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Central nervous system-associated macrophages modulate the immune response following stroke in aged mice

Nature Neuroscience volume 27, pages 1721–1733 (2024)Cite this article

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Abstract

Age is a major nonmodifiable risk factor for ischemic stroke. Central nervous system-associated macrophages (CAMs) are resident immune cells located along the brain vasculature at the interface between the blood circulation and the parenchyma. By using a clinically relevant thromboembolic stroke model in young and aged male mice and corresponding human tissue samples, we show that during aging, CAMs acquire a central role in orchestrating immune cell trafficking after stroke through the specific modulation of adhesion molecules by endothelial cells. The absence of CAMs provokes increased leukocyte infiltration (neutrophils and CD4+ and CD8+ T lymphocytes) and neurological dysfunction after stroke exclusively in aged mice. Major histocompatibility complex class II, overexpressed by CAMs during aging, plays a significant role in the modulation of immune responses to stroke. We demonstrate that during aging, CAMs become central coordinators of the neuroimmune response that ensure a long-term fine-tuning of the immune responses triggered by stroke.

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Fig. 1: Age-associated immune responses and BBB leakage in the naive mouse brain occur without changes in the number or morphology of CAMs.
Fig. 2: CAMs change their transcriptomic phenotype during aging and overexpress genes implicated in the regulation of innate and adaptive immune responses, including antigen presentation and leukocyte cell–cell adhesion.
Fig. 3: Upregulation of HLA and CD74 expression by CAMs in the cortex of older humans.
Fig. 4: CAM depletion increases functional deficit after stroke in aged mice but not in young mice.
Fig. 5: CAM depletion increases the expression of the P-sel adhesion molecule and leukocyte trafficking in cerebral vessels after stroke in aged mice but not in young mice.
Fig. 6: CAM depletion increases neutrophil and CD4+ and CD8+ T cell infiltration after stroke in aged mice.
Fig. 7: Blockade of MHC class II mimics some of the features of global CAM depletion by CLO treatment in aged mice.

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Data availability

Mouse macrophage RNA-seq data are available at the Gene Expression Omnibus under accession number GSE267623. All data necessary for the conclusions of the study are available in the main text, figures and Extended Data figures. Source data are provided with this paper.

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Acknowledgements

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement number 813294 (ENTRAIN). This work was also supported by grants from the Ministère de l’Enseignement Supérieur et de la Recherche and INSERM (French National Institute for Health and Medical Research; HCERES U1237-2017/2022), Throne (ANR-22-CE14-002), Eranet-Neuron MeniPSYs (ANR-22-NEU2-0005) and the Fondation pour la Recherche Médicale (ARF202005011926; E.L.). The work of A.M. is supported by the UK Dementia Research Institute (DRI), which receives its funding from UK DRI, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. A.M. also holds a UKRI Medical Research Council fellowship (Career Development Award MR/V032488/1) and a Foundation for Research on Alzheimer’s disease award. A.P.F. is funded by the WINNINGNormandy Program supported by the Normandy Region and the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement number 101034329. Some illustrations were created using BioRender.com.

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Authors and Affiliations

  1. Normandie University, UNICAEN, Université Caen Normandie, INSERM UMR-S U1237, Physiopathology and Imaging of Neurological Disorders (PhIND), GIP Cyceron, Institut Blood and Brain @ Caen-Normandie (BB@C), Caen, France

    Damien Levard, Célia Seillier, Mathys Bellemain-Sagnard, Antoine Philippe Fournier, Eloïse Lemarchand, Chantal Dembech, Denis Vivien & Marina Rubio

  2. INSERM U1234 ‘PAn’THER’, Flow Cytometry Core-IRIB, Rouen, France

    Gaëtan Riou

  3. Academic Neuropathology, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK

    Karina McDade & Colin Smith

  4. Dementia Research Institute at the University of Edinburgh, Centre for Clinical Brain Sciences, Chancellor’s Building, Edinburgh Medical School, Edinburgh, UK

