Comprehensive analysis of complement activation in a hydroxyurea-treated patient cohort with sickle cell disease

Sickle cell disease (SCD) is an understudied, life-threatening genetic disorder affecting ∼300 000 infants yearly with limited treatment options. The complement system, a critical part of innate immunity, has emerged as a contributor to SCD pathophysiology, thus presenting a potential new treatment target. Our aim was to assess complement activity in children with SCD receiving hydroxyurea (HU) therapy during vaso-occlusive crisis (VOC) and steady state. Blood samples were collected from 46 pediatric patients with SCD during VOC (early and late) and steady state, with control samples from healthy volunteers. Clinical data were obtained from patient records, and patient heme levels were measured using colorimetric assay. Complement deposition on endothelial cells (ECs) was quantified using high-throughput automated immunofluorescence imaging. Complement protein concentration was measured using enzyme-linked immunosorbent assay and multiplex assays. We found that, in vitro, heme drove C3b and C5b-9 deposition on ECs. Patients had increased heme levels during both VOC and steady state compared with healthy controls. However, complement activation correlated with total hemoglobin (Hb) concentration in patients during steady state, but not heme levels. C5b-9 deposition on ECs was significantly higher in patients during early crisis compared with late crisis, suggesting heightened complement activity early in VOC, with C5b-9 deposition also strongly correlating with circulating soluble C5b-9 levels. A significant increase in the C3b/C3 ratio further indicated early complement activation during VOC. In conclusion, complement activity is likely highest in early VOC in patients with SCD, and presents a critical potential treatment target, but is overall attenuated by HU therapy despite elevated heme or Hb levels.