Neutrophil extracellular traps are a heterogeneous feature in sickle cell disease

Abstract

Abstract Sickle cell disease (SCD) is an inherited hemoglobin disorder caused by a single base change (A-T) in the beta-globin gene of hemoglobin with ensuing recurring inflammation that results in progressive end-organ damage and early mortality. Production of neutrophil extracellular traps (NETs) have been shown to be a key component of this inflammatory process in SCD pathophysiology. We sought to determine the relevance of NETosis versus a different type of programmed cell death (apoptosis) in SCD. We used an indirect assay using healthy neutrophils exposed to paired plasma samples (n=16) from SCD patients in steady-state and crisis for 30 min to 7 hrs, and a direct approach of observing neutrophils (3 healthy and 3 SCD) themselves up to 4 hrs. Features of NETosis (histone citrullination, nuclear decondensation, string nets) and apoptosis (piknotic nuclei, c-Parp, membrane damage) were assessed by imaging flow cytometry and fluorescence microscopy. Additionally, plasma titers for 20 inflammatory analytes were determined for the 16 paired SCD samples and 4 healthy controls. High circulating IL-6 levels at crisis found in 9/16 of the paired samples correlated with an increased number of NETs at crisis compared to steady-state. NETs were also observed directly in SCD neutrophils at steady state but not consistently. When NETs or histone citrullination were absent or low, c-Parp was typically detected. Additionally, membrane damage was observed in neutrophils from all SCD donors. The data suggest that the SCD environment disrupts neutrophil homeostasis and activate multiple cell death programs. SCD NETosis appears to be heterogeneous, its contribution to the disease pathophysiology remains to be determined.