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Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain

Nature Medicine volume 28, pages 517–527 (2022)Cite this article

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Abstract

Hematopoietic stem cell transplantation (HSCT) is a therapy used for multiple malignant and nonmalignant diseases, with chemotherapy used for pretransplantation myeloablation. The post-HSCT brain contains peripheral engrafted parenchymal macrophages, despite their absence in the normal brain, with the engraftment mechanism still undefined. Here we show that HSCT chemotherapy broadly disrupts mouse brain regenerative populations, including a permanent loss of adult neurogenesis. Microglial density was halved, causing microglial process expansion, coinciding with indicators of broad senescence. Although microglia expressed cell proliferation markers, they underwent cell cycle arrest in S phase with a majority expressing the senescence and antiapoptotic marker p21. In vivo single-cell tracking of microglia after recovery from chemical depletion showed loss of their regenerative capacity, subsequently replaced with donor macrophages. We propose that HSCT chemotherapy causes microglial senescence with a gradual decrease to a critical microglial density, providing a permissive niche for peripheral macrophage engraftment of the brain.

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Fig. 1: HSCT causes the accumulation of brain parenchymal donor macrophages that become resident and surveillant.
Fig. 2: HSCT busulfan chemotherapy causes loss of host microglia cells and increase in individual microglial process area correlating with donor macrophage density.
Fig. 3: HSCT causes loss of adult neurogenesis and host microglial cell cycle arrest.
Fig. 4: Busulfan chemotherapy causes broad senescence in the brain.
Fig. 5: HSCT host microglial senescence and incapability to recover from PLX3397-induced depletion cause donor macrophage brain parenchymal engraftment.
Fig. 6: Summary of results and busulfan−induced microglial depletion/senescence model of macrophage brain engraftment.

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Data availability

The data used in the figures for this study are provided in Supplementary information and are covered by the Creative Commons Attribution 4.0 International License (no. CC BY 4.0): https://creativecommons.org/licenses/by/4.0/

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Acknowledgements

We thank P. Aubourg for his inspiration to initiate this study; J. Strauch, I. Bernstein and G. Dufayet-Chaffaud for experimental support; E. Gomez Perdiguero, G. Lepousez, I. Gabanyi, S. Voytek, F. Koukouli, H. Song, G. Eberl, L. Peduto, F. Jagot-Brunner, G. Parsons and A. Giniatullina for advice; and T. Sailor for two-photon stage fabrication. This work was partly funded by bluebird bio, Inc. as part of a collaboration (K.A.S., P.-M.L. and N.C.), Agence Nationale de la Recherche (no. ANR-15-CE37-0004-01, to K.A.S. and P.-M.L.), Agence Nationale de la Recherche (no. ANR-15-NEUC-0004, Circuit-OPL, to K.A.S. and P.-M.L.), the life insurance company ’AG2R-La-Mondiale’ (to K.A.S. and P.-M.L.), the Agence Nationale de la Recherche ‘Investissements d’avenir’ program (no. ANR-19−P3IA-0001, PRAIRIE 31A Institute, to C.G., J.-B.M. and C.L.V.) and a pre- and postdoctoral fellowship from the Laboratory for Excellence (LabEx) ‘Revive’ (no. ANR-10-LABX-73, to C.G. and K.A.S.). S.L.-M, S.T., B.G.-L. and N.C. funding from the program “Investissements d’avenir” ANR-10-IAIHU-06 and ANR-11-INBS-0011–NeurATRIS: Translational Research Infrastructure for Biotherapies in Neurosciences. Part of this work was carried out in the PHENOPARC core facility, CELIS core facility and ICAN core facility of ICM.

Author information

Author notes

  1. Nathalie Cartier

    Present address: Asklepios Biopharmaceutical, Inc., Institut du Cerveau (ICM), Paris, France

  2. These authors contributed equally: Sergio López-Manzaneda, Satoru Tada, Beatrix Gillet-Legrand, Pierre-Marie Lledo, Nathalie Cartier.

