Neutrophil-specific expression of JAK2-V617F, but not CALRdel, induces thrombocytosis and megakaryocyte hyperplasia

Aside from their crucial role in innate immunity, neutrophils have an important and previously underestimated function in the regulation of hematopoietic stem cell and erythropoietic niches in both normal physiologic conditions and MPN pathophysiology [51, 52]. To examine the effects of neutrophil-restricted expression of JAK2-V617F and of CALRdel in inflammation, Ly6G-Cre positive JAK2+/VF, JAK2+/+, CALR+/del and CALR+/+ mice were generated as described under Material and Methods. Cre recombinase-depending expression of tdTomato and of the transgene was achieved through a CAG promoter located in the ROSA26 locus [41]. Control experiments demonstrated that JAK2-V617F or CALRdel is only expressed in tdTomato positive neutrophils of Ly6G-Cre JAK2+/VF and CALR+/del mice, respectively (Additional file 3: Fig. S1). The neutrophil-specificity of the Ly6G-Cre model (Catchup model) has been described previously [41]. Importantly, Ly6G-Cre directed tdTomato expression was absent in CD11b + Ly6G− macrophages or other Ly6G− leukocytes and in GMPs [41]. Additional control experiments in hematopoietic progenitors, in monocytes, in the erythrocyte lineage, platelets and in megakaryocyte progenitors (MKPs) demonstrated almost complete tdTomato-negativity in Ly6G-Cre JAK2+/VF and Ly6G-Cre CALR+/del mice, respectively (Additional file 3: Fig. S2-4). In Ly6G-Cre JAK2+/VF and CALRdel (CALR+/del) mice, white blood cell (WBC) counts and red blood cell (RBC) counts remained within the normal range (Fig. 1, A and D). However, there was a tendency towards an increase in neutrophil counts in JAK2+/VF mice, while hematocrit (HCT) and spleen weight tended to decrease in these mice (Fig. 1, B, C and E). Interestingly, there was a moderate but highly significant increase in PLT counts of JAK2+/VF mice (JAK2+/VF: 1519 ± 59 109/L; JAK2+/+: 1146 ± 58 109/L) in both male and female mice (Fig. 1F and Additional file 3: Fig. S5, A-C). Increased PLT counts were also observed in aged JAK2+/VF mice (Additional file 3: Fig. S5F). BM sections of JAK2+/VF mice exhibited megakaryocyte (MK) hyperplasia and formation of MK clusters, which are regarded as a hallmark of MPN [40, 53] (Fig. 1, G and I). Nonetheless, MK progenitor counts were comparable (Fig. 1H). Serum thrombopoietin (TPO) concentrations showed no discrepancies between the two mouse strains (Additional file 3: Fig. S5G). MK hyperplasia in JAK2+/VF mice was not caused by an increase in megakaryocyte/erythroid progenitors (MEP) in the BM (Additional file 3: Fig. S6A and C). Phenotypic analysis of HSPCs revealed that expression of JAK2-V617F or CALRdel in neutrophils did not result in any significant numerical or compositional changes of HSPCs in the BM or the splenic compartment (Additional file 3: Fig. S6, A-D). Additionally, examination of spleens did not reveal any discernible differences in spleen size or composition between both Ly6G-Cre JAK2+/VF and CALR+/del mice and their respective WT controls (Additional file 3: Fig. S7).

Fig. 1

Neutrophil-specific expression of JAK2-V617F, but not CALRdel, induces thrombocytosis and megakaryocyte hyperplasia. (A-F) White blood cell (WBC) count, neutrophil (NEUT) count, spleen weight, red blood cell (RBC) count, hematocrit, and platelet (PLT) count of Ly6G-Cre JAK2+/+, JAK2+/VF, CALR+/+ and CALR+/del mice (each n = 14). (G) Representative hematoxylin-eosin staining of bone marrow sections of JAK2+/+ (n = 5), JAK2+/VF (n = 3), CALR+/+ (n = 8) and CALR+/del mice (n = 10). (H) Megakaryocyte progenitor (MKP) counts in bone marrow of Ly6G-Cre JAK2+/+ (n = 5), JAK2+/VF (n = 5), CALR+/+ (n = 7) and CALR+/del mice (n = 7) shown as fold change versus control. (I) Quantitative analysis (performed in a blinded fashion) of megakaryocytes (MKs) in bone marrow of Ly6G-Cre JAK2+/+, JAK2+/VF, CALR+/+ and CALR+/del mice in 200x high-power fields (HPF) showing numbers of MKs/HPF as fold change versus control. Data are shown as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 (unpaired, two-tailed t-test)

