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Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：中性粒细胞在骨髓增殖性肿瘤（MPN）的炎症和血栓风险增加中发挥关键作用。我们已经研究了JAK2-V617F或CALRdel的中性粒细胞特异性表达如何重新编程中性粒细胞的功能。
方法:产生Ly6G-CreJAK2-V617F和Ly6G-CreCALRdel小鼠。MPN参数如血细胞计数，将脾肿大和骨髓组织学与野生型小鼠进行比较。在与TPO/IL-1β体外孵育后，使用谱系阴性骨髓细胞研究了巨核细胞的分化。通过小鼠细胞因子阵列测定小鼠血清中的细胞因子浓度。通过细胞内FACS分析确定各种造血细胞群体中的IL-1α表达。在来自JAK2-V617F和CALR突变的小鼠和患者的分离的嗜中性粒细胞中进行RNA-seq以分析炎性细胞因子的基因表达。在海马细胞外通量分析仪上记录嗜中性粒细胞的生物能量学。体外监测嗜中性粒细胞的细胞运动（延时显微镜），和体内（双光子显微镜）在创建一个炎症环境。细胞粘附于整合素，体外研究了E-选择素和P-选择。使用GraphPadPrism进行统计分析。数据显示为平均值±SEM。不成对,采用双尾t检验。
结果：引人注目的是，中性粒细胞特异性表达JAK2-V617F,但不是CALRdel,足以在小鼠血清中诱导包括IL-1的促炎细胞因子。来自JAK2-V617F小鼠和患者的嗜中性粒细胞中的RNA-seq分析揭示了在CALR突变型嗜中性粒细胞中不表达的独特的炎性趋化因子特征。此外,与CALR突变患者相比,IL-1反应基因在JAK2-V617F患者的嗜中性粒细胞中显著富集。因此，JAK2-V617F阳性中性粒细胞，但不是CALR突变的中性粒细胞,是MPN炎症的致病驱动因素。与此相符,JAK2-V617F或CALRdel的表达引起中性粒细胞代谢表型的显着差异，提示JAK2-V617F细胞具有更强的炎症活性。此外，JAK2-V617F,但不是CALRdel,在中性粒细胞中诱导VLA4整合素介导的粘附表型。这导致体外和发炎血管中的嗜中性粒细胞迁移减少。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。
结论：综合来看，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突变型疾病的病理生理学差异有影响.
方法:产生Ly6G-CreJAK2-V617F和Ly6G-CreCALRdel小鼠。MPN参数如血细胞计数，将脾肿大和骨髓组织学与野生型小鼠进行比较。在与TPO/IL-1β体外孵育后，使用谱系阴性骨髓细胞研究了巨核细胞的分化。通过小鼠细胞因子阵列测定小鼠血清中的细胞因子浓度。通过细胞内FACS分析确定各种造血细胞群体中的IL-1α表达。在来自JAK2-V617F和CALR突变的小鼠和患者的分离的嗜中性粒细胞中进行RNA-seq以分析炎性细胞因子的基因表达。在海马细胞外通量分析仪上记录嗜中性粒细胞的生物能量学。体外监测嗜中性粒细胞的细胞运动（延时显微镜），和体内（双光子显微镜）在创建一个炎症环境。细胞粘附于整合素，体外研究了E-选择素和P-选择。使用GraphPadPrism进行统计分析。数据显示为平均值±SEM。不成对,采用双尾t检验。
结果：引人注目的是，中性粒细胞特异性表达JAK2-V617F,但不是CALRdel,足以在小鼠血清中诱导包括IL-1的促炎细胞因子。来自JAK2-V617F小鼠和患者的嗜中性粒细胞中的RNA-seq分析揭示了在CALR突变型嗜中性粒细胞中不表达的独特的炎性趋化因子特征。此外,与CALR突变患者相比,IL-1反应基因在JAK2-V617F患者的嗜中性粒细胞中显著富集。因此，JAK2-V617F阳性中性粒细胞，但不是CALR突变的中性粒细胞,是MPN炎症的致病驱动因素。与此相符,JAK2-V617F或CALRdel的表达引起中性粒细胞代谢表型的显着差异，提示JAK2-V617F细胞具有更强的炎症活性。此外，JAK2-V617F,但不是CALRdel,在中性粒细胞中诱导VLA4整合素介导的粘附表型。这导致体外和发炎血管中的嗜中性粒细胞迁移减少。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。
结论：综合来看，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突变型疾病的病理生理学差异有影响.
