Mechanism of ruxolitinib is better in treating hydrxyurea resistan polycythemia vera (PV) than essential thrombicythemia (ET)

Abstract

Abstract Introduction.Studies have shown that ruxolitinib is more effective in treating hydroxyurea-resistant PV than ET (Vannucchi et al., N Engl J Med 2015; Harrison et al., Blood 2017). The underlying mechanism is unclear. We found that TLR-2 was significantly elevated in PV compared to ET (Wang et al., Mediators of Inflammation, 2024), and the underlying mechanism of ruxolitinib's effects was through the TLR2 pathway and dendritic cells to inhibit cytokine production (Heine et al., Blood 2013). Therefore, we postulate that the differential effects of ruxolitinib between PV and ET occur through this mechanism. Methods. 1) TLR-2 Assay. Mononuclear cells (MNC) were isolated from peripheral blood (PB) by gradient centrifugation with Ficoll–Paque. The quantification of TLR-2 was performed by immunostaining 1 × 106 MNC that were incubated with fluorescence-conjugated anti-TLR2 and assayed by flow cytometry. 2) Inflammatory Cytokines. Cytokine ELISA Assay. The Meso Scale Discovery Multi-Spot Assay system was used for the multiplex ELISA assay. Human plasma and dendritic cell culture supernatants, after stimulation with PamCSK4 (TLR-2 agonist), were analyzed in duplicates for 10 cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α. 3) Dendritic Cell Cultures. CD14+ cells were isolated using isolation kits (Miltenyi Biotec) and cultured in RP10 medium supplemented with granulocyte macrophage–colony-stimulating factor (GM-CSF; 100 ng/mL) and IL-4 (20 ng/mL; R&D Systems) for 7-8 days. Cytokines were assayed after adding PamCSK4. Results.1) TLR-2 levels were significantly elevated in MNC cells from PV patients compared to ET patients . A total of 37 PV patients, 51 ET patients, and 21 controls were studied. The mean TLR-2 values were 358.5 ± 26.01 in PV, 292.3 ± 13.13 in ET, and 220.6 ± 9.8 in controls, respectively. TLR-2 was significantly elevated in PV (P < 0.001) and ET (P < 0.01) compared to controls, and TLR-2 was significantly elevated in PV compared to ET (P < 0.01). 2) PV patients with elevated TLR-2 (n=10; TLR-2-E) produced more plasma cytokine (IL-1β) than ET patients with normal values (n=10; TLR-2- N). The values (pg/mL) (mean ± SE) were 1.55 ± 1.02 in PV and 0.93 ± 0.29 in ET, respectively (P < 0.05). Other plasma cytokines were elevated in PV with TLR-2 E compared to ET with TLR-2 N but were not significantly elevated. 3) Cytokines produced by dendritic cells. Cytokines were significantly elevated in PV with TLR-2 E compared to ET with TLR-2 N for IL-8 and TNF-α. The TNF-α values (pg/mL) (mean ± SE) were 47.55 ± 16.78 in PV, 15.5 ± 4.0 in ET, and 13.90 ± 3.29 in controls, respectively (P < 0.05 for PV vs. ET; P = NS for ET vs. CTR). The IL-8 levels were 47.56 ± 16.78 in PV with TLR-2 E, 15.54 ± 4.04 in ET with TLR-2 N, and 13.90 ± 3.29 in controls. P values were significant for PV vs. ET (P < 0.05) and not significant for ET vs. CTR (P = NS). Conclusion.1) TLR-2 values are significantly elevated in patients with PV compared to ET. 2) TLR-2 level elevated patients secrete more cytokines than TLR-2 level normal patients in both plasma and cultured dendritic cells. These two facts likely explain that ruxolitinib's effects occur through the TLR-2 inflammatory pathway, having more pronounced effects in PV than in ET. Studies involving more patients in the future will be necessary to substantiate these findings .