Exploring the Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

项目总结 过度溶血与单核细胞、巨噬细胞和血管内皮细胞的炎症激活有关。 并经常导致失控的无菌炎症，从而增加感染和 输精管闭塞。溶血产物，血红素，激活caspase-1，这是确保正确调控的一种酶。 促炎症细胞因子、白介素1β和 IL-18。我们的初步数据显示，血红素促进原代人巨噬细胞释放IL-1β，并 SCD巨噬细胞对血红素刺激更敏感。我们的数据还显示caspase-5被激活 通过血红素和Caspase-5在单核细胞系中缺失导致IL-1β释放显著增加 在血红素治疗上。这些结果表明，caspase-5调节caspase-1的活性。此外，我们还拥有 鉴定了SCD患者炎性半胱氨酸酶基因的五种变体。Caspase-1变异体与 中风风险降低，而caspase-4变异与风险增加相关。不管是不是 这些变种对促炎刺激的反应仍然难以捉摸。我的目标是把 亚铁血红素激活炎性半胱氨酸天冬氨酸酶的机制及其临床意义 SCD患者的预后。我的中心假设是，血红素激活了炎症性半胱氨酸酶，这两种酶 正、负性调节SCD的炎症反应。具体目标是：1)确定后果 血红素诱导的炎性半胱氨酸天冬氨酸酶的激活；2)确定血红素诱导的分子要求 炎症性caspase的激活；以及3)决定炎症性caspa的自然遗传变异。 SES影响SCD的炎症反应。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caspase的上游需求 激活。为了完成目标1，我将对比IL-1β、炎性caspase和底物切割的水平，以及 A)健康捐献者和SCD患者的免疫细胞中的血红素引起的炎性细胞死亡；以及 B)野生型或缺乏caspase-1、-4或-5的免疫细胞。对于目标2，我将使用BIFC来表征血红素- 诱导半胱氨酸天冬氨酸氨基转移酶的激活，并确定BIFC在单细胞中的发病动力学和定位。COM- BIFC实验将确定血红素诱导的caspase-1、-4和-5激活的影响因素 寡聚-破坏突变体和炎症体基因沉默。炎性小体的相互作用将是持续的 通过免疫沉淀或直接结合血红素，将使用无细胞分析进行探索。最后，我们要- 完整的目标3，每个变体对caspase激活、蛋白质结构和血红素结合的影响将被阻止- 使用生化和细胞分析以及硅学研究来开采。来自SCD患者的数据将被评估 以确定这些结果如何与携带这些变异的SCD患者的生理症状相关联。 这些研究将为深入了解caspase激活、血红素诱导 炎症和细胞死亡作为阐明这些机制在SCD中如何改变和放大的方法。

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.