Fig 2: Inflammatory nature of the old BM niche.a, Differentially secreted cytokines in young and old BM fluid measured by 200-plex array and clustered based on broad biological functions (n = 5). b, Changes in pro-inflammatory cytokines in young and old BM fluids. Results are from 3 independent cohorts. c, qRT-PCR-based analyses of Il1a, Il1b and Tnf expression in old BM cells and stromal fractions (n = 6). Results are represented as violin plots and are expressed as fold change compared to their respective young counterpart. Neu, Mac-1+/Ly-6G+/Ly-6Cmid neutrophil; Mac, Mac-1+/Ly-6G-/Ly-6C- macrophage; B cell, B220+/CD19+ B cell; T cell, CD4+/TCRß+ T cell; Marrow, Ter119-/CD45- central marrow fraction; Endo, Ter119-/CD45- endosteal fraction. d, Violin plots representation of Il1a, Il1b, and Il1rn expression in the indicated young and old mesenchymal and endothelial like groups. Results are from the plate-based scRNAseq dataset; *padj = 0.05, ** padj = 0.01, *** padj = 0.001. e, Caspase 1 activity (aCASP1) in the indicated young and old BM and stromal populations with representative FACS plots (top) and quantification (bottom). f, Experimental scheme for the indicated co-culture experiments showing the effects of young and old stroma on young HSC expansion as dependent on IL-1ß. Data are means ± S.D.; *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 3: Age-related changes in blood and BM populations and altered lineage bias in old MPPs.a, Overlap between cytokines upregulated in old BM fluids and published SASP profile40. b, Representative SA-b-gal staining in control irradiated mouse embryonic fibroblasts (MEF) and isolated young and old MSC-S and OBC. Scale bar, 20 µm. c, Absolute expression of Il1b in different mature hematopoietic cell types and unfractionated (CD45-/Ter119-) endosteal and central marrow stroma from pooled young and old samples. Results are expressed as -log(dCt) relative to gapdh. d, Complete blood count (CBC) parameters in young and old mice. Results are from 7 independent cohorts. WBC, white blood cell; My, myeloid (neutrophil + basophil + eosinophil); Ly, lymphocyte; RBC, red blood cell; Pt, platelet. e, Cellularity and quantification of mature populations in young and old BM. Results are from 3 independent cohorts. Gr, Mac-1+/Gr-1+ granulocyte; B cell, B220+ B cell; T cell, CD3+ T cell. f, Quantification of progenitor populations in young and old BM. Results are from 3 independent cohorts. CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte-erythrocyte progenitor; CLP, common lymphoid progenitor; CFU-E, erythroid colony-forming unit; PreGM, pre-granulocyte/macrophage; Pre-MegE, pre-megakaryocyte/erythrocyte; MkP, megakaryocyte progenitor. g, Representative staining and quantification of CD34, CD41, CD62P and vWF levels on young and old HSCs. Results are from 2 independent cohorts, with data represented as box and whiskers (min to max) and expressed as fold changes in mean fluorescence intensity (MFI) relative to young HSCs. h, Characteristic expression patterns of lineage determinant genes in the droplet-based scRNAseq young and old LK/LSK dataset shown in Fig. 4b. Cells in the UMAP were colored according to the expression levels of the indicated genes. Color scheme is based on ln scale of normalized counts from the indicated minimum (gray) to maximum (red) value in the scale. i, IPA Upstream Regulators analysis of young and old populations from the droplet-based scRNAseq young and old LK/LSK dataset shown in Fig. 4b filtered on cytokines and growth factors. Data are means ± S.D. except when indicated; *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 4: Key role of IL-1 in the aging of both BM niche and blood system.a-b, GSEA results in old Il1r1-/- HSPC population for the Gene Ontology pathways affected in either old WT HSPCs (a) or young WT HSPCs (b) identified by droplet-based scRNAseq analyses. n/a, non-available; ns, non-significant; *nominal p value = 0.05, ** nominal p value = 0.01, *** nominal p value = 0.001. c, Peripheral blood CD45.1+ donor chimerism (left) and number of donor-derived GMPs (right) in young or old WT and Il1r1-/- CD45.2+ recipient mice at 4 months (mo) after lethal irradiation and transplantation (Tplx) with 2x106 young WT CD45.1+ donor BM cells. d, Il1r1 expression in the droplet-based scRNAseq of young and old WT stroma and LK/LSK datasets. Cells in the UMAP were colored according to the expression levels of the indicated genes. Color scheme is based on ln scale of normalized counts from the indicated minimum (gray) to maximum (red) value in the scale. e-i, Unchanged aging features in old Tnf-/- mice with: (e) color scheme; (f) blood parameters; (g) endosteal (left) and central marrow (right) mesenchymal population frequencies; (h) BM hematopoietic population frequencies; and (i) engraftment over time (left) and lineage reconstitution (right) at 4 mo post-Tplx of the indicated HSC populations. Results are from 3 independent cohorts of young and old WT and age-matched Tnf-/- mice, with HSCs isolated from the pooled BM of mice of the same genotype and transplanted into 3 to 5 recipients, each. Data are means ± S.D. except for engraftment results shown in (i) (± S.E.M.); *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 5: MC-derived CCL2 sustains a homeostatic MΦ phenotype across the vascular tree(A and B) UMAP based dimensionality reduction of single-cell RNA-seq of FACS-sort enriched CD45+ CD11bhi CD64hi F4/80hi cells in kidney (A) and lung (B) of Ccl2MCΔ/Δ and Ccl2MC+/+ mice (n = 4/group).(C–G) Volcano and violin plots depicting selected significantly differentially regulated genes in (C) kidney resident MΦ cluster 2, (D) kidney resident MΦ cluster 0, (E) lung monocyte cluster 1, (F) lung alveolar MΦ cluster 2, (G) lung Folr2hi Mrc1hi interstitial MΦ cluster 3.(H) Frequency of lung Zeb2hi interstitial MΦ cluster 4 cells among all analyzed cells.(I) Significantly differentially regulated genes, associated with a functionally differentiated, efferocytotic MΦ phenotype in Ccl2MC+/+ and Ccl2MCΔ/Δ chimera mice. Low-input RNA-seq of FACS-sorted Cx3cr1+ MΦs from Ccl2MC+/+ or Ccl2MCΔ/Δ chimera mice with MCRFP-ep; Cx3cr1-MΦGFP-rep bone marrow after 20 weeks western diet (experimental setup further depicted in Figure S3) (n = 3–4 chimera mice). Student’s t test was used. Expression levels of depicted genes normalized to sample with highest expression (set as 1) across all samples. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression. ∗ p < 0.05.