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Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms

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Science Translational Medicine  11 Apr 2018:
Vol. 10, Issue 436, eaan8292
DOI: 10.1126/scitranslmed.aan8292
  • Fig. 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 (MPNs) (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 dimethyl sulfoxide (DMSO) for 2 hours. Patients receiving a JAK inhibitor are indicated by JAKI. Data are shown as individual values and medians. (B) Representative IF images of human neutrophils after stimulation with 4 μM IO or DMSO for 2 hours. 4′,6-Diamidino-2-phenylindole (DAPI) is shown in blue, and citrullinated histone H3 (H3cit) is shown in green. Scale bar, 50 μm. (C) Percentages of human neutrophils with evidence of NET formation after stimulation with phorbol 12-myristate 13-acetate (PMA) (10 nM) with and without ruxolitinib pretreatment (n = 4). Neutrophils are derived from controls. (D) Representative images of human neutrophils from healthy controls stimulated with PMA (10 nM) after 150 min of ex vivo pretreatment with DMSO, ruxolitinib (300 nM), or GSK484 (PAD4 inhibitor,10 μM). Scale bar, 50 μm. (E) Lung tissue sections from mice expressing the Jak2V617F mutation as compared to Jak2WT mice. Scale bar, 200 μm. (F) Characterization of clot content in the lungs of Jak2V617 mice. Hematoxylin and eosin (H&E) stain. Scale bar, 50 μm. VWF, von Willebrand factor. (G) Lung tissue sections from mice expressing the Jak2V617F mutation as compared to Jak2WT mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3cit (green), respectively. Scale bar, 100 μm. (H) Percentages of mouse neutrophils with evidence of NET formation by morphological criteria (left) (n = 9 for all genotype/treatment combinations) or H3cit-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 IF images of mouse neutrophils derived from Jak2WT and Jak2V617F mice after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue, and H3cit is shown in green. Scale bar, 50 μm.

  • Fig. 2 Jak2V617F is associated with increased venous thrombosis tendency that is reversed with ruxolitinib.

    (A) Rates of thrombosis at 2 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: Jak2WT vehicle, n = 8; Jak2V617F vehicle, n = 11; Jak2V617F ruxolitinib, n = 8. At 4 hours: Jak2WT vehicle, n = 15; Jak2V617F vehicle, n = 14; Jak2V617F ruxolitinib, n = 14. (B) Representative image at 2 hours after IVC stenosis in a Jak2WT mouse and a Jak2V617F mouse. (C) dsDNA plasma concentration in Jak2WT (n = 13) and Jak2V617F (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 H3cit (green), respectively. DAPI is shown in blue. Scale bar, 100 μm. (E) Percentage of cells (DAPI) staining positively for H3cit in thrombi harvested at 4 hours after IVC stenosis. (F) HCT, neutrophil count, and platelet count (PLT) in Jak2V617F mice after 72 hours of treatment with vehicle (n = 36) or ruxolitinib (90 mg/kg twice a day; n = 29).

  • Fig. 3 PAD4 is overexpressed in MPNs and is essential for the NET-driven prothrombotic phenotype in Jak2V617F-driven MPN mouse models.

    (A) Lung sections from mice 10 weeks after transplantation with Pad4+/+ or Pad4−/− c-Kitpositive cells transduced with Jak2V617F vector. H&E stain. Scale bar, 200 μm. (B) IF studies of lung sections from mice 10 weeks after transplantation with Pad4+/+ or Pad4−/− c-Kit cells transduced with Jak2V617F vector. IF studies demonstrate H3cit depositions in the background of a hypercellular lung section in Pad4+/+/Jak2V617F mice as compared to Pad4−/−/Jak2V617F mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3cit (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 PV harboring the JAK2V617F mutation (β-actin used as loading control; representative image of three technical replicates; n = 5 for both groups).

  • Fig. 4 JAK2V617F-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 CHIP and/or JAK2V617F mutation. (C) VAF of individuals with JAK2V617F CHIP separated according to the incidence of venous thrombosis.

  • Table 1 Comparison of incidence of venous thrombosis between groups according to the presence of CHIP and JAK2V617F mutation.

    Analysis group*Control
    P value
    Schizophrenia
    P value
    All
    P value
    Non-CHIP versus
    CHIP
    0.0006 (0.003)0.110.0004 (0.002)
    Non-CHIP versus
    non-JAK2V617F
    CHIP
    0.008 (0.04)0.570.025 (0.125)
    Non-CHIP versus
    JAK2V617F CHIP
    0.0009 (0.0045)0.120.0003 (0.0015)

    *Individuals with fewer than three mutations of unknown significance were excluded from further analysis. Individuals with three or more mutations of unknown significance are classified as having clonal hematopoiesis with unknown driver (see also table S5).

