Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

中文翻译：

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JAK2-V617F 或 CALRmut 的中性粒细胞特异性表达在骨髓增生性肿瘤中诱导不同的炎症特征


中性粒细胞在骨髓增生性肿瘤 （MPN） 的炎症和血栓形成风险增加中起着至关重要的作用。我们研究了 JAK2-V617F 或 CALRdel 的中性粒细胞特异性表达如何重编程中性粒细胞的功能。生成 Ly6G-Cre JAK2-V617F 和 Ly6G-Cre CALRdel 小鼠。将血细胞计数、脾肿大和骨髓组织学等 MPN 参数与野生型小鼠进行比较。与 TPO/IL-1β 体外孵育后，使用谱系阴性骨髓细胞研究巨核细胞分化。通过 Mouse Cytokine Array 测定小鼠血清中的细胞因子浓度。通过细胞内 FACS 分析测定各种造血细胞群中 IL-1α 的表达。在 JAK2-V617F 和 CALR 突变小鼠和患者的分离中性粒细胞中进行 RNA-seq 分析炎性细胞因子的基因表达。在 Seahorse 细胞外通量分析仪上记录中性粒细胞的生物能量学。在产生炎症环境时，在体外（延时显微镜）和体内（双光子显微镜）监测中性粒细胞的细胞运动。在体外研究细胞对整合素、 E-选择素和 P 选择的粘附。使用 GraphPad Prism 进行统计分析。数据显示为 SEM ±平均值。应用未配对的双尾 t 检验。引人注目的是，JAK2-V617F 的中性粒细胞特异性表达，而不是 CALRdel，足以诱导小鼠血清中的促炎细胞因子，包括 IL-1。JAK2-V617F 小鼠和患者的中性粒细胞中的 RNA-seq 分析揭示了独特的炎症趋化因子特征，该特征在 CALR 突变的中性粒细胞中不表达。 此外，与 CALR 突变患者相比，IL-1 反应基因在 JAK2-V617F 患者的中性粒细胞中显著富集。因此，JAK2-V617F 阳性中性粒细胞，而不是 CALR 突变的中性粒细胞，是 MPN 炎症的致病驱动因素。与此一致，JAK2-V617F 或 CALRdel 的表达引起了中性粒细胞代谢表型的显着差异，表明 JAK2-V617F 细胞的炎症活性更强。此外，JAK2-V617F（而不是 CALRdel）在中性粒细胞中诱导 VLA4 整合素介导的粘附表型。这导致中性粒细胞在体外和发炎血管中的迁移减少。与 CALR 突变个体相比，这种机制可能导致 JAK2-V617F 患者血栓形成风险增加。综上所述，我们的研究结果强调了 MPN 中性粒细胞的基因型特异性差异，这些差异对 JAK2-V617F 与 CALR 突变疾病的差异病理生理学有影响。