Authors and Affiliations

  1. Institut Pasteur, Université de Paris, CNRS UMR 3571, Perception and Memory Unit, Paris, France

    Kurt A. Sailor, George Agoranos, Corentin Guerinot & Pierre-Marie Lledo

  2. INSERM UMR1169, Université Paris-Sud, Université Paris-Saclay, Orsay, France

    Sergio López-Manzaneda, Satoru Tada, Beatrix Gillet-Legrand & Nathalie Cartier

  3. Thérapie Cellulaire et Génique des Maladies Neurologiques de l’Enfant et de l’Adulte, Institut du Cerveau et de la Moelle Épinière, Hôpital Pitié-Salpêtrière, Paris, France

    Sergio López-Manzaneda, Satoru Tada, Beatrix Gillet-Legrand & Nathalie Cartier

  4. Decision and Bayesian Computation, USR 3756 & Neuroscience Department CNRS UMR 3751, Institut Pasteur, Université de Paris, CNRS, Paris, France

    Corentin Guerinot, Jean-Baptiste Masson & Christian L. Vestergaard

  5. Sorbonne Université, Collège doctoral ED3C, Paris, France

    Corentin Guerinot

  6. bluebird bio, Inc., Cambridge, MA, USA

    Melissa Bonner, Khatuna Gagnidze & Gabor Veres

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Contributions

K.A.S. undertook conceptualization, investigation, methodology and writing of the original draft. G.A. carried out conceptualization, investigation, methodology, writing review and editing. S.L.-M., B.G.-L. and S.T. were responsible for conceptualization and investigation. C.G. and C.L.V. carried out methodology. J.-B.M. performed methodology and supervision. M.B. and K.G. were responsible for conceptualization, writing of the review and editing. G.V. undertook conceptualization. P.-M.L. carried out supervision, conceptualization, writing of the review and editing. N.C. carried out supervision, conceptualization, writing of the review and editing.

Corresponding authors

Correspondence to Kurt A. Sailor, Pierre-Marie Lledo or Nathalie Cartier.

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Competing interests

This project was partially funded by bluebird bio, Inc. for reagents and supplies, with partial salaries provided to G.A., B.G.-L., S.L.-M. and S.T.; M.B. and K.G. are current employees, and G.V. a former employee, of bluebird bio, Inc.; K.A.S., C.G., J.-B.M., C.L.V., P.-M.L. and N.C. declare no competing interests.

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Nature Medicine thanks Joanne Kurtzberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Jerome Staal was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team

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Extended data

Extended Data Fig. 1 HSCT donor cell macrophage characteristics within brain.

(a) Plot of donor cells that are negative for macrophage or microglia markers (tdTom+/Iba-1-/TMEM119−; mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; ***P < 0.001, **P < 0.01, *P < 0.05; P = 0.0025, 0.0019, 0.0006 and 0.0128 for 2, 4, 6 12 and 24 weeks, respectively, vs. 24 weeks; n = 4 mice for 2 and 4 weeks, n = 6 mice for 6, 12 and 24 weeks). (b) Plot of density of donor cells (tdTom+) in brain regions of olfactory bulb (OB), hippocampus, dorsal cerebral cortex (DorCtx), lateral cerebral cortex (LatCtx) and striatum at 6, 12 and 24 weeks post-HSCT. The OB has a significantly higher density of cells compared to all other brain regions at 12 and 24 weeks. (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; P < 0.0001 for OB 12 and 24 weeks vs. 6 weeks, ****P < 0.0001 for non-OB regions vs. OB at respective week with **P = 0.0095 24 weeks VentCtx vs. OB.; n = 3, 4 and 3 mice for 6, 12 and 24 weeks post-HSCT, respectively). (c) Surface renderings of three example donor cells at 17, 19 and 23 weeks post-HSCT. Scale bar = 15 µm. (d) Cell surface area plot of the same donor cells in vivo imaged from 12 to 25 weeks post-HSCT. (Light red plot of individual cells, black plot mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; n = 5, 10, 11, 9, 11, 11, 9, 7 cells for 12, 15.3, 16.3, 17.4, 18.9, 19.1, 23.1 and 24.4 weeks post-HSCT, respectively).

