Neutrophil extracellular trap formation in patients with chronic myeloproliferative neoplasms

MARIN OYARSUN C; GLEMBOSTKY AC; MOIRAHI B; LEV PR; CASTRO RIOS M; HELLER PG; CARESTIA A; MARTA RF; SCHATTNER M

Estoril

Workshop; European School of Hematology (ESH) 7th International Conference on Myeloproliferative Neoplasms; 2016

European School of Hematology (ESH)

The key role of leukocytes in the pathogenesis of chronic myeloproliferative neoplasms (MPN) has been highlighted in recent years. Neutrophil extracellular traps (NETs) are web-like structures made of DNA/histones decorated by neutrophil granular proteins, such as myeloperoxidase and elastase, released by activated neutrophils in the setting of infection or inflammation. NETs may be triggered by pathogens, proinflammatory cytokines, activated platelets and autoantibodies. Based on the presence of neutrophil activation and a proinflammatory environment, we asessed whether NETs, which promote thrombosis, play a role in MPN patients, including essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF). Although MPN neutrophils were activated, as evidenced by increased CD11b expression, and showed increased basal reactive oxygen species (ROS), unstimulated NET formation ex vivo did not differ significantly between patients and controls, as assessed by fluorescence microscopy for DNA and neutrophil elastase, and enhanced NETosis remained limited to a subset (19%) of patients, who did not share distinctive clinical features. There was a trend towards decreased NETosis when neutrophils were stimulated by the proinflammatory cytokine TNFα and a significant reduction in NET formation after stimulus with PMA, which represents a potent NET inducer, as demonstrated both by microscopy (P=0.004) and by fluorometry of released DNA after digestion of NETs with nuclease (P=0.001). The impaiment in PMA-triggered NETosis was most severe in MF patients and was due to decreased PMA-induced ROS generation and ERK1/2 phosphorylation, which are intermediate steps in NET formation. This defect may reflect neutrophil dysfunction and represent an intrinsic cell defect or be due to cell exhaustion secondary to in vivo activation. PMA-induced CD11b upregulation was preserved, indicating that, despite lower levels of PMA-induced NETosis, neutrophil responses to PMA were not globally impaired. Our results do not seem to support a major role for NETs in MPN pathogenesis, although availability of standardized NET biomarkers may contribute to further research in this field. Further study of other neutrophil functions may help determine whether innate immunity is disturbed in MF.