ABSTRACT Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals

from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives

both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+ mesenchymal stromal cells

associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of

deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche

inflammation is a tractable strategy to improve blood production during ageing. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS MICRO-ENVIRONMENTAL SENSING BY BONE MARROW STROMA IDENTIFIES IL-6 AND TGFΒ1 AS REGULATORS OF

HEMATOPOIETIC AGEING Article Open access 14 August 2020 THE ROLE OF THE HAEMATOPOIETIC STEM CELL NICHE IN DEVELOPMENT AND AGEING Article 10 September 2024 NICHE DERIVED NETRIN-1 REGULATES

HEMATOPOIETIC STEM CELL DORMANCY VIA ITS RECEPTOR NEOGENIN-1 Article Open access 27 January 2021 DATA AVAILABILITY The RNA-seq and microarray data that support the findings of this study

have been deposited in the Gene Expression Omnibus under accession code GSE169162. The _M._ _musculus_ genome GRCm38.p4 is available from the National Center for Biotechnology Information

(https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.24/). The chip annotation MSigDB.v7.2.chip is available from the Broad Institute

(https://software.broadinstitute.org/cancer/software/gsea/wiki/index.php/MSigDB_v7.2_Release_Notes). Gene sets used for GSEA h.all.v7.2.symbols.gmt and c2.cp.reactome.v7.2.symbols.gmt are

available from the Broad Institute (https://data.broadinstitute.org/gsea-msigdb/msigdb/release/7.2/). All other data are available from the corresponding author upon reasonable request.

Source data are provided with this paper. CODE AVAILABILITY All code and packages used to support the findings of this study are either publicly available or available from the corresponding

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the ionizing radiation response. _Proc. Natl Acad. Sci. USA_ 98, 5116–5121 (2001). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank S. Villeda (UCSF) for providing

some of the old C57BL/6 mice; D. Reynaud (UCSF) for initial analyses of the old mice; A. Valencia (UCSF) for technical assistance with various experiments; S. Kinston (Cambridge University)

for scRNA-seq library preparations; A. Li (Bone Imaging Research Core, UCSF) and both E. Guo and P. T. Shyu (CUIMC) for microCT analyses; M. Lee (UCSF) and M. Kissner (CUIMC) for management

of our Flow Cytometry Core facilities; and all members of the Passegué Laboratory for critical insights and suggestions. C.A.M. was supported by NIH F31HL160207 and a NYSTEM training grant;

E.V.V. by a Rubicon Grant from The Netherlands Organization for Scientific Research, a Stem Cell Grant from BD Biosciences and a NYSTEM training grant; O.C.O by CRI/Margaret Dammann Eisner

postdoctoral fellowship CRI3617; J.W.S. by long-term EMBO postdoctoral fellowship ALTF-2021-196; P.V.D. by NIH F31HL151140; E.M.P. by NIH F32HL106989 and K01DK09831; S.T.B. by a CIRM

postdoctoral fellowship; and T.T.H. by an AHA and Hillblom Center for the Biology of Aging predoctoral fellowship. F.J.C-N., X.W. and B.G. were supported by grants from the Wellcome

(206328/Z/17/Z), CRUK (C1163/A21762) and core funding by the Wellcome to the Cambridge Stem Cell Institute. This work was funded by NIH R01CA184014, NIH R35HL135763, Glenn Foundation

Research Award and LLS Scholar Award to E.P., and supported in part through the NIH/NCI Cancer Center Support Grant P30CA013696 to CUIMC. The funders of this work had no role in study

design, data collection and analysis, decision to publish or preparation of the manuscript. Clipart in figures was created using BioRender. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Columbia Stem Cell Initiative, Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, USA Carl A. Mitchell, Evgenia V. Verovskaya, Oakley C. Olson, 

James W. Swann, Paul V. Dellorusso & Emmanuelle Passegué * The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Medicine, Division

Hematology/Oncology, University of California San Francisco, San Francisco, CA, USA Evgenia V. Verovskaya, Aurélie Hérault, Si Yi Zhang, Arthur Flohr Svendsen, Eric M. Pietras, Sietske T.

