Neutrophils remain detrimentally active in hydroxyurea-treated patients with sickle cell disease

Abstract

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment.

Detection and Quantification of Histone H4 Citrullination in Early NETosis With Image Flow Cytometry Version 4

“…In comparison with healthy neutrophils, IFC competently detected changes cell membrane texture in neutrophils isolated from patients with sickle cell disease at steady state. This environment, known for its high inflammatory potential and high levels of free-circulating heme, was also directly linked with an increased potential for NETs production ( 15 ). Thus, while it does appear that NETs-associated cell membrane damage is stimulus-dependent, it might nevertheless be a useful parameter to follow up while characterizing NETosis.…”

Neutrophil Extracellular Trap-Driven Occlusive Diseases

“…They serve as dynamic cytokine reservoirs even under healthy conditions [ 52 ]. The intravascular hemolysis of abnormal hemoglobin was discovered to initiate vascular inflammation due to the release of cytokines and, further, inflammatory mediators, thus inducing excessive NETs formation during the VOC of SCD [ 53 , 54 ]. A recent study confirmed the parallel induction of both NETs and inflammatory agents in plasma collected during VOC compared to the steady-state conditions [ 55 ].…”

Targeting Neutrophil Adhesive Events to Address Vaso-Occlusive Crisis in Sickle Cell Patients

“…demonstrated the importance of elevated plasma heme in a humanized SCD mouse model. In fact, unstimulated neutrophils isolated from SCD patients rapidly begin forming NETs compared to almost no NET formation in age-matched healthy controls, even when under treatment with hydroxyurea ( 67 ). This elevated level of heme induces arrested neutrophils to release NETs that have been associated with hypothermia and rapid death.…”