Extended Data Fig. 2 Host microglia density decline with HSCT, donor and host macrophage properties.

(a) The same registered brain region 2-photon in vivo imaged. Scale bar = 100 µm. (b) Microglia density comparing between in vivo 2-photon imaged CX3CR1-GFP mice and histology (tdTom−/Iba-1+/TMEM119+) analysis. (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; n = 4 animals all groups). (c) Immunohistochemistry at 24 weeks showing donor macrophages (tdTom+/Iba-1+/TMEM119−, white closed arrowhead), host microglia (tdTom−/Iba-1+/TMEM119+, white open arrowhead) and host macrophages (tdTom−/Iba-1+/TMEM119−, blue open arrowhead). Scale bar = 20 μm. (d) Plot of host macrophage density at control (Ctrl), 1 day (1d), 1, 2, 4, 6, 12 and 24 weeks post-HSCT (mean ± SEM; two-sided Kruskal-Wallis with Dunn’s multiple comparisons test; **P < 0.01; P = 0.0063 12 weeks vs. Ctrl; n = 7, 7, 4, 4, 4, 6, 6 and 6 mice for Ctrl, 1d, 1, 2, 4, 6, 12 and 24 weeks, respectively). (e) Plot of donor macrophage density vs. host microglia density at 12 and 24 weeks post-HSCT. (Mean of individual brain sections; simple linear regression [black and orange dashed lines] significance from slope = 0, P = 0.5382 F = 0.3930 R2 = 0.02026 and P = 0.0007 F = 13.86 R2 = 0.2958 for 12 and 24 weeks, respectively; n = 24 and 35 brain sections for 12 and 24 weeks, respectively). (f) Macrophage convex hull plot at 12 and 24 weeks post-HSCT (mean ± SEM; two-sided unpaired t-test; n = 5 and 6 mice for 12 and 24 weeks post-HSCT, respectively). (g) Convex hull plot of donor and host parenchymal macrophages (mean ± SEM; two-sided unpaired t-test; n = 1, 5 and 6 mice for 6, 12 and 24 week donor cells post-HSCT, respectively [binned], and n = 4 and 6 mice for 12 and 24 week host cells post-HSCT, respectively [binned]).

Extended Data Fig. 3 Effect of HSCT on SVZ proliferation, adult neurogenesis neural progenitors and derived immature neurons.

(a) lateral ventricle (LV) sub-ventricular zone with immunohistochemistry for Ki67+ cells (open arrowhead). Scale bar = 100 μm. (b) Plot of Ki67+ density in subventricular zone of the lateral ventricles (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; ****P < 0.0001 Ctrl vs. all; n = 4, 3, 4, 4, 4, 6, 6 and 3 mice for Ctrl, 1d, 1, 2, 4, 6, 12 and 24 weeks, respectively). (c) Adult hippocampus dentate gyrus region showing doublecortin (DCX) positive adult neurogenesis neural progenitor cells/immature neurons in the subgranule zone (open arrowhead). Scale bar = 100 µm. (d) Adult olfactory bulb in control (Ctrl) doublecortin labeling (open arrowhead). Scale bar = 200 µm. (e) Plot of doublecortin adult neurogenesis neural progenitor cell density in dentate gyrus subgranule zone of hippocampus with orange circles representing modeled normal age-related decline of adult neurogenesis DCX. (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; **** p < 0.0001 Ctrl vs. 6, 12 and 24 weeks; n = 4, 3, 4, 3 mice for Ctrl, 6, 12 and 24 weeks, respectively). (f) Plot of doublecortin relative fluorescent intensity (% max control) in sub-ventricular zone of lateral ventricles (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; **** p < 0.0001 Ctrl vs. 6, 12 and 24 weeks; n = 4, 3, 4, 3 mice for Ctrl, 6, 12 and 24 weeks, respectively). (g) Plot of doublecortin relative fluorescent intensity (%max control) in olfactory bulb (mean ± SEM; two-sided unpaired t-test; ***P = 0.0001 Ctrl vs. 24 weeks; n = 4, 3 mice for Ctrl and 24 weeks, respectively).