Bakker, Theodore T. Ho & Emmanuelle Passegué * Wellcome and MRC Cambridge Stem Cell Institute, Department of Haematology, Jeffrey Cheah Biomedical Centre, Cambridge University,

Cambridge, UK Fernando J. Calero-Nieto, Xiaonan Wang & Berthold Göttgens Authors * Carl A. Mitchell View author publications You can also search for this author inPubMed Google Scholar *

Evgenia V. Verovskaya View author publications You can also search for this author inPubMed Google Scholar * Fernando J. Calero-Nieto View author publications You can also search for this

author inPubMed Google Scholar * Oakley C. Olson View author publications You can also search for this author inPubMed Google Scholar * James W. Swann View author publications You can also

search for this author inPubMed Google Scholar * Xiaonan Wang View author publications You can also search for this author inPubMed Google Scholar * Aurélie Hérault View author publications

You can also search for this author inPubMed Google Scholar * Paul V. Dellorusso View author publications You can also search for this author inPubMed Google Scholar * Si Yi Zhang View

author publications You can also search for this author inPubMed Google Scholar * Arthur Flohr Svendsen View author publications You can also search for this author inPubMed Google Scholar *

Eric M. Pietras View author publications You can also search for this author inPubMed Google Scholar * Sietske T. Bakker View author publications You can also search for this author

inPubMed Google Scholar * Theodore T. Ho View author publications You can also search for this author inPubMed Google Scholar * Berthold Göttgens View author publications You can also search

for this author inPubMed Google Scholar * Emmanuelle Passegué View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.V.V. initiated the studies

and contributed to most of the experiments with help from A.H. for BM fluid isolation and immunophenotyping. S.Y.Z. performed GMP cluster analyses and ELISA assays. A.F.S. performed

immunofluorescence and whole-mount staining. S.T.B. performed initial 5-FU analyses. T.T.H. performed cell collection and provided technical assistance. C.A.M. re-analysed all the generated

data, contributed to various stromal and molecular analyses and, with help from O.C.O., J.W.S. and P.V.D, completed the chronic IL-1β exposure studies and performed all the analyses of

_Il1r1_-deficient mice. F.J.C-N., X.W. and B.G. prepared and analysed the scRNA-seq samples. E.M.P. prepared the microarray samples. E.V.V. and E.P. designed the initial experiments, and

C.A.M. and E.P. revised the experiments. E.V.V., C.A.M. and E.P. wrote and edited the manuscript. C.A.M. and E.P. handled the resubmission. CORRESPONDING AUTHOR Correspondence to Emmanuelle

Passegué. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks Iannis Aifantis and the other,

anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 GROSS ANALYSIS OF THE REMODELED OLD BM CAVITY. A, H&E staining of humeri and sterna of 2 individual

young and old mice. Scale bar, 100 µm. B, TPO and TGF-β levels in young and old BM fluids. Results are from 2 independent cohorts. C, μCT analyses of young and old tibia with representative

images of cortical and trabecular regions (left) and quantification of bone volume/total volume (BV/TV) and connectivity density (right). D, Representative image of bone lining ALCAM+

osteoblasts immunofluorescence staining in young and old mice. Representative of 3 independent experiments. Scale bar, 100 µm. E, Representative images and quantification of

immunofluorescence staining of vascular volume (laminin) and vascular leakage by dragon-green beads (DGB) diffusion assay in young and old BM. Scale bar, 50 µm. F, Representative images and

quantification by flow cytometry of DGB endocytosis in young and old marrow SEC; scale bar, 5 µm. Results are from 3 independent cohorts. G, Von Kossa staining in young and old endosteal

MSC-S. H, Experimental scheme for the indicated co-culture experiments showing the effects of young or old BM cells on young MSC-S (top right), and young BM cells on young and old MSC-S

