Interferon-α Inhibits NET Formation in Neutrophils Derived from Patients with Myeloproliferative Neoplasms in a Neutrophil Sub-Population-Specific Manner

Abstract

Neutrophils and neutrophil extracellular traps (NETs) contribute to thrombosis and hyperinflammation in myeloproliferative neoplasms (MPN). High-density neutrophils (HDNs) and low-density neutrophils (LDNs) have recently been characterized as distinct neutrophil sub-populations with distinct morphological and functional properties. We aim to study the kinetics of NET formation and inhibition with interferon-α (IFNα) in neutrophils derived from patients with MPN as compared to matched healthy controls. Ex vivo NET formation was assessed by neutrophil-elastase activity, neutrophil-associated nucleosomes, myeloperoxidase (MPO), and citrullinated histone H3 content. IFNα significantly inhibited NET formation in neutrophils derived from MPN patients. Neutrophil sub-population analysis demonstrated that HDNs drive the increase in NET formation as compared to LDNs in patients and in healthy controls and are effectively inhibited by IFNα, an effect that is lost in LDNs. In conclusion, we demonstrate that in MPN, HDNs drive excess NET formation and are more sensitive to IFNα inhibition. These observations uncover unique neutrophil sub-population biology and dynamics in MPN.

Keywords: high-density neutrophils; interferon-α; myeloproliferative neoplasia; neutrophil extracellular traps.

Figure 1

IFNα decreases NET formation in neutrophils derived from patients with MPN. (a) ELISA quantification of released nucleosomes by neutrophils from 10 MPN patients and from 3 healthy controls following activation with PMA with or w/o IFNα treatment. * p ≤ 0.03. (b) Confocal microscopy images of neutrophils from a representative PV patient and from a healthy control exposed to PMA with or w/o IFNα. Neutrophils were immunostained with anti-MPO (pink) and DAPI (blue). Magnification ×4. Scale bar = 100 μm.

Figure 2

HDNs are more susceptible to NET formation and are inhibited by IFNα. (a) Expression of CXCR4, as measured by FACS analysis, in HDNs and LDNs from a representative PV patient (upper panel) and a healthy control (lower panel). (b) Change in neutrophil elastase activity in HDNs and LDNs derived from MPN patients (n = 13) and matched healthy controls (n = 4) following stimulation with PMA for 4 h with or w/o exposure to IFNα. Blue arrows designate a decrease and red arrows designate an increase in elastase activity following exposure to IFNα. (c) Neutrophil elastase activity in MPN neutrophil-sub-population (n = 13) compared with healthy controls neutrophil-sub-population quantified by ELISA. (d) ELISA quantification of released nucleosomes by HDNs and LDNs from MPN patients (n = 12) and from healthy controls (n = 4) with or w/o treatment of IFNα. * p ≤ 0.03, ** p ≤ 0.002.

Figure 3

IFNα reduces citrullinated histone H3 burden in HDNs derived from patients with MPN. (a) Representative images of HDNs and LDNs derived from a patient with PV and a healthy control, exposed to PMA with and w/o IFNα. Neutrophils were immuno-stained with anti-histone H3 (citrulline R2 + R8 + R17, green) and with DAPI (blue). Magnification ×20. Scale bar = 100 μm. (b) Bar graphs showing the mean values of binary area fraction of citrullinated histone staining in HDNs and LDNs from patients and from healthy controls. The results are presented as the surface covered by citrullinated histone relative to the surface covered by cells (n =  6), * p ≤ 0.05.