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)-1? and IL-18. Our preliminary data shows that heme promotes the release of IL-1? 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-1? 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-1?, 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.
The objective of these studies is to characterize the activation mechanism of the inflammatory caspases, caspase-1, 4, and -5, in response to extracellular heme. This project will delineate the roles of inflammatory caspases in mediating inflammation in sickle cell disease (SCD). Altogether, this work has the potential to iden- tify a novel inflammatory pathway relevant to SCD, potentially leading to the discovery of new targets and strategies for therapeutic intervention in SCD.