Clonal hematopoiesis related TET2 loss-of-function impedes IL1β-mediated epigenetic reprogramming in hematopoietic stem and progenitor cells

Abstract

Clonal hematopoiesis (CH) is defined as a single hematopoietic stem/progenitor cell (HSPC) gaining selective advantage over a broader range of HSPCs. When linked to somatic mutations in myeloid malignancy-associated genes, such as TET2-mediated clonal hematopoiesis of indeterminate potential or CHIP, it represents increased risk for hematological malignancies and cardiovascular disease. IL1β is elevated in patients with CHIP, however, its effect is not well understood. Here we show that IL1β promotes expansion of pro-inflammatory monocytes/macrophages, coinciding with a failure in the demethylation of lymphoid and erythroid lineage associated enhancers and transcription factor binding sites, in a mouse model of CHIP with hematopoietic-cell-specific deletion of Tet2. DNA-methylation is significantly lost in wild type HSPCs upon IL1β administration, which is resisted by Tet2-deficient HSPCs, and thus IL1β enhances the self-renewing ability of Tet2-deficient HSPCs by upregulating genes associated with self-renewal and by resisting demethylation of transcription factor binding sites related to terminal differentiation. Using aged mouse models and human progenitors, we demonstrate that targeting IL1 signaling could represent an early intervention strategy in preleukemic disorders. In summary, our results show that Tet2 is an important mediator of an IL1β-promoted epigenetic program to maintain the fine balance between self-renewal and lineage differentiation during hematopoiesis.

Fig. 1. Chronic IL1β exposure enhances the expansion of Tet2-KO cells and myeloid bias with the elevation of Ly6c^hi over Ly6c^lo monocytes/macrophages.

a–c Lineage-depleted BM cells from wild-type (WT) CD45.1 and Tet2-KO CD45.2 mice were transplanted into WT CD45.1/2 mice which after 3 weeks were treated with IL1β or vehicle daily. a Experimental design, b percentage of WT CD45.1 or Tet2-KO CD45.2 cells out of all CD45⁺ cells (chimerism, significance shown between Tet2-KO with and without IL1β treatment) and the percentage WT CD45.1 and Tet2-KO CD45.2⁺ cells which are myeloid in the PB (significance shown between Tet2-KO and WT with IL1β treatment, n = 5 mice/group for Veh, 6 for IL1β) and c myeloid subsets (CD11b⁺Gr1^hi, CD11b⁺Gr1^lo) at week 15 (n = 5 mice/group for Veh, 4 for IL1β). d–f Lineage-depleted BM cells from WT CD45.1⁺ and Rosa-rtTA-driven inducible Tet2 knockdown (shTet2) CD45.2 mice were transplanted into WT CD45.1⁺CD45.2⁺ mice, treated with doxycycline (dox) two weeks after transplantation and IL1β or vehicle three weeks after transplantation for increasing intervals up to 10 weeks and then with and without doxycycline (withdrawal; w/d) for an additional two weeks. Arrows indicate the time relative to the initial treatment (week: 0, 3, 6, 8) and duration (days: 2, 4, 7, and 28) of daily treatment with IL1β or vehicle. Green indicates time and duration of doxycycline treatment. d Experimental design, e percentage of GFP⁺CD45.2 cells out of donor-derived CD45⁺ cells and the percentage of WT CD45.1 and shTet2 CD45.2 cells which are myeloid (CD11b⁺) in the PB for doxy and IL1β or vehicle-treated experimental arms (significance shown between shTet2 and WT with IL1β treatment, right panel, n = 5 mice/group), and f neutrophils (CD11b⁺Ly6c^midLy6g⁺), Ly6c^hi monocytes/macrophages (CD11b⁺Ly6c^hiLy6g^-), and Ly6c^lo monocytes/macrophages (CD11b⁺Ly6c^loLy6g^-) in the spleen with doxy or without doxy (w/d) for IL1β or vehicle treatments (n = 5 mice/group for Veh, IL1β, Dox w/d IL1β, 4 for Dox w/d mice/group). Error bars represent mean ± SEM. For panels c and f two-factor ANOVA determined family-wise error rate (FWER) adjusted p values. For panels b and e, a student’s two-tailed t-Test determined the significance between single comparisons. For FWER adjusted and regular p values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. IL1β drives expansion, myeloid bias, and enhances self-renewal of Tet2-KO HSPCs.