Neutrophil-specific expression of JAK2-V617F, but not CALRdel, up-regulates inflammatory cytokines in serum

A chronic non-resolving inflammatory syndrome with a pro-inflammatory cytokine signature in the serum is a significant disease feature in MPNs [54, 55]. However, the role of granulocytes in the inflammatory condition is not well-understood. To investigate whether expression of JAK2-V617F or CALRdel specifically in neutrophils induces pro-inflammatory cytokines, we measured serum concentrations of a panel of cytokines in Ly6G-Cre JAK2+/VF and CALR+/del mice (Fig. 2A). Compared to the WT controls, the cytokine levels of IL-1α, IL-12 (p40), and M-CSF cytokines were significantly (p < 0.05) elevated in JAK2+/VF mice by factors of 1.8, 1.6 and 2.1, respectively (Fig. 2B and Additional file 1: Tab. S2). Furthermore, in Ly6G-Cre JAK2+/VF mice, although not statistically significant, levels of five additional cytokines in serum were observed to be upregulated by more than 1.5-fold: IL-1β, IL-2, IL-10, IL-17, and TNFα (Additional file 1: Tab. S2). In contrast to the JAK2-V617F mutation, statistically significant changes in the serum cytokine levels were not induced by neutrophil-specific expression of CALRdel and levels of only two cytokines were up-regulated by more than 1.5-fold: IL-5 and LIX (CXCL5) (Additional file 1: Tab. S3). IL-5 has a significant role in type-2 adaptive immunity, which includes atopic diseases, but has not yet been implicated in cancer-associated inflammation [56]. Together, this suggests that expression of JAK2-V617F in neutrophils is sufficient to induce typical MPN pro-inflammatory cytokines in serum [17,18,19,20]. Conversely, expression of CALRdel in neutrophils seems to have no significant impact on the induction of a pro-inflammatory cytokine signature in serum.

Recent studies revealed that IL-1β has a pivotal role in clonal expansion, BM fibrosis and amplified megakaryopoiesis associated with JAK2-V617F-driven MPN-like disease in mice [17, 18]. Both, the genetic deletion of IL-1β and of IL-1R1, respectively and administration of anti-IL-1β antibody and anti-IL-1R1 antibody, respectively suppressed increased PLT and BM CD41+ cell counts in JAK2-V617F driven MPN-mice [17, 18]. Therefore, we hypothesized that the higher PLT counts observed in the Ly6G-Cre JAK2-V617F model were due to the increased IL-1β serum levels (Additional file 1: Tab. S2). Thus, we studied the impact of IL-1β on the differentiation of lineage-negative (lin−) BM cells into MKs. Figure 2C displays that IL-1β increased TPO-driven megakaryopoiesis in vitro using lin− BM cells isolated from Ly6G-Cre JAK2+/+ and JAK2+/VF mice. Ploidy analysis additionally indicated an increase in 8 N-cells following IL-1β stimulation (Supplementary Figure S8). Considering the published evidence [17, 18] regarding the significant role of IL-1β in JAK2-V617F-induced thrombocytosis and elevated megakaryopoiesis, our findings support the concept that increased IL-1β serum levels are responsible for the observed elevation of PLT and MK counts in Ly6G-Cre JAK2+/VF mice.

