NIV Congres

Introduction: Sickle cell disease (SCD) is characterized by recurrent acute vaso-occlusive painful crisis frequently leading to SCD related complications, like acute chest syndrome. The pathophysiology of painful crisis is complex, with an important role for polymorphonuclear neutrophils (PMN). Recently, PMN have been demonstrated to form NETs upon activation. Nucleosomes and histones, together with neutrophil proteases, exposed on these NETs have been shown to be bactericidal, but can also be strongly procoagulant and cytotoxic to endothelial cells. In sepsis, the level of circulating nucleosomes has been reported to correlate with severity of inflammation, organ dysfunction and mortality.

The Aim of this case-control study was to assess plasma levels of circulating nucleosomes and human neutrophil elastase-α₁-antitrypsin (EA) complexes, as measure of NET formation and systemic PMN activation.

Methods: Blood samples of healthy controls (24) and sickle cell patients during steady state (74) and painful crisis (70) were analyzed using ELISA. For statistical analysis, patients were divided in two groups with patients with the relatively severe genotypes HbSS and HbSβ⁰-thalassemia grouped together (HbSS/HbSβ⁰-thal) and patients with the relatively milder HbSC and HbSß⁺-thalassemia genotypes in the other group (HbSC/HbSß⁺-thal).

Results: Plasma levels of nucleosomes in both patients with HbSS/HbSβ⁰-thal and HbSC/HbSβ⁺-thal were significantly higher during painful crisis (median; IQR, 20.2 U/ml; 8.9-129.0, p < 0.001, and 11.7 U/ml; 5.1-67.7, p = 0.045) as compared to those in steady state (6.0 U/ml; 3.0-9.8 and 7.1 U/ml; 4.6-9.6). An increment in EA levels was seen in HbSS/HbSβ⁰-thal and HbSC/HbSβ⁺-thal patients during painful crisis (75.1 ng/ml; 56.5-102.4, p 0.001; 62.0 ng/ml; 48.0-96.7; p = 0.051) as compared to levels during steady state (45.7 ng/ml; 34.7-59.7 and 50.2 ng/ml; 33.3-67.7), which just failed to reach statistical significance in HbSC/HbSβ⁺-thal patients. During painful crisis, EA levels correlated strongly with levels of nucleosomes in both HbSS/HbSβ⁰-thal (Sr = 0.55, p < 0.001) and HbSC/HbSβ⁺-thal patients (Sr = 0.90, p < 0.001). In steady state HbSS/HbSβ⁰-thal patients, levels of nucleosomes correlated with endothelial markers sVCAM-1 and vWF:Ag (Sr = 0.421, p = 0.003; Sr = 0.452, p<  0.001). Six patients who developed an acute chest syndrome during painful crisis were among the patients with the highest nucleosome and EA levels. A moderate (Sr = 0.431, p < 0.001) correlation was found between levels of nucleosomes and duration of hospitalization.

Conclusion: We demonstrate for the first time increased levels of circulating nucleosomes in sickle cell patients with painful crisis reflecting NET formation which strongly correlates with PMN activation and disease severity.