(bottom). I, Frequency of endosteal and marrow mesenchymal populations in young and middle age (13-month-old) mice with changes in CFU-F from endosteal OLCs (bottom right). Data are means ±

S.D; P-values were obtained by two-tailed Welch’s t-test without adjustment for multiple comparisons. Source data EXTENDED DATA FIG. 2 MOLECULAR FEATURES OF OLD MESENCHYMAL POPULATIONS. A,

UMAP visualization of the entire plate-based scRNAseq dataset of niche populations of mesenchymal and endothelial populations shown in Fig. 2a. B, Global changes in MSC-L gene identity in

clusters M2 vs. M1. C, Representative flow cytometry staining (top) and quantification of LepR and PDGF-Rα levels (bottom) in young and old MSC-L. Results are from 6 independently analyzed

groups of 1 or 2 young or old mice. D, HSC niche factors with violin plots representation of _Kitl_ and _Cxcl12_ expression in the indicated young and old mesenchymal populations (left) and

SCF and SDF1α levels in young and old BM fluids (right). E, GSEA results for Hallmark biological processes significantly affected in old MSC-L-like, OPr-like and MSC-S-like groups (FDR < 

0.05). Data are means ± S.D. except for (b) and (d); P-values were obtained by two-tailed Student’s t-test without adjustment for multiple comparisons (b), or by Welch’s t-test without

adjustment for multiple comparisons (c,d). Source data EXTENDED DATA FIG. 3 MOLECULAR FEATURES OF OLD ENDOTHELIAL POPULATIONS AND FURTHER CHARACTERISTICS OF THE OLD NICHE. A, ICGS output of

young and old endothelial populations from the plate-based scRNAseq dataset shown in Fig. 2a with 7 clusters of cells (E1 to E7, horizontal) defined according to the expressing pattern of

the 7 clusters of genes (a to g, vertical). Examples included in gene clusters a to g are shown. Star, contaminating mesenchymal/endothelial doublets; P, pericytes; A, arterioles; T,

transition vessels.Color scheme is based on normalized gene expression level from the indicated minimum(blue) to maximum (yellow) value in the scale. B, Ingenuity Pathway Analysis (IPA)

canonical pathways enriched in old AEC-like and SEC-like groups (Z-score > 1; p < 0.01). C, Characteristic expression patterns for the indicated genes in the droplet-based scRNAseq

dataset of young and old endosteal and central marrow stromal fractions shown in Fig. 2c. 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 0 (gray) to the indicated maximum value in the scale (red). Source data EXTENDED DATA FIG. 4 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-β-gal staining in control

irradiated mouse embryonic fibroblasts (MEF) and isolated young and old MSC-S and OLC. Representative of 3 independent experiments. Scale bar, 20 µm. C, Absolute expression of _Il1b_ in

different mature hematopoietic cell types and unfractionated 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; 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, granulocyte. 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; Pre-GM, pre-granulocyte/macrophage; Pre-MegE, pre-megakaryocyte/erythrocyte; MkP, megakaryocyte progenitor. G, Representative staining and

quantification of young and old HSC markers. 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. Cells in the UMAP were

colored according to the expression levels of the indicated genes. I, IPA Upstream Regulators analysis of young and old populations from the droplet-based scRNAseq young and old LK/LSK

dataset filtered on cytokines and growth factors. Data are means ± S.D. except for (g) where box ranges from 25th to 75th percentile with center line at the median, and whiskers range from

minima to maxima; P-values were obtained by two-tailed Welch’s t-test without adjustment for multiple comparisons. Source data EXTENDED DATA FIG. 5 ALTERED FUNCTIONALITY OF OLD HSCS AND