Fig. 2

Neutrophil-specific expression of JAK2-V617F, but not CALRdel, up-regulates inflammatory cytokines. (A) Cartoon depicting the experimental design to study serum cytokine concentrations in JAK2+/VF and CALR+/del mice in comparison to their corresponding WT controls. Cytokine protein concentrations in serum of Ly6G-Cre JAK2+/+ (n = 6) and JAK2+/VF mice (n = 6), and in serum of Ly6G-Cre CALR+/+ (n = 8) and CALR+/del mice (n = 10) were analyzed by Eve Technologies, Canada (Mouse Cytokine Array/Chemokine Array 32-Plex, duplicate testing). Created with Biorender.com. (B) Bar graphs of significantly elevated serum cytokine concentrations of IL-1α, IL-12(p40) and M-CSF in Ly6G-Cre JAK2+/VF mice. Data are shown as median ± IQR. *p ≤ 0.05 (unpaired, two-tailed t-test). (C) IL-1β induced megakaryocytic differentiation of lineage-negative cells isolated from bone marrow. Left and middle panel: Impact of IL-1β (25 ng/ml) on the number of formed immature (CD41+) and mature (CD41+ CD42d+) megakaryocytes upon TPO-driven differentiation of lineage-negative cells isolated from bone marrow of Ly6G-Cre JAK2+/+ and JAK2+/VF mice (each n = 4). Data are shown as mean ± SEM. *p ≤ 0.05 (unpaired, two-tailed t-test). Right panel: Representative images of megakaryocytes differentiated from lineage-negative bone marrow cells isolated from Ly6G-Cre JAK2+/+ and JAK2+/VF mice at baseline and upon four-day TPO-driven differentiation with or without IL-1β (n = 2). (D) Neutrophil-specific expression of JAK2-V617F increases IL-1α expression in megakaryocyte progenitors (MKP). Intracellular staining for IL-1α levels in various hematopoietic cell populations using anti-IL-1α antibody and isotype control antibody, respectively was performed as described under Supplemental Methods. Mean fluorescence intensity (MFI) was measured by flow cytometry. The specific MFI (MSFI) was calculated by subtracting the MFI of the isotype control from the MFI of the anti-IL-1α antibody stained sample. Data are shown as mean ± SEM. *p ≤ 0.05 (unpaired, two-tailed t-test with Welch correction). Cartoon created with Biorender.com

Next, we investigated which hematopoietic cell population is the source of the elevated serum levels of inflammatory cytokines in JAK2-V617F induced MPN-like disease. We focused on IL-1α and performed intracellular IL-1α staining in various hematopoietic cell types including hematopoietic progenitors. The results showed that granulocytes, B-cells, T-cells, monocytes and macrophages from Ly6G-Cre JAK2+/+ and JAK2+/VF mice expressed similar IL-1α protein levels, respectively (Fig. 2D). However, MKP cells from Ly6G-Cre JAK2+/VF mice displayed significantly higher IL-1α protein levels (Fig. 2D). Together, this data suggests that MKPs are a major source of the elevated IL-1α serum levels in Ly6G-Cre JAK2+/VF mice. However, non-hematopoietic stromal cells may also participate in this process.

Cytokine gene expression signatures in JAK2-V617F and CALRmut neutrophils isolated from mice and humans

Next, we performed RNA-seq analysis on neutrophils isolated from Ly6G-Cre JAK2+/+, JAK2+/VF, CALR+/+, and CALR+/del mice. In JAK2-V617 neutrophils, 53 genes out of a total of 14,923 genes analysed were significantly (adjusted p-value < 0.05) regulated (Additional File 1, Tab. S6), whereas in CALRdel granulocytes no significant alteration were noted (data not shown). In order to determine whether the elevated cytokines observed in the serum of JAK2-V617F mice originated from JAK2-V617F granulocytes, the gene expression of a cluster of neutrophil-derived cytokines, previously reported by C. Tecchio, was examined [57]. The heatmap of relative changes induced by JAK2-V617F is depicted in Fig. 3A, left panel. Notably, four chemokine genes (Cxcl2, Cxcr4, Cxcl3 and Ccl6) showed a statistically significant change (adjusted p-value < 0.05), whereas this signature was not present in neutrophils from CALR+/del mice (Fig. 3A, middle panel). In addition, Il1rl1 and Csf2rb2 genes were also significantly (adjusted p-value < 0.05) regulated by JAK2-V617F, but not by CALRdel. This data suggests that the up-regulation of inflammatory cytokines in serum of Ly6G-Cre JAK2+/VF mice (IL-1α, IL-12 (p40), and M-CSF) does not derive directly from JAK2-V617F neutrophils. Instead, indirect mechanisms are involved employing other hematopoietic and possibly non-hematopoietic cells. An example for this is the IL-1α production in MKPs illustrated in Fig. 2D. Potentially this is regulated via signals from the above mentioned chemokine signature (Cxcl2, Cxcr4, Cxcl3 and Ccl6), which needs to be further investigated. Regarding the mechanism of action of CALRdel in neutrophils, it is worth mentioning that polymorphonuclear cells (PMN) were reported to express low levels of the TPO receptor c-Mpl and that TPO transiently induces low level STAT1 tyrosine phosphorylation in granulocytes [58]. Additionally, Terada and colleagues [59] found that TPO stimulates ex vivo expansion of neutrophils during the early stages of differentiation. Thus, the available literature suggests that neutrophils or a fraction of neutrophils possess functional MPL, indicating that mutated CALR may bind to MPL thereby exhibiting its oncogenic functions. Therefore, we investigated MPL expression on BM-derived neutrophils in our CALRmut model. FACS analysis of BM-derived neutrophils of Ly6G-Cre CALR+/+ and CALR+/del mice detected low level MPL expression (Additional file 3: Fig. S9A). Furthermore, following incubation with TPO, a slight increase of p-STAT5 was observed in the neutrophils of Ly6G-Cre CALR+/del mice (Additional file 3: Fig. S9B). However, CALRdel is able to activate the G-CSFR as demonstrated by Chachoua et al. [8]. Therefore, CALRdel may dysregulate intrinsically some processes in granulocytes via its action on G-CSFR.

