Program: Oral and Poster Abstracts
Session: 635. Myeloproliferative Syndromes: Basic Science: Poster II
Multiplex analysis of a 29 cytokine/chemokine panel in normal donor (n=8) and PV patient (n=10) serum, indicated significant upregulation of inflammatory cytokines through both JAK2-dependent and independent signalling, including IFNγ, interleukin (IL)-5, IL-12 (p< 0.05; JAK2 dependent), and CXCL10, MIP-1α and TNF-α (p< 0.001; JAK2 independent). PV patients also had significant deregulation of JAK2 dependent and IFN response genes (n=84 genes) especially upregulation of GBP1 (p< 0.05), MGST3 and SUMO3 (p< 0.001) and downregulation of PML (p< 0.01) and NFκB (p< 0.001). Using Duolink® in situ proximity ligation assays in both JAK2 V617F cell lines (UKE1 and SET2) and PV patient/normal donor monocytes, we demonstrate that JAK2 is SUMOylated and actively co-localises with PML in the cell nucleus. Importantly, there was a significant increase in the degree of interaction between JAK2 and PML in PV monocytes compared to normal donors (n=3, p< 0.001). Treatment of UKE1 and SET2 cell lines with the JAK1/2 inhibitor ruxolitinib led to a decrease in JAK2 dependent and IFN response genes as well as the degree of JAK2/PML co-localisation indicating active JAK2 mediates these responses.
To determine whether the deregulation of these cellular processes is manifested by a functional alteration in the myeloid lineages of patients with PV, we analysed their monocyte profiles and the ability of monocytes to form M1 and M2 macrophages as well as neutrophil function. Monocytes exist in different subsets defined by the expression of CD14 and CD16. In PV, the proinflammatory intermediate subset was significantly increased (16.4% ±2.04 vs 6.5% ±1.08, p< 0.05, n=5) at the expense of the classical subset of monocytes (79% ±2.04 vs 88.9% ±1.07, p< 0.05, n=5). The non-classical, IL-1RA producing subset remains unchanged (3.6% ±0.38 vs 4.6% ±0.48 p= 0.1). Macrophages generated from these monocytes were polarized for M1 or M2 differentiation using GM-CSF and M-CSF respectively. They were then cultured in the presence of normal or PV patient serum with or without stimulation; IFNγ and lipopolysaccharide (LPS) for M1 macrophages and IL-4 for M2 macrophages. Culture supernatant was then assessed for the same 29 cytokine/chemokine panel as above and showed significant alterations including an increase in IL-6 and CXCL10 in PV derived M1 macrophages (p< 0.001), while M2 macrophages had a decrease in CXCL1 and increase in IL-1RA (p< 0.01).
Neutrophils were cultured for 24 hours in the presence of normal or PV patient serum with or without stimulation with LPS and showed significant alteration in the production of several proinflammatory cytokines/chemokines compared to neutrophils cultured in Promocell Base Media DXF alone including IFNγ, VEGF, and CXCL10 (p< 0.001), indicating that serum from PV patients can elicit neutrophil activation.
In conclusion, we have shown that inflammation is a major pathophysiological process in PV patients resulting in increased PML/JAK2 co-localisation, increased transcription of direct JAK2 target and IFN response genes, high circulating proinflammatory cytokine/chemokine levels, altered proinflammatory monocyte profiles, and significant change in the secretory production of mature granulocytes following stimulation. Treatment of JAK2 V617F positive cell lines with ruxolitinib significantly alters the inflammasome supporting its efficacy in the treatment of PV patients to alleviate the symptoms and complications of chronic inflammation.
Disclosures: Drummond: Gilead: Membership on an entity’s Board of Directors or advisory committees ; Celgene: Membership on an entity’s Board of Directors or advisory committees , Speakers Bureau ; Novartis: Membership on an entity’s Board of Directors or advisory committees , Speakers Bureau ; Baxalta: Membership on an entity’s Board of Directors or advisory committees . Copland: BMS: Honoraria , Membership on an entity’s Board of Directors or advisory committees , Research Funding ; Novartis: Honoraria , Membership on an entity’s Board of Directors or advisory committees , Research Funding ; Ariad: Honoraria , Membership on an entity’s Board of Directors or advisory committees ; Pfizer: Honoraria , Membership on an entity’s Board of Directors or advisory committees .
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