中性粒细胞特异性表达 JAK2-V617F 或 CALRmut 可导致骨髓增殖性肿瘤中不同的炎症特征。

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.

背景：中性粒细胞在炎症和骨髓增生性肿瘤（MPN）中血栓形成风险增加中起着至关重要的作用。我们研究了中性粒细胞特异性表达 JAK2-V617F 或 CALRdel 如何重新编程中性粒细胞的功能。

方法：生成了 Ly6G-Cre JAK2-V617F 和 Ly6G-Cre CALRdel 小鼠。与野生型小鼠相比，比较了 MPN 参数，如血细胞计数、脾肿大和骨髓组织学。使用 TPO/IL-1β 在体外孵育时，研究了谱系阴性骨髓细胞中的巨核细胞分化。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中的 IL-1α 表达。使用从 JAK2-V617F 和 CALR 突变小鼠和患者中分离的中性粒细胞进行 RNA-seq 分析以研究炎症细胞因子的基因表达。使用 Seahorse 细胞外通量分析仪记录中性粒细胞的生物能量。通过在体内（双光子显微镜）和体外（延时显微镜）创建炎症环境来监测中性粒细胞的迁移。在体外（时间 lapse 显微镜）和体内（双光子显微镜）中监测中性粒细胞与整合素、E-选择素和 P-选择素的黏附。使用 GraphPad Prism 进行统计分析。数据表示为平均值±SEM。使用未配对、双尾 t 检验。

结果：令人惊讶的是，中性粒细胞特异性表达 JAK2-V617F，但不是 CALRdel，足以诱导包括小鼠血清中 IL-1 在内的促炎细胞因子。来自 JAK2-V617F 小鼠和患者的中性粒细胞的 RNA-seq 分析揭示了一种独特的炎症趋化因子特征，而在 CALR 突变型中性粒细胞中未表达。此外，与 CALR 突变型患者相比，JAK2-V617F 患者的 IL-1 反应基因显著富集在 JAK2-V617F 阳性中性粒细胞中。因此，JAK2-V617F 阳性中性粒细胞而不是 CALR 突变中性粒细胞是 MPN 中炎症的致病性驱动因素。与此一致的是，表达 JAK2-V617F 或 CALRdel 导致中性粒细胞代谢表型发生显著差异，表明 JAK2-V617F 细胞具有更强的炎症活性。此外，JAK2-V617F 而非 CALRdel 诱导中性粒细胞中 VLA4 整合素介导的黏附表型。这导致体外中性粒细胞迁移减少，并在炎症血管中减少。这种机制可能导致 JAK2-V617F 患者与 CALR 突变个体相比，血栓形成风险增加。

结论：总之，我们的研究结果突出了 MPN 中性粒细胞中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具有重要意义。