Fig. 3

RNA-seq in granulocytes from JAK2-V617F and CALR-mutated mice and patients shows distinct inflammatory cytokine signatures (A) Left and middle panel: RNA-seq of granulocytes obtained from Ly6G-Cre JAK2+/+, JAK2+/VF, CALR+/+ and CALR+/del mice (each n = 3) was performed by GENEWIZ Inc. (Leipzig, Germany). Using DESeq2, a comparison of gene expression between the JAK2+/+ versus JAK2+/VF and CALR+/+ versus CALR+/del groups of samples was performed. The heatmaps represent fold changes in mean normalized counts of cytokine RNA abundances relative to the WT controls. Left panel: Ly6G-Cre JAK2+/+ versus JAK2+/VF mice; middle panel: Ly6G-Cre CALR+/+ versus CALR+/del mice. *adjusted p-value ≤ 0.05, **adjusted p-value ≤ 0.01, ****adjusted p-value ≤ 0.0001; right panel: RNA-seq was performed on peripheral blood granulocytes isolated from JAK2-V617F positive (n = 4), healthy donors (n = 3) and CALR-mutated (n = 2) patients by GENEWIZ Inc. (Leipzig, Germany). Using DESeq2, a comparison of RNA expression between JAK2-V617F positive patients versus age-matched healthy donors and CALR-mutated patients versus healthy donors was performed. The heatmaps depict fold changes of mean normalized counts of cytokine RNA abundances relative to the healthy donor controls. Cartoon created with Biorender.com. (B) GSEA of IL-1 pathways in granulocytes isolated from JAK2-V617F positive patients compared to CALR-mutated patients. Positive NES in the heatmap indicate substantial (FDR q-values < 0.15) enrichment in granulocytes from JAK2-V617F positive patients. The values point out NES for each pathway. All gene sets were obtained from the GSEA website (UC San Diego and Broad Institute; https://www.gsea-msigdb.org). (C) RNA-seq data from JAK2-V617F positive patients (n = 4 consecutive patients) compared to CALR-mutated (n = 2 consecutive patients) patients is tested for enrichment of genes related to the “Interleukin 1 Signaling Pathway” by Gene Set Enrichment Analysis (GSEA). GSEA was performed using the GSEAv4.3.2 (UC San Diego and Broad Institute; https://www.gsea-msigdb.org). Comparisons exhibiting a p-value < 0.05 and FDR q-value < 0.15 were considered significant. The enrichment map was used for visualization of the GSEA results. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) p-values were calculated upon 10,000 gene set permutations

