SUMMARY New, inexpensive, accessible treatments are urgently needed to regulate chronic inflammation in sickle cell dis- ease (SCD). SCD is a devastating red blood cell disorder that impacts millions of people worldwide. The majority of clinical symptoms in SCD, including stroke, vaso-occclusive crisis (VOC), and acute chest syndrome (ACS) are largely attributable to uncontrolled inflammation driven by excessive hemolysis, due to the release of heme that triggers the innate immune response. As the precise mechanisms are unclear, closing this knowledge gap is essential for identifying new druggable targets to mitigate symptoms of chronic inflammation in SCD. Building on prior research from the applicant showing that the inflammatory caspases, caspase-1, -4, and -5 are essential mediators of heme-induced inflammation, and that caspase-4 is the apical caspase in this pathway, the central hypothesis of this application is that heme-induced caspase-4 activation is the apical step in the inflamma- tory caspase cascade inducing chronic inflammation that leads to organ injury in SCD. This hypothesis will be tested by determining: 1) the mechanisms of heme-induced caspase-4 activation; 2) the consequences of heme-induced caspase-4 activation in SCD; and 3) the contributions of inflammatory caspases to clinical outcomes in children with SCD. Under the first Aim, heme binding sites on caspase-4 will be identified to test the working hypothesis that heme binding to caspase-4 is an initiating activation event of systemic inflammation in SCD. Under the second Aim, a caspase-4 knockout SCD mouse will be used to investigate the role of caspase- 4 in heme-induced inflammation, vaso-occlusion, and lethality in SCD. Under the third Aim, patient data and samples will be assessed for markers of hemolysis (free heme/heme scavenger levels) and inflammatory caspase pathways (plasma cytokine levels, macrophage markers, inflammasome gene expression) to determine how they relate to clinical outcomes (VOC, ACS, abnormal TCD, and stroke). By analyzing coding variants of caspases and direct inhibition of caspase-4 in patient cells, the consequences of disrupting these pathways in individual patients will be evaluated. Upon completion of these aims, the mechanisms, and consequences of heme-induced inflammatory caspase activation at the cellular, organ, and patient level will be uncovered. These investigations will provide essential information for developing efficient, specific, and accessible drugs to coun- teract the often-fatal consequences of inflammation in SCD.