Sleep modulates haematopoiesis and protects against atherosclerosis

Abstract

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin—a stimulatory and wake-promoting neuropeptide—in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

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Fig. 1: Sleep fragmentation aggravates atherosclerosis, increases haematopoiesis and decreases production of hypocretin in the hypothalamus.
Fig. 2: Hypocretin suppresses haematopoiesis and atherosclerosis.
Fig. 3: Hypocretin controls production of CSF1 by pre-neutrophils in the bone marrow.
Fig. 4: Haematopoietic CSF1 deletion or hypocretin supplementation protects against sleep fragmentation-induced haematopoiesis and atherosclerosis.

Data availability

All relevant data are included in the paper and its Supplementary Information. Source Data for Figs. 14 are available in the online version of the paper.

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Acknowledgements

This work was supported in part by NIH grants R35 HL135752, R01 HL128264, P01 HL131478, an American Heart Association Established Investigator Award, and the Patricia and Scott Eston MGH Research Scholar (to F.K.S.); NIH grant R35 HL139598 (to M.N.); Swiss National Science Foundation grants 31003A_125323 and 31003A_144282 (to A.V.); a CIHR postdoctoral fellowship and a Banting postdoctoral fellowship (to C.S.M.); the doctoral program Cell Communication in Health and Disease (CCHD) funded by the Austrian Science Fund (to M.G.K.); a Swedish Research Council postdoctoral fellowship (to S.R.); an American Heart Association postdoctoral fellowship (to S.H.); a postdoctoral fellowship from the Fondation pour la Recherche Medicale (to C.V.); the German Research Foundation (DFG; 331536185 to F.K. and 398190272 to W.C.P.); and a Boehringer-Ingelheim-Fonds MD fellowship (to L.H.). We thank A. Lichtman for providing the PCSK9 adenovirus, D. Scadden for providing stromal cell reporter mice and K. Joyes for editing the manuscript.

Reviewer information

Nature thanks P. S. Frenette, V. Papayannopoulos, A. R. Tall and A. Yamanaka for their contribution to the peer review of this work.

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Authors

Contributions

C.S.M. conceived the project, designed and performed experiments, analysed and interpreted data, made the figures and wrote the manuscript; M.G.K. designed and performed experiments, and analysed and interpreted data; S.R., S.H., A.V., A.A., C.V., C.T.C., J.E.M., F.K., W.C.P., V.F., A.M.F., A.F.G., L.H., Y.I. and F.F.H. performed experiments; A.V., C.J.B., P.L., M.T., T.E.S. and M.N. provided intellectual input and edited the manuscript; A.V., M.T. and T.E.S. provided materials; F.K.S. conceived the project, designed experiments, interpreted data and wrote the manuscript.

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Correspondence to Filip K. Swirski.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Effects of sleep fragmentation on metabolic and cellular parameters.

a, Image of a sleep fragmentation cage. b, Body weight (n = 10 per group). c, Plasma cholesterol at ZT3 (n = 5 per group). d, Plasma glucose at ZT3 (n = 5 per group). e, Glucose tolerance test (GTT) beginning at ZT3 (light period) and ZT12 (dark period) (n = 4 per group). fh, Apoe/ mice were placed in sleep fragmentation chambers where the sweep bar operated during the dark period (ZT12–0) when mice are normally awake. Control mice were maintained in sleep fragmentation chambers with a stationary sweep bar. f, Assessment of atherosclerosis and lesion area (n = 5 per group). g, Assessment of blood Ly-6Chigh monocytes and neutrophils (n = 5 per group). h, Assessment of bone marrow LSK cells and proliferation (n = 5 per group). i, Aortic macrophage proliferation in Apoe/ and Apoe/ SF mice after 16 weeks of sleep fragmentation at ZT3 and ZT14 (n = 5 Apoe/ mice; n = 4 Apoe/ SF mice). j, Quantification of B cells, CD4+ T cells and CD8+ T cells in the blood of Apoe−/− and Apoe−/− SF mice at ZT3 (n = 10 Apoe/ mice; for B and CD4 T cells, n = 6 Apoe/ SF mice and for CD8 T cells, n = 7 Apoe/ SF mice). k, Quantification of B cells, CD4+ T cells and CD8+ T cells in the spleen of Apoe−/− and Apoe−/− SF mice at ZT3 (n = 10 Apoe/ mice; n = 7 Apoe/ SF mice). l, Quantification of B cells in the bone marrow of Apoe−/− and Apoe−/− SF mice at ZT3 (n = 10 Apoe−/− mice; n = 7 Apoe/ SF mice). Data are mean ± s.e.m.