Extended Data Fig. 4 Ventral to dorsal brain pattern of NG2 depletion and partial recovery with HSCT.

(a) Confocal images of NG2 immunohistochemistry of dorsal cortex in control (Ctrl), 4 and 24 weeks post-HSCT with NG2+ cells indicated (open arrowhead). Scale bar = 50 µm. (b) Plot of NG2+ cell density at control (Ctrl), 1 day, 1, 2, 4, 6, 12 and 24 weeks post-HSCT (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; ****P < 0.0001 for 1 to 12 weeks vs. Ctrl and **P = 0.0021 for 24 weeks vs. Ctrl; n = 7, 3, 4, 4, 4, 6, 6, and 3 mice for Ctrl, 1d, 1, 2, 4, 6, 12, 24 weeks post-HSCT, respectively). (c) Tiled brain hemispheres at Control, 2, 4, 6, 12 and 24 weeks post-HSCT of NG2 cells. Control brain section has dense labeling of NG2 cells throughout cortex. Two weeks post-HSCT most NG2 cells are lost in cortex but small population of dense labeled NG2 cells found in thalamic regions (open arrowhead). By four weeks the section is devoid of NG2 cells. For 6, 12 and 24 weeks the NG2 cells returned but with a ventral reconstitution and a clear demarcation (dashed black line) from dorsal cortical regions. This pattern was observed in most mice, with a few individual outliers having dorsal near-complete recovery (Extended Data Fig. 4b at 6-12 weeks). Scale bar = 1 mm.

Extended Data Fig. 5 Effect of HSCT on donor cell proliferation and host microglia Ki67 modeling.

(a) Plot of donor macrophage proliferation at 2, 4, 6, 12 and 24 weeks post-HSCT (mean ± SEM; one-way two-sided ANOVA with Tukey multiple comparisons test; n = 4, 4, 6, 6, and 3 for 2, 4, 6, 12, and 24 weeks post-HSCT, respectively). (b) Plot of microglia density (green dashed line and spots; from Fig. 2a) with density model (orange line) at a 5%/day loss rate from Ctrl to 2 weeks post-HSCT and 10%/day from 2-4 weeks post-HSCT as an adjustment for inferred Ki67 increased proliferation. Using this 10%/day loss rate, there would be a complete depletion of host microglia by 12 weeks (orange dotted-line) due to loss of assumed Ki67 driven cell proliferation.

Extended Data Fig. 6 Single cell tracking of in vivo 2-photon image microglia.

Scatter plot above of total number of microglia in a FOV for each mouse, with color matched to tracks below, during PLX recovery period. Bottom plot of individual cell tracks (horizontal colored lines) for individual mice from HSCT group for 9 days of PLX3397 recovery. Each line represents one tracked cell and its survival duration.

Extended Data Fig. 7 Donor cell re-population of brain with effective loss of host microglia, proliferation of donor cells and migration front.

(a) In vivo 2-photon projected image of mouse 1 with successful donor cell engraftment after PLX-recovery and mouse 2, unsuccessful donor cell engraftment after PLX-recovery showing higher density of host microglia. Scale bar = 50 µm. (b) In vivo detected mitotic spheres of proliferating cells segregated into days after PLX3397 recovery from Fig. 4f (mean ± SEM; n = 5 mice). (c) Migration front at 0 min (red dashed line) and 120 min (green dashed line) at 6d post-PLX3397 showing migration direction (white arrows). Scale bar = 50 µm. (d) Same FOV (green dashed line) as in c, at 6, 7 and 8 days post-PLX. Scale bar = 100 µm.

Extended Data Fig. 8 PLX/busulfan efficient replacement of brain microglia population with donor macrophages and effect on donor and host motility and cortical neuron density.

