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Exploring the Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

探索镰状细胞病中炎症性半胱天冬酶激活的机制

基本信息

批准号：
10318116
负责人：
Beatriz Elena Bolivar-Vega
金额：
$ 4.22万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2022-06-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318116
关键词：
Affect Binding Biochemical Biological Assay CASP1 gene CASP3 gene CASP5 gene Caspase Cell Death Cell Line Cells Cellular Assay Child Clinical Code Complement Complex Data Disease Endothelial Cells Ensure Enzymes Fluorescence Functional disorder Gene Proteins Gene Silencing Genetic Variation Hematological Disease Heme Hemoglobin Hemolysis Homologous Gene Human Image Immune Immunoprecipitation Infection Inflammasome Inflammation Inflammatory Interleukin-1 beta Interleukin-18 Kinetics Laboratories Measures Mediating Mendelian disorder Molecular Mus Mutate Mutation Outcome Pathogenicity Pathway interactions Patients Phenotype Physiological Play Predisposition Proteins Publishing Regulation Risk Role Sickle Cell Sickle Cell Anemia Signal Transduction Sterility Stimulus Stroke Symptoms Techniques Testing Therapeutic Intervention VDAC1 gene Variant Work base beta Globin cytokine druggable target exome sequencing experimental study extracellular improved in silico insight macrophage monocyte mutant novel prevent protein structure recruit response stroke risk

项目摘要

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.

项目总结