IL-1β enhances inflammatory cytokine and chemokine responsiveness in JAK2-mutant HSCs. (A) Schematic drawing of the experimental setup for competitive transplantation. BM cells from VF;GFP, VF;IL-1β^−/−;GFP, VF;IL-1R1^-/;GFP, or WT;GFP donor mice that were not induced by tamoxifen were mixed with competitor BM cells from a WT donor in a 1:1 ratio. Recipient mice were induced with tamoxifen 16 weeks after transplantation to induce JAK2-V617F expression. Mice from each genotype (n = 3-4 per group) were euthanized 2 or 4 weeks after tamoxifen induction and LT-HSCs (Lin⁻Sca1⁺cKit⁺CD48^–CD150⁺) were purified by fluorescence-activated cell sorter for RNA sequencing. (B) The time course of blood counts of recipient mice after tamoxifen induction. (C) Human JAK2-V617F messenger RNA gene expression in LT-HSCs (normalized log2 counts per million [CPM]) at 2 and 4 weeks after tamoxifen induction. Multiple t tests were performed for statistical analyses. (D) Pathway analysis 4 weeks after tamoxifen induction performed with WebGestalt online tool⁴⁰, using Gene Ontology as the functional database. Minimum and maximum number of genes for a category was set to 15 and 500, respectively. TOP method (TOP means the categories will be first ranked based on the false discovery rate [FDR] and then the top N most significant categories will be selected) was used to identify significance levels of the enriched categories, and number of permutations was 1000. Bars representing significant pathways (FDR of <0.05) are colored according to their normalized enrichment score and the FDR as indicated. The names of the pathways upregulated in VF vs all 3 other genotypes are colored in red or orange according to their FDRs. (E) Expression of genes from the top 3 pathways identified by Gene Ontology in panel D to be commonly upregulated in LT-HSCs from VF mice vs mice from all 3 other genotypes. All data are presented as mean ± SEM; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. See also supplemental Figure 10.