Sickle red blood cell/Siglec-9 interactions contribute to neutrophil activation in SCD and are associated with survival

• Sickle RBCs abnormally engage neutrophil Siglec-9 and induce Siglec-9 shedding, ineffectively inhibiting neutrophil activation. • Soluble Siglec-9 may be a marker of mortality risk in sickle cell disease. Sialic acid-binding Ig-like lectin-9 (Siglec-9) is a neutrophil inhibitory receptor. Prior studies have shown that sickle red blood cells (SS RBCs) abnormally engage neutrophil Siglec-9, suggesting that SS RBCs may not be able to effectively inhibit neutrophil activation. We examined SS RBC/Siglec-9 interactions and determined the effect of SS RBCs on Siglec-9 expression and neutrophil function. Using a novel monoclonal antibody-specific immobilization of erythrocyte antigen (MAIEA) assay, we demonstrated that SS RBCs have significantly decreased glycophorin A (GPA) and glycophorin B (GPB)-mediated binding to Siglec-9. Furthermore, exposure of healthy neutrophils to SS RBCs but not healthy AA RBCs induced shedding of Siglec-9 from neutrophil surfaces in vitro . Ex vivo , sickle cell disease (SCD) patients had fewer Siglec-9 positive neutrophils and elevated plasma soluble Siglec-9. Disruption of SS RBC/Siglec-9 interactions, both by desialylating RBCs and by blocking neutrophil Siglec-9, enhanced SS RBC-induced but not healthy AA RBC-induced neutrophil extracellular trap (NET) release. Finally, in a cohort of 32 SCD patients, soluble Siglec-9 was correlated with fetal hemoglobin (HbF), soluble intercellular adhesion molecule-1 (sICAM-1), and overall mortality. These studies demonstrate that unlike AA RBCs, SS RBCs not only directly activate neutrophils, but they also induce loss of neutrophil Siglec-9 and abnormally engage residual Siglec-9. Our findings suggest that neutrophil activation in SCD is a result of both direct activation and ineffective inhibition of neutrophils by SS RBCs.