MPPS. A, Colony formation in methylcellulose for young (Y) and old (O) HSCs, MPP3 and MPP4. Results are from 2 independent experiments. Mix: all lineages; GM: granulocyte/macrophage; G(or)M:

granulocyte (or) macrophage; MegE: megakaryocyte/erythrocyte; CFU: colony-forming units. B, Myeloid differentiation in liquid culture for young and old MPP3 with quantification (right) of

immature Sca-1+/c-Kit+ cells (left) and mature Mac-1+/FcγR+ macrophage (right). C, Representative flow cytometry staining of CD19+ lymphoid vs. Mac-1+ myeloid differentiation in OP9 + IL7

culture conditions for young and old HSCs, MPP3 and MPP4. Results are representative of 3 independent experiments. D, Representative histograms of CFSE staining of cultured young and old

HSCs, MPP3 and MPP4. Results are representative of 3 independent experiments. E, Cleaved caspase 3/7 (CC3/7) activity in cultured young and old HSCs, MPP3 and MPP4. F, Short-term lineage

tracking following transplantations of young and old HSCs, MPP3 and MPP4 in sub-lethally irradiated recipients with experimental scheme (left) and quantification of overall blood donor

chimerism (top graphs) and myeloid chimerism among donor cells (bottom graphs). Results are from 3 independent cohorts. Data are means ± S.D. except for (f) (± S.E.M.); P-values were

obtained by two-tailed Welch’s t-test without adjustment for multiple comparisons (a, d, f), or by two-tailed Student’s t-test without adjustment for multiple comparisons (b). Source data

EXTENDED DATA FIG. 6 IMPROVED AGING FEATURES WITH IL-1 SIGNALING BLOCKADE. A–C, Short-term blockade of IL-1 signaling upon Anakinra (Ana) treatment in young (Y) and old (O) mice with: (a)

experimental scheme; (b) changes in HPSC frequency; and (c) engraftment over time (left) and lineage reconstitution (right) at 4 months (4 mo) post-transplantation (Tplx) of the indicated

HSC populations. Results are from 3 independent cohorts of young and old mice injected with either PBS or Anakinra, with HSCs isolated from the pooled BM of mice from the same treatment

group and transplanted into 3 to 5 recipients, each. D, E, Additional characterization of the effects of Anakinra blockade of IL-1 signaling during 5FU-mediated regeneration in young and old

mice with: (d) changes in IL-1α, IL-1β and MIP1α levels in BM fluids; and (e) platelet (Plt) levels in peripheral blood. Results are from 3 independent cohorts started with 15 young and 11

old mice treated once with 5FU, injected daily with either PBS or Anakinra, and analyzed at day 12 post-5FU treatment. F, GSEA results for Hallmark biological processes significantly

enriched in MSC-L2 vs. MSC-L1 groups (FDR < 0.05). Data are from the droplet-based scRNAseq analyses of endosteal and central marrow stromal fractions in young (n = 2) and old (n = 2)

_Il1r1__+/+_ wild type (WT) mice and old (n = 1) _Il1r1__−/−_ mice shown in Fig. 7b. G, H, Droplet-based scRNAseq analyses of Lin−/c-Kit+ (LK) and Lin−/Sca-1+/c-Kit+ (LSK) BM fractions

isolated from young (n = 2) and old (n = 2) _Il1r1__+/+_ wild type (WT) and old (n = 1) _Il1r1__−/−_ mice with (g) UMAP visualization and (h) quantification of percent of HSPCs and

progenitors. Data are means ± S.D. except for engraftment results shown in (c) (± S.E.M.); P-values were obtained by one-way Anova adjusted for multiple comparisons using the Holm-Šídák

method. Source data EXTENDED DATA FIG. 7 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, not 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 2 × 106 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-values were obtained by Kolmogorov-Smirnov test

(a,b), by one-way Anova adjusted for multiple comparisons using the Holm-Šídák method (c,i), or by two-tailed Student’s t-test without adjustment for multiple comparisons (f, g, h). Source

data EXTENDED DATA FIG. 8 SCHEMATIC OF THE CROSSTALK BETWEEN THE BM NICHE AND HEMATOPOIETIC SYSTEM DURING PHYSIOLOGICAL AGING. In youth, HSCs reside primarily in the central marrow where

they are maintained by peri-sinusoidal MSC-L and produce a balanced output of all mature cell lineages (Mk, megakaryocytes; Ery, erythrocytes, My, myeloid cells; Ly, lymphoid cells).