Extended Data Fig. 2 Sleep and circadian migration of leukocytes.

ad, Quantification of Ly-6Chigh monocytes and neutrophils at ZT3 and ZT14 in the spleen (a), bone marrow (b), lung (c) and liver (d) of Apoe−/− mice and Apoe−/− mice after 16 weeks of sleep fragmentation. Group sizes are indicated in the figure. Data are mean ± s.e.m., *P < 0.05, **P < 0.01, two-way ANOVA.

Extended Data Fig. 3 Sleep-mediated haematopoiesis and extramedullary haematopoiesis.

a, Gating strategy and quantification of haematopoietic progenitor cells at ZT3 in the bone marrow (n = 10 Apoe−/− mice, except for GMPs, n = 11 mice; n = 10 Apoe/ SF mice, except MPP3 and MPP4, n = 9 mice). LtHSCs, long-term haematopoietic stem cells; StHSC, short-term haematopoietic stem cells. b, Gating strategy and quantification of haematopoietic progenitor cells at ZT3 in the spleen (n = 9 Apoe−/− mice, except CMPs, n = 10 mice; n = 9 Apoe−/− SF mice). c, C57BL/6 wild-type mice that were fed a regular chow diet were subjected to sleep fragmentation for 16 weeks after which Ly-6Chigh monocytes, neutrophils and LSK cells were quantified at ZT3 (n = 8 wild-type mice, except n = 9 mice for spleen Ly-6Chigh monocytes, n = 5 mice for bone marrow neutrophils, n = 10 mice for bone marrow LSK cells; n = 4 wild-type SF mice, except n = 9 mice for bone marrow LSK cells). Data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney U-tests.

Extended Data Fig. 4 Sleep fragmentation does not alter bone structure and does not depend on the microbiome.

a, b, μCT analysis of trabeculae (a) and cortical bone structure (b) of Apoe−/− mice and Apoe−/− mice after 16 weeks of sleep fragmentation. The bone volume fraction (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), structural model index (SMI), cortical tissue mineral density (Ct.TMD), cortical area (Ct.Ar), total area (T.Ar), cortical thickness (Ct.Th) and cortical porosity (Ct.Porosity) were analysed (n = 9 per group). c, d, Analysis of leukocytosis in SF (c) and Hcrt−/− (d) mice at ZT3 after receiving a cocktail of antibiotics in drinking water for 4 weeks (n = 3 per group). Data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney U-tests.

Extended Data Fig. 5 Sleep fragmentation does not activate the peripheral sympathetic nervous system but has effects on hypothalamic gene transcription and food consumption.

a, Plasma corticosterone levels in Apoe−/− mice and Apoe−/− mice after 16 weeks of sleep fragmentation at ZT3 and ZT14 (n = 4 mice per group, except n = 5 Apoe−/− mice at ZT3). b, Systolic and diastolic blood pressure at ZT3 (n = 4 mice per group). c, Immunohistochemical analysis and quantification of tyrosine hydroxylaze (TH) staining in the bone marrow of Apoe−/− mice, Apoe−/− SF mice and Apoe−/− mice subjected to 3 weeks of chronic variable stress (n = 4 Apoe−/− mice; n = 4 Apoe−/− SF mice; n = 3 Apoe−/− stressed mice). d, Quantification at ZT3 of blood Ly-6Chigh monocytes and neutrophils, bone marrow LSK cells and proliferation in Apoe−/− mice and Apoe−/− SF mice after antagonism of the β3 receptor for 4 weeks (n = 3 Apoe−/− mice treated with β3 blocker; n = 4 Apoe−/− SF mice treated with β3 blocker). e, Quantification of time in outer zone during open field test (n = 9 Apoe−/− mice; n = 8 Apoe−/− SF mice). f, Quantification of time spent in light box during light–dark box test (n = 6 per group). g, Quantification of time in new arm during Y-maze test (n = 8 Apoe−/− mice; n = 5 Apoe−/− SF mice). h, Analysis of neuropeptide expression in the hypothalamus at ZT3 (n = 5 Apoe−/− mice, except n = 6 for Pmch, Tph2, Gad1and Npy; n = 5 Apoe−/− SF mice, except n = 4 for Npy). i, Neuropeptide receptor expression in the hypothalamus at ZT3 (n = 6 Apoe−/− mice, except Hcrtr1, n = 10 mice; n = 5 Apoe−/− SF mice, except Hcrtr2, n = 6 mice). j, Circadian gene expression in the hypothalamus at ZT3 and ZT14 (n = 3 Apoe−/− mice; n = 4 Apoe−/− SF mice). k, Mouse food consumption during the course of sleep fragmentation (n = 4 Apoe−/− mice at ZT3, except n = 6 Apoe−/− mice on HFD for 16 weeks at ZT3; n = 4 Apoe−/− SF mice at ZT3, except n = 6 Apoe−/− SF mice on HFD for 16 weeks at ZT3; n = 5 Apoe−/− mice at ZT14, except n = 4 Apoe−/− mice on HFD for 10 weeks at ZT14 and n = 6 Apoe−/− mice on HFD for 16 weeks at ZT14; n = 5 Apoe−/− SF mice at ZT14, except n = 4 Apoe−/− SF mice on HFD for 10 weeks at ZT14 and n = 6 Apoe−/− SF mice on HFD for 16 weeks at ZT14). Data are mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed Mann–Whitney U-tests. ns, not significant.