(a) Tiled images of histological sections at 21 weeks post-HSCT of mice from PLX recovery experiment (Fig. 4) showing pattern of tdTom (red) and CX3CR1-GFP (green) cells having an average of 91.27 ± 3.67 % replacement of brain area with donor macrophages. Mouse 5 FOVs highlight typical morphology of host microglia (FOV 1) with large cell area and complex, fine process pattern compared to host peripheral macrophages (FOV 2) having a smaller cell area with less complex process pattern. FOV 3 demonstrates efficient white matter engraftment of donor macrophages. Scale bar for whole-brain images = 500 µm and for FOV images scale bar = 20 µm. (b) Plot of process area change (%/10 min) between control, no HSCT microglia (Ctrl microglia), sparse post-HSCT host microglia (sparse HSCT microglia), post-HSCT/post-PLX macrophages (dense donor macrophages) and post-HSCT macrophages (sparse donor macrophages). (mean ± SD; one-way two-sided ANOVA with Tukey multiple comparisons test; *P = 0.0408 Ctrl microglia vs. sparse HSCT microglia, ***P = 0.0002 sparse HSCT microglia vs. dense donor macrophages, and ****P < 0.0001 for dense donor macrophages and control microglia vs. sparse donor macrophages; n = 40, 28, 40 and 20 cells for Ctrl microglia, sparse HSCT microglia, dense donor macrophages and sparse donor macrophages, respectively). (c) Plot of NeuN+ cell density of layer 2/3 in dorsal cortex between control (Ctrl) and 21 weeks post-HSCT, PLX withdrawal donor macrophage enriched (HSCT) brains. (mean ± SEM; two-sided unpaired t-test; p = 0.9780).

Supplementary information

Reporting Summary

Supplementary Video 1

Donor cell tdTom+ peripherally derived meningeal phagocytes and intravascular cells. Two-photon in vivo imaged time-lapse video at 1 week post HSCT showing initially detected tdTom+ cells within the meningeal space as phagocytes, imaged at 10-s intervals (arrowhead in first half of video). Blood vessels show a continuous stream of circulating tdTom cells, as well as cells adhering to the lumen and migrating (arrowhead in second half of video).

Supplementary Video 2

Donor cell tdTom+ macrophages in brain parenchyma. Two-photon in vivo imaged z-stack movie (superficial to deep) at 2 months post HSCT showing initially detected tdTom+ macrophages in brain parenchyma (arrowhead), with depth from brain surface indicated. Also note high density of tdTom+ blood cells within the vasculature.

Supplementary Video 3

Sparse donor cell-derived tdTom+ macrophage process dynamics. Two-photon in vivo imaged time-lapse video of tdTom+ macrophage process dynamics imaged at 10-min intervals for 50 min. Left panel shows raw images, and right highlights new (green) and lost (red) processes.

Supplementary Video 4

Donor macrophage cell division within brain parenchyma and cell detection within vasculature. Two-photon in vivo imaged time-lapse video imaged at 10-min intervals for 2 h of tdTom+ macrophages (purple heatmap) undergoing massive cell division and migration at 6 days recovery from PLX3397 treatment, with blood vessels labeled in red. The first half of the video highlights mitotic spheres (arrowhead) and division events while the second half shows an example of a donor blood cell within a blood vessel for attempted tracking of transmigration events.

Supplementary Video 5

Donor macrophage migration front. Two-photon in vivo imaged time-lapse video of tdTom+ macrophage (purple heatmap) migration front at edge of brain region largely devoid of microglia/macrophages. The first half of the video highlights examples of mitotic spheres (arrowhead) behind the migration front, and the second half is at more accelerated speed with a line highlighting the edge of the migration front.

Supplementary Data 1

Data used in study figures.

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Sailor, K.A., Agoranos, G., López-Manzaneda, S. et al. Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain. Nat Med 28, 517–527 (2022). https://doi.org/10.1038/s41591-022-01691-9

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