a, b Lineage-depleted BM cells from wild-type (WT) CD45.1⁺ and Rosa-rtTA-driven inducible Tet2 knockdown (shTet2) CD45.2 mice transplanted into lethally irradiated WT CD45.1⁺CD45.2⁺ mice and treated with IL1β or vehicle as well as with and without doxycycline (withdrawal; dox w/d) as described in Fig. 1d and analyzed by flow cytometry (n = 5 for Veh, IL1β, Dox w/d IL1β, 4 for Dox w/d mice/group). a Percentage of live cells which are LT-HSCs and b MPP2, MPP3, and MPP4 in the BM and spleen of shTet2 and WT mice. c The ratio of the change in frequency between dox to dox w/d for MPP2s, MPP3s, and MPP4s in BM. d Colony-forming unit (CFU) assay of sorted LSK cells from WT or Tet2-KO BM (n = 9 technical replicates with 3 biological replicates); colonies were counted and the cells were re-plated every 7 days. M, macrophage; G, granulocyte; GM, granulocyte/macrophage; BFU-E, erythroid. e Simplified hematopoietic hierarchy with red text representing subsets elevated in frequency in Tet2-KO relative to WT mice treated with IL1β with red arrows connecting the most elevated subsets. Error bars represent mean ± SEM. For panels, a, b, and d, two-factor ANOVA was used to determine FWER-adjusted p values. For panel c, one factor ANOVA was used to determine the FWER-adjusted p values. For all FWER adjusted p values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. Tet2-KO HSCs exhibit transcriptional signatures for self-renewal capacity under inflammatory stress.

a WT and Tet2-KO mice were treated for 5 weeks with and without IL1β (n = 4 mice/group). BM cells were harvested and lineage-negative cells were enriched by magnetic selection. This enrichment increases the representation of progenitors but reduces the percentage of differentiated cells. Cells were analyzed by 10X single-cell RNA (scRNA) sequencing. b UMAP of 54,984 cells representing 17 clusters and bar graph representing their proportions including hematopoietic stem cell (HSC), progenitor (Prog), immature myeloid progenitor (IMP), granulocyte or monocyte progenitor (Gran/Mono Prog), monocyte (Mono), macrophage (Mac), neutrophil (Neut), basophil (Baso), megakaryocyte progenitor (MegP), common lymphoid progenitor (CLP), T cell (T-cell), plasmacytoid dendritic cells and B cell (pDc/B-cell), eosinophil (Eo), megakaryocyte-erythroid progenitor (MEP), colony-forming unit erythroid (CFU-E), erythroblast CD45^- EryB (EryB_CD45-) and erythroblast CD45⁺ EryB (EryB_CD45 + ). c Venn diagram of differentially expressed genes (DEGs) in Tet2-KO HSCs relative to WT treated with vehicle or IL1β. d Volcano plot of DEGs in Tet2-KO relative to WT HSCs with or without IL1β stimulation. Selected genes based on enriched pathways are labeled. q values were determined using DESeq2. e Gene-set enrichment analysis (GSEA) from HSCs in Tet2-KO relative to WT showing selected significantly enriched gene sets (FDR < 0.05), with IL1β (red) or vehicle (black) or pathways not significantly enriched in the vehicle (grey). q values were determined by GSEA v4.2.1 using an empirical phenotype-based permutation test procedure. f Heatmap of upregulated DEGs in Tet2-KO relative to WT HSCs with and without IL1β stimulation for HSCs, Prog, IMPs, and GMPs. The genes shown are the lead genes for enriched pathways by GSEA analysis within HSCs, categorized into “Inflammation only”, “Inflammation and Self-renewal”, or “Self-renewal only” using the GSEA subcategories assigned in Fig. 3e. q values were determined by DESeq2. g Enrichment score plots for the gene signature of the top 100 upregulated genes in Tet2-KO relative to WT with IL1β (top) or vehicle (bottom) using DEGs from human TET2 mutant (left) versus TET2 WT (right) acute myeloid leukemia with p values determined by GSEA v4.2.1. For q values: *q < 0.05, **q < 0.01, ***q < 0.001, ****q < 0.0001.

Fig. 4. Tet2-KO HSPCs resist IL1β-driven methylation reduction of lineage-specific enhancer regions and terminal differentiation promoting transcription factor binding sites.

