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