In order to compare cytokine gene expression in granulocytes obtained from healthy donors with JAK2-V617F and CALR mutant patients (clinical characteristics are depicted in Additional file 1: Tab. S4), we performed RNA-seq and generated a heatmap of cytokine gene expression in human neutrophils (Fig. 3A, right panel). Apparently, the fold-changes were more prominent as compared to the Ly6G-Cre mouse models (Fig. 3A, left and middle panel). However, when considering the adjusted p-values the changes in gene expression were not statistically significant. This is not unexpected, because there is high inter-individual variability which is influenced, among other factors, by inherited genetic predisposition factors and disease phenotype. Nevertheless, the overall observed changes high-lighted differences between the two groups of patients investigated (Fig. 3A, right panel): generally, JAK2-V617F positive patients displayed a greater number of upregulated cytokines in comparison to those carrying CALR mutations, where more cytokines were downregulated. Interestingly, in comparison to healthy donors CXCL3, CCL2, CCL4, CXCL8, CXCL1, and CCL3 showed up-regulation in JAK2-V617F positive patients, whereas these genes were unchanged or down-regulated in CALR mutant patients (Fig. 3A, right panel). Furthermore, gene expression of IL1RN, IL1B, and TNFA also displayed up-regulation in JAK2-V617F patients, whereas in CALR-mutant patients they showed a tendency to decrease (Fig. 3A, right panel). This data is interesting, given the crucial role of IL-1β in the development of JAK2-V617F positive disease [17, 18]. However, it requires further analysis in a larger cohort of patients.

To investigate whether we could differentiate between JAK2-V617F and CALR mutant patients based on gene expression profiling, we aimed to determine if there were any differentially expressed functional pathways. We hypothesized that IL-1 signaling could be an interesting candidate based on previously published results [17, 18] and on the increase in gene expression of IL1B in neutrophils of JAK2-V617F positive patients (Fig. 3A, right panel). Importantly, Gene Set Enrichment Analysis (GSEA) [60, 61] revealed a remarkable and highly significant enrichment of the “Interleukin 1 Signaling Pathway”, “IL-1 Structural Pathway” and “IL-1R Pathway” in neutrophils sampled from patients with JAK2-V617F, when compared with CALR-mutated patients (Fig. 3B and C). Analysis of JAK2-V617F positive patients versus healthy donors also indicated a trend towards an enrichment of these pathways (Additional file 1: Tab. S5). Together, this data portrays a marked influence of the JAK2-V617F mutation in generating an IL-1 response profile in granulocytes of MPN patients.

Pan-hematopoietic expression of JAK2-V617F induces a prominent inflammatory cytokine signature in neutrophils

Unlike in the Ly6G-Cre JAK2+/VF model, granulocytes in Vav-Cre JAK2+/VF mice interact with signals from various other JAK2-V617F positive cell populations (such as progenitor cells, MKs, endothelial cells, etc.). This interaction potentially leads to more significant differences in gene expression, compared to the more subtle changes seen in granulocytes from Ly6G-Cre JAK2+/VF mice. The Vav-Cre JAK2+/VF mouse model has been previously described and reliably recapitulates a PV-like phenotype of MPN [40, 43]. Neutrophils from Vav-Cre JAK2+/VF and JAK2+/+ mice were isolated and RNA-seq was performed (Fig. 4A). 3,508 genes out of a total of 13,881 genes were significantly (adjusted p-value < 0.05) regulated in neutrophils from Vav-Cre JAK2+/VF mice (data not shown). In comparison to the low number of regulated genes in the Ly6G-Cre JAK2+/VF model, this indicates that the gene expression signature of neutrophils in JAK2-V617F induced disease is not primarily regulated by cell-intrinsic JAK2-V617F expression. Instead, JAK2-V617F neutrophils are more sensitive to extrinsic signals originating from other JAK2-V617F positive hematopoietic cells.