Extended Data Fig. 6 Hypothalamic expression of hypocretin and dynorphin.

a, Hypothalamic expression of hypocretin and quantification of blood Ly-6Chigh monocytes and neutrophils in Apoe−/− mice after 6, 8 and 12 weeks of sleep fragmentation (for Hcrt, n = 4 Apoe−/− mice, except n = 5 for Apoe−/− mice after 12 weeks; for Hcrt, n = 4 Apoe−/− SF mice; for blood cells at 6 weeks, n = 5 mice per group; for blood cells at 8 weeks, n = 4 mice per group; for blood cells at 12 weeks, n = 9 mice per group). b, Sections of the hypothalamus stained for dynorphin and hypocretin. c, Quantification of hypothalamic dynorphin+ cells per high-powered field of view (n = 5 Apoe−/− mice; n = 4 Apoe−/− SF mice, of two independent experiments). d, e, Dynorphin (Pdny) mRNA expression in the hypothalamus of SF mice (d; n = 6 Apoe−/− mice; n = 5 Apoe−/− SF mice) and Hcrt−/− mice (e; n = 4 wild-type mice; n = 5 Hcrt−/− mice). f, TUNEL staining of hypothalamic sections from Apoe−/− and Apoe−/− SF mice (representative of four biological replicates) along with a positive control of TUNEL-stained myocardium 1 day after myocardial infarction (MI) (n = 1). gi, Apoe−/− mice were sleep-fragmented for 16 weeks then allowed to recover and sleep normally for 10 weeks. Control mice slept normally for 26 weeks. g, Analysis of hypothalamic hypocretin expression (n = 5 Apoe−/− mice; n = 4 Apoe−/− SF mice). h, Blood Ly-6Chigh monocytes and neutrophils (n = 5 mice per group). i, Quantification of bone marrow LSK cells and proliferation of LSK cells (n = 5 mice per group). Data are mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed Mann–Whitney U-tests.

Extended Data Fig. 7 Haematopoiesis in hypocretin-deficient mice.

ac, Quantification of the number of leukocytes in wild-type and Hcrt−/− mice at ZT3 in blood (a; n = 5 mice per group), spleen (b; for Ly-6Chigh monocytes and neutrophils, n = 7 wild-type mice and n = 8 Hcrt−/− mice; for B, CD8+ T, and CD4+ T cells, n = 5 wild-type mice and n = 6 Hcrt−/− mice) and bone marrow (c; for Ly-6Chigh monocytes and neutrophils, n = 7 wild-type mice and n = 8 Hcrt−/− mice; for B and T cells, n = 5 wild-type mice and n = 6 Hcrt−/− mice; for CMPs, GMPs and MDPs, n = 7 wild-type mice and n = 8 Hcrt−/− mice; for LSK populations, n = 5 wild-type mice and n = 6 Hcrt−/− mice). Data are mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney U-tests.

Extended Data Fig. 8 Hypocretin and hypocretin receptor-1 expression and production.

a, b, Relative Hcrt mRNA expression in tissues (a; n = 3) and sorted bone marrow cells (b; n = 4). c, Hcrt expression in the bone marrow and bone in Apoe−/− mice and in Apoe−/− mice subjected to sleep fragmentation for 16 weeks (n = 5 Apoe−/− mice; n = 4 Apoe−/− SF mice). d, Hypocretin-1 protein levels in cerebrospinal fluid (CSF), plasma and bone marrow (BM) fluid of wild-type and Hcrt−/− mice (n = 4 mice per group). e, Hypocretin-1 protein levels in the plasma and bone marrow fluid of Hcrt−/− mice 3 h after intra-cisterna magna (i.c.m.) injection of HCRT-1 or PBS. (n = 4 mice per group). f, Relative Hcrtr1 mRNA expression in tissues (n = 4 mice, except aorta and spleen, n = 3). g, Hcrtr2 expression in sorted bone marrow cells (n = 4 mice). h, Granulocyte–macrophage colony forming units (CFU-GM) from bone marrow cells of wild-type mice exposed to hypocretin-1 ex vivo in culture medium (n = 3 per group). i, Assessment of hypocretin receptor-1 protein in hypothalamus and sorted bone marrow neutrophils by western blot. Data are mean ± s.e.m.