Abundant peri-arteriolar MSC-S at the endosteum efficiently produce OPr cells that support osteoblast development, ECM deposition and bone formation. With age, numerical loss and functional

decline of MSC-S and OPr leads to bone thinning, with the remaining OPr constitutively producing IL-1. Chronic IL-1, in turn, reinforces niche degradation at the endosteum and contributes to

dysfunction of the sinusoidal vasculature. Chronic IL-1 also acts in trans on central marrow MSC-L and HSPCs, driving the appearance of an inflammatory iMSC-L subset and steady-state

engagement of emergency myelopoiesis (EM) programs with GMP cluster (cGMP) formation. Strikingly, acute IL-1 blockade with Anakinra enables more youthful blood production during 5FU-mediated

regeneration, and life-long removal of IL-1 signaling in _Il1r1__−/−_ mice maintains MSC-L in a more youthful cell state associated with improved blood production and HSC function.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figs. 1–6 REPORTING SUMMARY SUPPLEMENTARY TABLES Supplementary Table 1: ICGS of young and old niche cells. Plate-based scRNA-seq

gene expression data of mesenchymal and ECs were separated and subjected to unsupervised single-cell population identification using ICGS. The relative expression of every guide gene in

each cluster, as well as the relative expression of every guide gene in each pooled group of clusters corresponding to the indicated cell types, was calculated by averaging the relative

expression of each gene across individual cells within a cluster or group. Supplementary Table 2: DEGs and pathway analyses of young versus old niche cells. Plate-based scRNA-seq gene

expression data between young and old cells in pooled identity groups as defined by ICGS were analysed for DEGs using the DESeq2 package. Overlap with Hallmark gene sets was evaluated, and

significant (FDR < 0.05) overlaps are indicated in bold for mesenchymal populations. As no significant overlaps were observed for endothelial populations, IPA software (Qiagen) was used

to determine IPA canonical pathways with absolute _z_-score ≥ 1 and –log10(_P_ value) ≥ 2 enriched in young and old AEC-like and SEC-like cells. Supplementary Table 3: Quantibody array-based

measurements of cytokine concentration in young and old BM fluids. BM fluids isolated from young (10 weeks of age) and old (27–29 months of age) mice (_n_ = 5) were analysed using

Quantibody Testing Service (Raybiotech). Samples were diluted fourfold before analyses. Results are mean ± s.d. and are expressed as pg ml–1 concentration; *_P_ < 0.05, **_P_ _<_ 0.05,

***_P_ < 0.05. Supplementary Table 4: DEGs and pathway analyses of young versus old HSCs. SAM was performed on young and old cells within each population to determine SAM delta scores.

The top 1,000 most highly DEGs for each group were collected. SAM scores for these genes were used for GSEA and overlap with Reactome gene sets was evaluated for each population. The top 5

statistically significant (FDR < 0.05) overlaps for each group are in bold. SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA

FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA EXTENDED DATA

FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4

Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 7 Statistical

source data. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mitchell, C.A., Verovskaya, E.V., Calero-Nieto, F.J. _et al._ Stromal niche inflammation

mediated by IL-1 signalling is a targetable driver of haematopoietic ageing. _Nat Cell Biol_ 25, 30–41 (2023). https://doi.org/10.1038/s41556-022-01053-0 Download citation * Received: 09

November 2021 * Accepted: 15 November 2022 * Published: 17 January 2023 * Issue Date: January 2023 * DOI: https://doi.org/10.1038/s41556-022-01053-0 SHARE THIS ARTICLE Anyone you share the

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