Fig. 4

Pan-hematopoietic expression of JAK2-V617F up-regulates pro-inflammatory cytokines in neutrophils and hematopoietic progenitors (A) Cartoon depicting the experimental design to study gene expression signatures in neutrophils and intracellular IL-1α expression in Vav-Cre JAK2+/VF mice in comparison to their corresponding WT controls. Created with Biorender.com. (B) RNA-seq of granulocytes obtained from Vav-Cre JAK2+/VF and JAK2+/+ mice (each n = 4) was performed by GENEWIZ Inc. (Leipzig, Germany). Using DESeq2, a comparison of gene expression between the Vav-Cre JAK2+/+ versus JAK2+/VF groups of samples was performed. The heatmaps represent fold changes in mean normalized counts of cytokine RNA abundances relative to the WT controls. Vav-Cre JAK2+/+ versus JAK2+/VF mice; *adjusted p-value ≤ 0.05, **adjusted p-value ≤ 0.01, ****adjusted p-value ≤ 0.0001 (C) GSEA of “Hallmark Inflammatory Response” genes in granulocytes isolated from Vav-Cre JAK2+/VF and JAK2+/+ mice. The enrichment map was used for visualization of the GSEA results. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) p-values were calculated upon 10,000 gene set permutations. The positive NES of 1.39 in the figure indicates substantial (FDR q-value = 0.03; nominal p-value = 0.02) enrichment in genes linked with the inflammatory response. The “Hallmark Inflammatory Response” gene set was obtained from the GSEA website (UC San Diego and Broad Institute; https://www.gsea-msigdb.org). (D, E, F) Intracellular staining for IL-1α levels in various hematopoietic cell populations using anti-IL-1α antibody and isotype control antibody, respectively was performed as described under Supplemental Methods. Mean fluorescence intensity (MFI) was measured by flow cytometry. The specific MFI (MSFI) was calculated by subtracting the MFI of the isotype control from the MFI of the anti-IL-1α antibody stained sample. Data are shown as mean ± SEM. *p ≤ 0.05 (unpaired, two-tailed t-test with Welch correction)

Next, we focused on the cytokine gene expression levels induced by JAK2-V617F and generated a heatmap of relative changes (Fig. 4B). Significant changes (adjusted p-values < 0.05) were detected in nine genes by DESeq2 analysis. Il6 (2.3-fold change), Cxcl4 (2.0-fold change), Cxcl2 (1.8-fold change), Il1rl1 (1.4-fold change) and Mif (1.4-fold change) were identified as the five up-regulated cytokine genes in JAK2-V617F positive neutrophils, while Cxcr4 (0.8-fold change), Ltb (0.7-fold change), Il1rn (0.6-fold change) and Ccl6 (0.58-fold change) showed a decrease. These changes in gene expression did not overlap with the panel of upregulated cytokines in serum of Vav-Cre JAK2+/VF mice which comprises CCL2, CCL11, CXCL5, CXCL9, CXCL10 and IL-1α (as published previously [43]), again suggesting that neutrophils are not the primary cellular source of upregulated serum cytokines in JAK2-V617F induced disease.

Since neutrophils are recognized as an important pathogenic link to inflammation we performed Gene Set Enrichment Analysis (GSEA) on RNA-seq data from neutrophils of Vav-Cre JAK2+/VF and JAK2+/+ mice utilizing the “Hallmark Inflammatory Response” signature from the Molecular Signature Database (MSigDB) hallmark gene set collection [62] (Fig. 4C). GSEA indicated a highly significant enrichment of genes linked to “Hallmark Inflammatory Response” in neutrophils from Vav-Cre JAK2+/VF mice (Fig. 4C). Of note, this signature was not enriched in neutrophils obtained from Ly6G-Cre JAK2+/VF mice (data not shown). Based on this data, we conclude that the inflammatory gene expression signature of JAK2-V617F neutrophils is particularly prone to external signals originating from other JAK2-V617F positive cells.

Next, we examined which hematopoietic cell populations are the source of the elevated serum levels of inflammatory cytokines in the Vav-Cre JAK2-V617F model. We focused on IL-1α (upregulated by a factor of 4.8 in serum of Vav-Cre JAK2+/VF mice) and performed intracellular IL-1α staining in various hematopoietic cell types including hematopoietic progenitors. The results showed that granulocytes, B-cells, T-cells, monocytes and macrophages from Vav-Cre JAK2+/+ and JAK2+/VF mice expressed similar IL-1α protein levels, respectively (Fig. 4D). However, CD41+ cells from Vav-Cre JAK2+/VF mice displayed significantly higher IL-1α protein levels (Fig. 4E). Elevated IL-1α levels were also found in CMP (Fig. 4F). MEP, MKP and LSK cells from Vav-Cre JAK2+/VF mice also trended in this direction (Fig. 4F). Together, this data suggests that MKs and myeloid progenitors but not granulocytes are major contributors to the elevated IL-1α serum levels in Vav-Cre JAK2+/VF mice. However, in myelofibrosis, it has been shown that both, myeloid progenitors and mature myeloid cells, may produce inflammatory cytokines, yet with distinct cytokine secretion profiles [63].

