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Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal

Nature Cell Biology volume 18, pages 607–618 (2016)Cite this article

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Abstract

Haematopoietic stem cells (HSCs) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory ‘emergency’ signal, directly accelerates cell division and myeloid differentiation of HSCs through precocious activation of a PU.1-dependent gene program. Although this effect is essential for rapid myeloid recovery following acute injury to the bone marrow, chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges such as transplantation. Importantly, these damaging effects are transient and fully reversible on IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and is likely to underlie deregulated blood homeostasis in chronic inflammation conditions.

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Figure 1: IL-1 accelerates HSC differentiation along the myeloid lineage.
Figure 2: IL-1 induces precocious activation of a PU.1 gene program in HSCs.
Figure 3: PU.1 activation requires direct and sustained IL-1R signalling.
Figure 4: IL-1 enforces HSC myeloid differentiation in vivo.
Figure 5: IL-1 accelerates myeloid regeneration following acute BM injury.
Figure 6: Chronic IL-1 exposure forces myeloid cell production at the expense of HSC engraftment.
Figure 7: Chronic IL-1 exposure impairs HSC self-renewal.
Figure 8: IL-1 effects are reversible on withdrawal.

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Change history

  • 28 April 2016

    In the version of this Article originally published online, in the Acknowledgements section, the NIH grant number 'K01 DK09831' should have read 'K01 DK098315'. This has been corrected in all versions of the Article.

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Acknowledgements

We thank M. Kissner and M. Lee for management of our Flow Cytometry Core Facility and members of the Passegué laboratory for insights and suggestions. E.M.P. was supported by NIH F32 HL106989 and K01 DK098315, C.M.-B. by an Institute of Health Carlos III (ISCIII) fellowship, R.L. by a CIRM Bridges internship and M.G.M. by the Swiss National Science Foundation (310030_146528/1). E.P. and research were supported by a LLS Scholar Award and NIH R01 HL092471.

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  1. Eric M. Pietras

    Present address: Division of Hematology, University of Colorado Denver, Aurora, Colorado, 80045, USA

Authors and Affiliations

  1. Department of Medicine, Division of Hematology/Oncology, The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California 94143, USA

    Eric M. Pietras, Cristina Mirantes-Barbeito, Sarah Fong, SiYi Zhang, Ranjani Lakshminarasimhan, Chih Peng Chin, José-Marc Techner & Emmanuelle Passegué

  2. Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland

    Dirk Loeffler & Timm Schroeder

  3. Division of Hematology, University Hospital and University of Zurich, 8091, Zurich, Switzerland

    Larisa V. Kovtonyuk & Markus G. Manz

  4. Department of Cell Biology, Albert Einstein Medical College, Queens, New York, 10461, USA

    Britta Will & Ulrich Steidl

  5. Weatherall Institute of Molecular Medicine, Oxford University, Oxford, OX3 9DS, UK

    Claus Nerlov

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Contributions

E.M.P. performed all of the experiments with assistance from C.M.-B. and S.F. D.L. helped with the single-cell continuous tracking experiments that were performed in T.S. laboratory. R.L. helped with the Il1r1−/− and 5-FU analyses, J.-M.T. with the in vitro cultures, C.P.C. with analysis of single-cell tracking experiments, L.V.K. and M.G.M. with the in vivo CFSE dilution experiments, B.W. and U.S. with the PU.1ΔURE experiments, and C.N. provided PU.1–EYFP reporter mice. E.M.P., C.M.-B., S.F. and E.P. designed the experiments and interpreted the results. E.M.P. and E.P. wrote the manuscript.

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Correspondence to Eric M. Pietras or Emmanuelle Passegué.

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Integrated supplementary information

Supplementary Figure 1 Gating strategy and effect of IL-1 on progenitor cell proliferation and survival.

(a) Gating strategy and representative FACS plots of stained c-Kit-enriched wild-type (WT) BM cells. MPP2 and MPP3 are often isolated as a combined MPP2/3 population for experiments. (b) Il1r1 expression in the indicated populations. Results are dye intensity levels from microarray analyses30. (n = 5 MPP4, n = 4 HSC and GMP, remainder n = 3). (c) Representative expansion in liquid culture in one of two independent experiments performed in triplicate. (d) Cleaved caspase-3 (CC3) activity expressed as relative luminescence units, RLU) after 12 hours culture in one of two independent experiments performed in triplicate (e) Experimental design and quantification of BrdU incorporation after 24 hours culture in one of two independent experiments performed in triplicate. (f) Mean absolute numbers of the indicated phenotypic cell populations in HSC cultures over time in two independent experiments. Source data for b are shown in Supplementary Table 1. Data are means, and shown in b as means ± S.D. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 2 Effect of IL-1 on progenitor cell differentiation.

