Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms

Abstract

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2^V617F, the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2^V617F-expressing neutrophils and that PAD4 is required for Jak2^V617F-driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2^V617F-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2^V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

Figure 1:. Neutrophils derived from patients with MPNs are associated with an increase in NET formation and a prothrombotic, NET-rich phenotype

(A) NET formation in patients with myeloproliferative neoplasms (MPN) (receiving a JAK inhibitor n=5, receiving other therapy n=14) compared to healthy controls (n=11) when stimulated with 4 μM ionomycin (IO) or DMSO for 2 hours. Patients receiving a JAK inhibitor are indicated by JAKI. Data shown as individual values and medians. (B) Representative immunofluorescence images of human neutrophils after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and citrullinated histone H3 (H3^cit) in green. Scale bar=50 μm. (C) The percentages of human neutrophils with evidence of NET formation after stimulation with PMA (10 nM) with and without ruxolitinib pre-treatment (n=4). Neutrophils are derived from controls. (D) Representative images of human neutrophils from healthy controls stimulated with PMA (10 nM) after 150 minutes of ex vivo pre-treatment with DMSO, ruxolitinib (300 nM), or GSK484 (PAD4 inhibitor,10 μM). Scale bar=50 μm. (E) Lung tissue sections from mice expressing the Jak2^V617F mutation as compared to Jak2^WT mice. Scale bar=200 μm. (F) Characterization of clot content in the lungs of Jak2^V617 mice. Hematoxylin and eosin (H&E) stain. Scale bar=50 μm. VWF – Von Willebrand factor. (G) Lung tissue sections from mice expressing the Jak2^V617F mutation as compared to Jak2^WT mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3^cit (green), respectively. Scale bar=100 μm. (H) The percentages of mouse neutrophils with evidence of NET formation by morphological criteria (left) (n=9 for all genotype/treatment combinations) or H3^cit positive staining (right) (n=6 for all genotype/treatment combinations) grouped by genotype after stimulation with 4 μM IO or DMSO for 2 hours. (I) Representative immunofluorescence images of mouse neutrophils derived from Jak2^WT and Jak2^V617F mice after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and H3^cit in green. Scale bar=50 μm.

Figure 2:. Jak2^V617F is associated with increased venous thrombosis tendency which is reversed with ruxolitinib.

(A) Rates of thrombosis at 2 hours and 4 hours after surgical stenosis of the IVC, with animals grouped according to genotype and in vivo treatment (vehicle or ruxolitinib 90 mg/kg twice a day for 72 hours). At 2 hours: Jak2^WT vehicle n=8, Jak2^V617F vehicle n=11, Jak2^V617F ruxolitinib n=8. At 4 hours: Jak2^WT vehicle n=15, Jak2^V617F vehicle n=14, Jak2^V617F ruxolitinib n=14. (B) A representative image at 2 hours after IVC stenosis in a Jak2^WT and a Jak2^V617F mouse. (C) dsDNA plasma concentration in Jak2^WT (n=13) and Jak2^V617F (n=10) mice subjected to partial stenosis of the IVC. (D) Neutrophil infiltration and NET content of sections of thrombi harvested at 4 hours after IVC stenosis, as shown by neutrophil-specific Ly6G (red) and H3^cit (green), respectively. DAPI is shown in blue. Scale bar=100 μm. (E) The percentage of cells (DAPI) staining positively for H3^cit in thrombi harvested at 4 hours after IVC stenosis. (F) The hematocrit (HCT), neutrophil count, and platelet count (PLT) in Jak2^V617F mice after 72 hours of treatment with vehicle (n=36) or ruxolitinib 90 mg/kg twice a day (n=29).

Figure 3:. PAD4 is overexpressed in MPNs and is essential for the NET-driven prothrombotic phenotype in Jak2^V617F-driven MPN mouse models.

(A) Lung sections from mice 10 weeks after transplantation with Pad4^+/+ or Pad4^−/− c-Kit positive cells transduced with Jak2^V617F vector. Hematoxylin and eosin (H&E) stain. VWF – Von Willebrand factor. Scale bar=200 μm. (B) Immunofluorescence studies of lung sections from mice 10 weeks after transplantation with Pad4^+/+ or Pad4^−/− c-Kit cells transduced with Jak2^V617F vector. Immunofluorescence studies demonstrate H3^cit depositions in the background of a hypercellular lung section in Pad4^+/+/Jak2^V617F mice as compared to Pad4^−/−/Jak2^V617F mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3^cit (green), respectively. DAPI is shown in blue. Scale bar=100 μm. (C) PAD4 protein expression and quantification in neutrophils isolated from healthy controls and patients with polycythemia vera (PV) harboring the JAK2^V617F mutation (actin used as loading control; representative image of 3 technical replicates; n=5 for both groups).

Figure 4:. JAK2^V617F positive clonal hematopoiesis is associated with increased thrombosis rates.

(A) CONSORT (Consolidated Standards of Reporting Trials) diagram of individuals in the population study. (B) Rates of venous thrombosis in patients with or without clonal hematopoiesis of indeterminate potential (CHIP) and/or JAK2^V617F mutation. (C) Variant allele frequency (VAF) of individuals with JAK2^V617F CHIP separated according to the incidence of venous thrombosis. *Individuals with either one or two mutations of unknown significance were excluded from further analysis. ^#Rates of thrombosis between groups by Fishers’ exact test ^$Bonferroni correction