Extended Data Fig. 9 Hypocretin, bone marrow neutrophils and HCRTR1.

a, Flow cytometry gating strategy for bone marrow pre-neutrophils, immature neutrophils and mature neutrophils. b, HCRTR1 (GFP) in bone marrow and blood neutrophils from WT;bmHcrtr1GFP/GFP mice. c, mRNA expression in cultured bone marrow pre-neutrophils exposed to LPS and/or HCRT-1 (for untreated n = 3 mice, except Mpo, n = 6 mice; for HCRT-1, n = 3 mice, expect Csf1, n = 4 mice and Mpo, n = 6 mice; for LPS, n = 3 mice except Csf1, n = 7 mice and Mpo, n = 6 mice; for LPS and HCRT-1, n = 3 mice, except Csf1, n = 11 mice, Csf2, n = 4 mice and Mpo, n = 6 mice). d, Csf1 expression in sorted bone marrow cells of wild-type and Hcrt−/− mice (n = 5 wild-type mice; n = 6 Hcrt−/− mice). e, Analysis of mRNA transcript expression in bone marrow leukocytes of Apoe−/− mice after 16 weeks of sleep fragmentation (for Apoe−/−, n = 5 mice, except Il10, Il34, Cxcl12, Csf3, n = 4 mice and Csf1, n = 9 mice; for Apoe−/− SF mice, n = 6 mice, except Il5, Il1β, Il6, Il34, Cxcl12, Csf3, n = 5, Il10, n = 5 mice and Csf1, n = 12). nd, not detected. f, Blood neutrophils in WT;bmHcrtr1GFP/GFP mice over 24 h (n = 3 per group). Data presented as mean ± s.e.m., **P < 0.01, one-way ANOVA.

Extended Data Fig. 10 Haematopoietic CSF1 deletion protects against haematopoiesis and atherosclerosis in hypocretin-deficient mice.

a, Schematic of chimeric models. b, c, Quantification of Ly-6Chigh monocytes and neutrophils in blood (b; n = 4 WT;bmWT mice; n = 6 Hcrt−/−;bmWT mice; n = 3 WT;bmCsf1−/− mice; n = 5 Hcrt−/−;bmCsf1−/− mice) and bone marrow (c; n = 4 WT;bmWT mice; n = 6 Hcrt−/−;bmWT mice; n = 3 WT;bmCsf1−/− mice; n = 5 Hcrt−/−;bmCsf1−/− mice). d, Quantification of the number of LSK cells (n = 4 WT;bmWT mice; n = 6 Hcrt−/−;bmWT mice; n = 3 WT;bmCsf1−/− mice; n = 5 Hcrt−/−;bmCsf1−/− mice) and proliferation (n = 4 WT;bmWT mice; n = 4 Hcrt−/−;bmWT mice; n = 3 WT;bmCsf1−/− mice; n = 5 Hcrt−/−;bmCsf1−/− mice) in bone marrow. e, Quantification of CMPs, GMPs and MDPs in chimeric mice (n = 4 WT;bmWT mice; n = 6 Hcrt−/−;bmWT mice; n = 3 WT;bmCsf1−/− mice; n = 5 Hcrt−/−;bmCsf1−/− mice). f, Bone marrow CSF1 levels (n = 4 WT;bmWT mice; n = 8 Hcrt−/−;bmWT mice; n = 4 WT;bmCsf1−/− mice; n = 7 Hcrt−/−;bmCsf1−/− mice). g, Schematic of chimeric models receiving Adv-PCSK9 and fed a HCD for 12 weeks. h, Plasma cholesterol levels (n = 7 WT;bmWT mice; n = 10 Hcrt−/−;bmWT mice; n = 5 WT;bmCsf1−/− mice; n = 6 Hcrt−/−;bmCsf1−/− mice). i, Images of cross-sections of aortic roots stained with oil red O and quantification of atherosclerosis in chimeric mice (n = 7 WT;bmWT mice; n = 9 Hcrt−/−;bmWT mice; n = 5 WT;bmCsf1−/− mice; n = 6 Hcrt−/−;bmCsf1−/− mice). Data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.

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Video 1

Video demonstrating the sleep fragmentation (SF) chambers used in the study (Lafayette Instrument, Lafayette, IN). The sweep bar moves along the bottom of the cage every 2 minutes gently waking the mice.

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McAlpine, C.S., Kiss, M.G., Rattik, S. et al. Sleep modulates haematopoiesis and protects against atherosclerosis. Nature 566, 383–387 (2019). https://doi.org/10.1038/s41586-019-0948-2

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