use BiFC to characterize the heme- induced caspase activation and to determine the kinetics and localization of the BiFC onset in single cells. Com- ponents of the heme-induced activation of caspase-1, -4, and -5 will be identified by BiFC experiments using oligomerization-disrupting mutants and inflammasome genes silencing. Inflammasome interactions will be con- firmed by immunoprecipitation or direct binding of heme will be explored using a cell free assay. Lastly, to com- plete Aim 3, the impact of each variant on caspase activation, protein structure, and heme-binding will be deter- mined using biochemical and cellular assays as well as in silico studies. Data from SCD patients will be assessed to determine how these results correlate with physiological symptoms of SCD patients harboring these variants. These studies will provide insight into the molecular mechanisms between caspase activation, heme-induced inflammation and cell death as ways to elucidate how these mechanisms may be altered and amplified in SCD.