(a,b) Myeloid differentiation in cultured MPP2/3, MPP4 and GMPs: (a) representative FACS plots, and (b) quantification of surface marker expression (n = 4 biological replicates/group). (c) Single cell clonogenic colony forming unit (CFU) assays in methylcellulose (n = 60 cells/group). Colonies were scored after 7 days. MkE: megakaryocyte/erythrocyte; M: macrophage; G: granulocyte; GM: granulocyte/macrophage; GMMkE: mix colony. (d) Representative colony-type (scale bar, 100 μm) and colony-morphology (scale bar, 10 μm; arrows indicate macrophages). (e) Representative FACS plots of myeloid differentiation in cultured HSCs and GMPs (n = 3 biological replicates). Source data for b are shown in Supplementary Table 1. Data in b are means ± S.D.; ∗p ≤ 0.05; ∗∗∗p ≤ 0.001. P-values in b were determined by Mann-Whitney u test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 3 PU.1 activation by IL-1.

(a,b) qRT-PCR analyses of myeloid lineage genes the indicated populations after 12 hours culture (n = 4 biological replicates/group for HSC, MPP4 and GMP, n = 3 for MPP2/3): (a) experimental design, and (b) expression of individual genes. Results are expressed as fold changes compared to levels in each-IL-1β population (set to 0). (c) PU.1 levels in individual PU.1-eYFP HSCs at 0 and 15 hours culture (n = 140 and 186 individual HSC/group). Results are expressed in arbitrary units (AU) with box plots showing median (lines) and 10–90th percentile (whiskers). (d) Division kinetics of PU.1-eYFP HSCs from Fig. 1e re-analysed based on pre-division PU.1lo (≤4 AU) and PU.1hi (>4 AU) expression levels. (e) Experimental design, representative histograms and quantification of BrdU incorporation in HSCs after 24 hours culture (n = 3 biological replicates/group). (f) PU.1 levels in PU.1-eYFP HSCs after 12 hours culture with the indicated concentrations of IL-1β. (g) Representative histograms showing PU.1 levels in PU.1-eYFP HSCs after 12 hours culture ± IL-1β (25 pg/ml) and the indicated inhibitors. Results with Il1r1−/−::PU.1-eYFP HSCs are shown as negative controls. (h) Validation of the blocking activity of the anti-M-CSFR antibody (αMR) in BM cells cultured for 5 days? M-CSF (M, 100 ng/ml). Source data for b and e are shown in Supplementary Table 1. Data are means ± S.D.; ∗p ≤ 0.05; ∗∗∗p ≤ 0.001. P-values in b and e were determined by paired Student’s t test, and in c,d by one-way ANOVA with Tukey’s test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 4 Gating strategies and effect of acute in vivo IL-1 treatment.

(a–c) Gating strategies with representative FACS plots for the different hematopoietic stem, progenitor and mature cell populations analysed in peripheral blood (PB) and bone marrow (BM): (a) PB of mice injected ± IL-1β for 20 days, (b) and (c) BM of mice injected ± IL-1β for 1 day. Number in each gate indicates population frequency. (d) BM cellularity and size of the indicated BM populations in mice injected ± IL-1β for 1 day (n = 15 −IL-1β and 13 +IL-1β mice/group). (e) Numbers of HSCLT and MPP1 in Il1r1+/+ and Il1r1−/− mice in mice injected ± IL-1β for 1 day (n = 15 −IL-1β and +IL-1β Il1r1+/+ mice, 5 −IL-1β and 6 +IL-1β Il1r1−/− mice/group) Data are means ± S.D.; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. P-values in d and e were determined by Mann-Whitney u test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 5 In vivo HSC division tracking and specificity of PU.1 activation.

(a) Representative FACS plots showing CFSE dilution in donor cells following 7 days treatment of the recipient mice ± IL-1β. (b) Gating strategy for identification of HSCLT in CFSE-labelled donor cells. (c) PU.1 levels in the indicated populations from PU.1-eYFP mice injected ± IL-1β for 1 or 20 days (n = 4 −IL-1β and 5 +IL-1β_ mice/group for day 1; n = 5 −IL-1β and 3 +IL-1β_ mice/group for day 20). Source data for c are shown in Supplementary Table 1. Data are means ± S.D.; ∗p ≤ 0.05. P-values in c were determined by Mann-Whitney u test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 6 Cytokine production following 5-FU myeloablation, identification of IL-1 producing cells and haematopoiesis in Il1r1−/− mice.