    †Rates of thrombosis compared between groups by Fisher’s exact test.

    ‡Nominal P values are given first. Adjusted P values after Bonferroni correction are given in parentheses.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/436/eaan8292/DC1

      Fig. S1. Complementary studies of NET formation in neutrophils derived from MPN patients.

      Fig. S2. Phenotype reminiscent of PV in mice with Jak2V617F-driven MPN.

      Fig. S3. Neutrophil, platelet, RBC, and fibrin thrombus content.

      Fig. S4. The effect of DNase treatment on thrombosis rate in an IVC partial ligation thrombosis model.

      Fig. S5. IVC full ligation thrombosis model in Pad4+/+ and Pad4−/− mice.

      Fig. S6. IVC full ligation thrombosis model in Jak2WT and Jak2V617F with and without ruxolitinib treatment.

      Fig. S7. Neutrophil activation and ROS production in neutrophils from Jak2WT and Jak2V617F mice.

      Fig. S8. Platelet function in Jak2WT and Jak2V617F mice.

      Fig. S9. NET formation in neutrophils derived from mice engrafted with Jak2V617F-expressing Pad4+/+ or Pad4−/− cells.

      Fig. S10. Blood cell counts from the Pad4/Jak2V617F retroviral bone marrow transplant model.

      Fig. S11. Differences in age between persons with and without CHIP.

      Table S1. Patient characteristics (not treated with JAK inhibitors).

      Table S2. Patient characteristics (JAK inhibitor–treated).

      Table S3. Comparing NET formation between groups.

      Table S4. ICD-10 codes used to identify thrombosis in the case-control cohort.

      Table S5. Summary of thrombosis rates in the case-control study broken down by cohort (schizophrenia versus healthy controls) and type of clonal hematopoiesis.

      Table S6. Primary data file (provided in Excel format).

    • Supplementary Material for:

      Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms

      Ofir Wolach, Rob S. Sellar, Kimberly Martinod, Deya Cherpokova, Marie McConkey, Ryan J. Chappell, Alexander J. Silver, Dylan Adams, Cecilia A. Castellano, Rebekka K. Schneider, Robert F. Padera, Daniel J. DeAngelo, Martha Wadleigh, David P. Steensma, Ilene Galinsky, Richard M. Stone, Giulio Genovese, Steven A. McCarroll, Bozenna Iliadou, Christina Hultman, Donna Neuberg, Ann Mullally, Denisa D. Wagner, Benjamin L. Ebert*

      *Corresponding author. Email: bebert{at}partners.org

      Published 11 April 2018, Sci. Transl. Med. 10, eaan8292 (2018)
      DOI: 10.1126/scitranslmed.aan8292

      This PDF file includes:

      • Fig. S1. Complementary studies of NET formation in neutrophils derived from MPN patients.
      • Fig. S2. Phenotype reminiscent of PV in mice with Jak2V617F-driven MPN.
      • Fig. S3. Neutrophil, platelet, RBC, and fibrin thrombus content.
      • Fig. S4. The effect of DNase treatment on thrombosis rate in an IVC partial ligation thrombosis model.
      • Fig. S5. IVC full ligation thrombosis model in Pad4+/+ and Pad4−/− mice.
      • Fig. S6. IVC full ligation thrombosis model in Jak2WT and Jak2V617F with and without ruxolitinib treatment.
      • Fig. S7. Neutrophil activation and ROS production in neutrophils from Jak2WT and Jak2V617F mice.
      • Fig. S8. Platelet function in Jak2WT and Jak2V617F mice.
      • Fig. S9. NET formation in neutrophils derived from mice engrafted with Jak2V617F-expressing Pad4+/+ or Pad4−/− cells.
      • Fig. S10. Blood cell counts from the Pad4/Jak2V617F retroviral bone marrow transplant model.
      • Fig. S11. Differences in age between persons with and without CHIP.
      • Table S1. Patient characteristics (not treated with JAK inhibitors).
      • Table S2. Patient characteristics (JAK inhibitor–treated).
      • Table S3. Comparing NET formation between groups.
      • Table S4. ICD-10 codes used to identify thrombosis in the case-control cohort.
      • Table S5. Summary of thrombosis rates in the case-control study broken down by cohort (schizophrenia versus healthy controls) and type of clonal hematopoiesis.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S6 (Microsoft Excel format). Primary data file (provided in Excel format).

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