WT and Tet2-KO mice were treated for 5 weeks with and without IL1β (n = 4 mice/group). BM cells were harvested and LSK, CMP, and GMPs were FACS purified and analyzed by whole genome bisulfite sequencing (Supplementary Fig. 10a). a Differentially methylated regions (DMRs) from 50 CpG long regions of the whole genome ( > 10% methylation difference, q < 0.05, statistical significance determined by SeqMonk) between Tet2-KO and WT LSKs with or without IL1β stimulation. The bar graphs below show the methylation level of all probes for each condition. b Percentage of 50 CpG segments overlapping each genomic element that are hypermethylated or hypomethylated DMRs in Tet2-KO relative to WT. c The percentage methylation difference between Tet2-KO and WT cells was analyzed using literature-derived ChIP-seq binding sites for each transcription factor (TF). d Top panel, MA plots showing methylation difference for LSK and CMP cells derived from Tet2-KO and WT mice treated with IL1β using literature-derived ChIP-seq binding sites of terminal differentiation promoting TFs. The percentage of sites with higher methylation in Tet2-KO than WT are shown in the top right corner of each graph. Bottom panel, the statistical significance of motif enrichment determined by hypergeometric distribution (HOMER analysis tool) within DMRs for TFs with and without IL1β stimulation. e Heatmap of the 150 most downregulated genes near hypermethylated TF binding motifs in Tet2-KO HSCs relative to WT treated with and without IL1β. Their differential expression is shown across HSC, progenitors, and IMP clusters. Error bars represent mean ± SEM. For panel 4a (lower), two-factor ANOVA was used to determine the FWER adjusted p values: ****p < 0.0001.

Fig. 5. Genetic and pharmacological inhibition of IL1R1 abrogates aberrant myeloid expansion in Tet2-KO mice.

a–d WT, Vav-cre Tet2^fl/fl (Tet2-KO), Il1r1-/- (Il1r1-KO), and Vav-cre Tet2^fl/flIl1r1^-/- (double knockout; DKO) mice were bled twice and 8 mice were sacrificed at greater than 60 weeks of age, the remaining cohort was followed to observe survival outcomes. a Experimental design. b Spleen weight in grams. c Survival of WT (n = 8), Tet2-KO (n = 16), Il1r1-KO (n = 15), and DKO (n = 18) mice. d The frequency of MHCII⁺ macrophages (CD45⁺CD11b⁺F4/80⁺MHCII⁺), F4/80^- Ly6c^hi monocytes (CD45⁺CD11b⁺F4/80^-Ly6c⁺Ly6g^-) and F4/80^- Ly6c^lo monocytes (CD45⁺CD11b⁺F4/80^-Ly6c^-Ly6g^-) in BM and spleen. e–g Lineage-depleted BM cells derived from WT CD45.1 and Tet2-KO CD45.2 mice were transplanted into lethally irradiated WT heterozygous CD45.1/2 mice. Three weeks after the transplantation mice were treated with IL1β (500 ng/mouse/day) or vehicle as well as with or without an IL1R1 antagonist (anakinra, 100 mg/kg) (n = 3 mice/group for Veh, Ana, and IL1β, and 4 for IL1β Ana). e Experimental design, f the percentage of Tet2-KO CD45.2 donor cells out of all CD45⁺ cells (chimerism) in PB, and g the frequency of LK and LSK cells in the spleen. h Human BM CD34⁺ progenitors were CRISPR-Cas9 edited for TET2 (TET2-sgRNA) alongside non-targeting control (NT-sgRNA), then 600 cells per well were plated with or without IL1β (25 ng/mL) and/or IL1RA (50 ng/mL). Colonies were counted after 2 weeks and individual colonies were picked for sanger sequencing to determine indel frequency within TET2. Total colony numbers at 2 weeks and CFU-M (monocyte) colony numbers, with the number of colonies identified with detectable indels shown. Error bars represent mean ± SEM. For panels a, d, g, f, and h two-factor ANOVA was used to determine the FWER-adjusted p values. For panel f, a student’s two-tailed t-Test was used to determine significance. For FWER adjusted and regular p values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6. Schematic depicting the impact of IL1β-driven inflammatory stress on hematopoiesis in TET2-mediated premalignancy.

Upon IL1β stimulation, Tet2-KO HSPCs showed enhanced self-renewal and bias towards MPP3s. Accordingly, Tet2-KO HSPCs (LSKs) resisted the loss of methylation observed in WT HSPCs upon IL1β stimulation within pro-differentiation transcription factors (TFs) binding motifs and lymphoid enhancers. Additionally, Tet2-KO HSCs exhibited enhanced transcriptional signatures for self-renewal and myeloid bias. Further downstream, in the hematopoietic lineage, Tet2-KO CMPs also resisted methylation loss observed in WT CMPs within erythroid enhancers upon IL1β stimulation. Consistent with this GMPs frequency was elevated over MEPs in Tet2-KO relative to WT mice under inflammatory stress, leading to the expansion of Ly6c^hi MHCII+ macrophages and neutrophils. Genetic or pharmacological inhibition of IL1R1 signaling suppressed myeloid expansion and delayed premalignant phenotype over the course of aging. Created with BioRender.com.