Neutrophil-specific expression of JAK2-V617F and CALRdel, respectively differentially affects the metabolic activity

It is well established that an inflammatory phenotype of immune and tissue-resident cells is reflected by, if not dependent on, an altered cellular metabolism for meeting the cells’ increased metabolic demands [45, 46]. Therefore, we compared the metabolic profile of neutrophils isolated from Ly6G-Cre JAK2+/VF and CALR+/del mice by means of real-time metabolic flux analyses (Fig. 5A). Neutrophils from JAK2+/VF mice displayed an overall increased metabolic activity (Fig. 5B-G). Both the glycolytic (Fig. 5B) as well as the respiratory (Fig. 5C) parameters tested were significantly higher compared to the neutrophils isolated from Ly6G-Cre CALR+/del mice. When compared to the respective wild type controls (Additional file 3: Fig. S10A and B), the metabolic parameters glycolysis (Fig. 5D), glycolytic capacity (Fig. 5E), basal respiration (Fig. 5F), maximal respiration (Fig. 5G), revealed higher activity in JAK2-V617F neutrophils and less activity in CALRdel neutrophils. Additional metabolic parameters are depicted in Additional file 3: Fig. S10 and are in line with these results. The OCR/ECAR ratio (Fig. 5H) as a surrogate for the balance between glycolysis and OXPHOS was similarly increased in both genotypes. However, the baseline overall metabolic phenotype (Fig. 5I) plotted as an ECAR/OCR map displayed a clear segregation between JAK2-V617F and CALRdel neutrophils with a more energetic profile for the former and a more quiescent profile for the latter. Thus, it appears that the higher inflammatory activity of JAK2-V617F neutrophils as characterized by the marked pro-inflammatory cytokine profiles described above is mirrored by a striking increase in the metabolic activity. It will be interesting to test this hypothesis in future studies by using inhibition experiments. Additional metabolic parameters including comparisons of the respective WT controls and the comparisons between JAK2-V617F versus WT and CALRdel versus WT are shown in Additional file 3: Fig. S10.

Fig. 5

Neutrophils isolated from Ly6G-Cre JAK2+/VF mice display enhanced metabolic activity. (A) Cartoon depicting the experimental design to study metabolic activity in neutrophils isolated from Ly6G-Cre JAK2+/VF and CALR+/del. Created with Biorender.com. (B) Glycolysis stress test (GST) and (C) mitochondrial stress test (MST) analyzing extracellular acidification rate (ECAR), as a surrogate for aerobic glycolysis, and oxygen consumption rate (OCR), as a surrogate for oxidative phosphorylation in neutrophils isolated from Ly6G-Cre JAK2+/VF and Ly6G-Cre CALR+/del (n = 3 with 3–6 technical replicates) were recorded in real-time upon sequential injection of compounds/inhibitors as described in the Supplemental Methods. The data presented were normalized to the background (phase 1 for GST and phase 4 for MST). Based on these and the corresponding wild-type data (Supplemental Fig. 7A and B), the metabolic parameters glycolysis (D), glycolytic capacity (E), basal respiration (F), maximal respiration (G), and the OCR/ECAR ratio as a surrogate for the balance between glycolysis and OXPHOS (H) were calculated and presented as the log2 fold change (log2 FC) of each individual relative to the respective wild-type average. (I) The baseline overall metabolic phenotype was calculated from (B and C) and plotted as an ECAR/OCR map. Data are shown as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired, two-tailed t-test). Abbreviations: OCR, oxygen consumption rate; ECAR, extracellular acidification rate; 2DG, 2-deoxyglucose; FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; AA/R, Antimycin A/Rotenone

Neutrophil-specific expression of JAK2-V617F, but not CALRdel, decreases migration of granulocytes in vitro

Inflammatory conditions cause granulocytes to be sequestered from the circulation and directed towards the endothelial layer of blood vessels via the le