Plasma heme overload in patients with SCD is a driver of defective phagocytosis. (A) Percentage and phagocytic index and representative flow cytometry dot plot of engulfing FITC⁺ phagocytes (i); percentage ATP⁺ KCs (ii); representative flow cytometry histograms of CD36, MerTk, and CD206 expression in KCs (iii); percentage of caspase-3⁺ apoptotic and 7AAD⁺ necrotic CD45⁻ hepatic parenchymal cells (iv); and serum ALT activity (v) in untreated and IL-4–treated HbS mice receiving 1 × 10⁷ FITC-labeled AC infusion. Data show a representative of 2 independent experiments. (B) FITC⁺ cell percentage, phagocytic index, and representative flow cytometry plot of BMDMs untreated or treated with 5 μM heme-albumin (heme) and 10% plasma from patients with SCD for 14 hours and then exposed to Escherichia coli bioparticles (pHrodo) for 2 hours (i). Correlation between FITC⁺ phagocytic cell percentage or phagocytic index and heme or Hx levels in plasma of the correspondent patient with SCD (ii). FITC⁺ cell percentage, phagocytic index, and representative flow cytometry plot of BMDMs untreated or treated with plasma from patients with SCD in presence or absence of 10 μM Hx plus 10 μM Hp (n = 17) (iii) or 50 ng/mL IL-4 (n = 9) (iv) for 15 hours, followed by exposure to pHrodo E. coli bioparticles for 2 hours. PI is calculated as (% FITC⁺ cells) × (FITC⁺ cell MFI) and expressed as PI fold change to untreated BMDMs. FITC⁺ phagocytic cell percentage changes caused by Hb/heme scavenging or IL-4 treatment are shown for plasma from each patients with SCD. Solid lines correspond to increased phagocytosis (13 patients [iii]; 7 patients [iv] and dashed lines correspond to decreased phagocytosis (4 patients [iii]; 2 patients [iv]) in presence of Hb/heme scavengers or IL-4. Data are average of 3 independent experiments. Values represent mean ± SEM (n = 2/3). Statistical analysis was performed by comparing 2 groups with 2-sided Welch t tests and 3 or more groups with a 1-way ANOVA followed by a Bonferroni posttest. ∗P < .05; ∗∗P < .01.