(a) Cytokine levels in BM plasma (top) and blood serum (bottom) of 5-FU-treated mice (mice/group: n = 7 day 0–8; n = 8 day 10; n = 6 day 12; n = 3 day 14; for M-CSF n = 4 day 0–12; n = 3 day 14). (b) Experimental design and gating strategy used to isolate BM stromal ECs, MSCs and OBCs, and hematopoietic granulocytes (Gr), monocytes (Mon), macrophages (MΦ), B cells and CD4 + T cells. Representative FACS plots of untreated d0 mice are shown. (c) Representative FACS plots and quantification of Ki67/DAPI cell cycle distribution in HSCs from Il1r1+/+ and Il1r1−/− mice (n = 4 Il1r1+/+ and 6 Il1r1−/− mice/group). (d) BM cellularity and size of the indicated BM populations in Il1r1+/+ and Il1r1−/− mice (n = 20 mice/group; for HSCLT and MPP1, n = 16 Il1r1+/+ and 13 Il1r1−/− mice/group) Source data for a are shown in Supplementary Table 1. Data are means ± S.D.; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. P-values in a were determined by one-way ANOVA with Dunnet’s test, and in c and d by Mann-Whitney u test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary Figure 7 Short-term in vitro and in vivo tracking of HSCs.

(a) Representative methylcellulose colony forming-unit (CFU) and cleaved caspase-3 (CC3; expressed as relative luminescence units: (RLU) assays. Results are representative of one of two independent experiments performed in triplicate. b-c, Terminal analyses of mice transplanted with HSCs exposed ± IL-1β for 20 days in donor mice and another 30 days in recipient mice shown in Fig. 6a-d): representative FACS plots showing donor chimerism, and myeloid (My) and lymphoid (Ly) donor lineage distribution in (b) PB and (c) BM HSCs 30 days post-transplant. (d) Representative FACS plots showing the frequency of GFP+ control and PU.1-transduced HSCs prior to transplantation (d0 input) and after 30 days post-transplant in the BM (d30). Data in a are shown as means.

Supplementary Figure 8 Functional analysis of IL-1-exposed HSCs.

(a–c) Limiting dilution analysis (LDA) of unfractionated BM cells from mice injected ± IL-1β for 20 days (106 cell dose: n = 4 −IL-1β and 4 +IL-1β mice/group; 105 cell dose: n = 4 mice/group; 104 cell dose: n = 4 −IL-1β and 3 +IL-1β mice/group): (a) experimental design, (b) LDA graph (values: estimated HSC frequency; solid lines: dose response curve; dashed lines: upper and lower confidence intervals), and (c) engraftment results at 16 weeks post-transplant, Mice with ≥0.5% donor chimerism were considered engrafted. (d–f) Analyses of mice injected ± IL-1β for 70 days n = 5 mice/group): (d) representative FACS plots and HSCLT number fractionated by CD41 expression (n = 4 −IL-1β and 5 +IL-1β mice/group), (e) size (means) of the indicated BM populations from two mice, and (f) methylcellulose colony forming-unit (CFU) assay in one experiment performed in triplicate. (g–i) Secondary transplantation (2° txpl) of HSCs re-isolated from primary transplanted mice (1° txpl, shown in Fig. 7d–f) reconstituted with HSCLT from mice injected ± IL-1β for 70 days (n = 5 mice/group): (g) experimental design, (h) donor chimerism and (i) lineage distribution in PB over time. Source data for b–d are shown in Supplementary Table 1. Data are means ± S.D. except when indicated. P-values in b were determined using Poisson statistical analysis, in c,d,h and i by Mann-Whitney u test. Exact P-values, number of replicates used to derive statistical data (n) and statistical tests used are shown in Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2632 kb)

Supplementary Table 1

Statistics source data. (XLSX 111 kb)

Supplementary Table 2

Exact P-values and statistical tests used in experiments. (XLSX 68 kb)

Supplementary Table 3

Antibodies used. (XLSX 51 kb)

Supplementary Table 4

Primers used for qRT-PCR and Fluidigm analysis. (XLSX 11 kb)

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Pietras, E., Mirantes-Barbeito, C., Fong, S. et al. Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal. Nat Cell Biol 18, 607–618 (2016). https://doi.org/10.1038/ncb3346

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