    Conor McQuaid & Axel Montagne

  5. Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany

    Lukas Amann & Marco Prinz

  6. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

    Marco Prinz

  7. Department of Clinical Research, Caen-Normandie University Hospital, CHU, Caen, France

    Denis Vivien

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Contributions

D.L. designed and performed the experiments, analyzed and interpreted the data, created the figures and wrote the manuscript. C. Seillier performed the flow cytometry experiments. M.B.-S. performed the quantification of human sample staining and analyzed and interpreted the data. A.P.F. performed the molecular MRI experiments. E.L. and C.D. performed immunohistological experiments and data analyses. G.R. performed the cell sorting experiments. A.M., K.M., C. Smith and C.M. performed the human sample staining experiments and participated in methods writing. A.M., M.P. and D.V. provided intellectual contributions. L.A. and M.P. performed the mouse RNA-seq data analyses and participated in methods writing. D.V. provided resources and intellectual contributions. M.R. designed and performed the experiments, analyzed and interpreted the data and wrote the manuscript.

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Correspondence to Denis Vivien or Marina Rubio.

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Extended data

Extended Data Fig. 1 Immunofluorescent characterization of CAMs in young and aged mice cortex.

a) Representative immunofluorescence images of CD206+ CAMs (red), P2Y12R+ microglia (green) and collagen IV in the basal lamina of blood vessels (gray) in young male mice brain cortex. Scale bar: 100 µm. b-d) Representative immunofluorescence images of CD206+ CAMs (red), P2Y12R+ microglia (green), GFAP+ astrocytes (cyan), AQP4+ astrocytic endfeet (cyan) in young mice brain cortex. Scale bar: 100 µm. e) Representative immunofluorescence images of CD206+ CAMs (red), Iba1+ microglia (green) and collagen IV in the basal lamina of blood vessels (gray) in young and aged male mice brain cortex. Scale bar: 100 µm. f) Representative immunofluorescence images of CD206+ and Lyve1+ CAMs (red) and podocalyxin-labelled blood vessels (gray), in young or aged male mice brain cortex. Scale bar: 100 µm. g) Representative immunofluorescence images of Iba1+ microglia (green) and CD68+ macrophage/phagocytic microglia (red) in young or aged male mice brain cortex. Scale bar: 100 µm. h) Immunohistological quantification of Iba1+ microglia and Iba1+/CD68+ phagocytic microglia or macrophages in young or aged male mice brain cortex. n=6 mice/groupe. Two-sided Mann-Whitney U-test. Data are presented as mean values +/− SEM.

Source data

Extended Data Fig. 2 Flow cytometry dot-plots and gating strategies.

a) Representative flow cytometry dot-plots and gating strategy used for quantification of CAMs, microglia or macrophages and neutrophils from young and aged male mice brain. b) Representative flow cytometry dot-plots and gating strategy used for quantification of CD4 and CD8 T cells from young and aged mice brain. c) Representative flow cytometry dot-plots and gating strategy used for quantification of CD19+ B220+ B cells from young and aged mice brain. d) Representative flow cytometry dot-plots and gating strategy used for isolation of CD11b+ CD45+ CD206+ CAMs by fluorescence activated cell sorting (FACS) from young and aged mice brain to analyze the RNA expression profile.

Extended Data Fig. 3 Immunohistochemical analysis of microglia in young and aged human brain cortex.

a) Table representing the information about the gender, the age and the Broadmann area of interest of the subjects within the two groups "Young controls" and "Aged controls". b) Representative IHC staining of human brain cortex within grey matter area. Hematoxylin (Blue), Iba1 (DAB), HLA (green). Scale bar: 50 µm. c) Representative IHC staining of human brain cortex within grey matter area. Hematoxylin (Blue), Iba1 (DAB), CD74 (green) Scale bar: 50 µm. d) Quantification of Iba1+, Iba1+ HLA+ and Iba1+ CD74+ microglia in the human grey matter. n= 5 young controls/ 5 aged controls. Two-sided Mann-Whitney Utest. Data are presented as mean values +/− SEM.

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Extended Data Fig. 4 CAMs depletion by intracerebroventricular injection of clodronate-liposomes.

a) Schematic representation of the experimental protocol for CAMs depletion and quantification. b) Representative flow cytometry dot-plots and gating strategy used for quantification of CAMs, microglia, activated microglia or macrophages and neutrophils from young and aged male mice brain treated with vehicle or clodronate-liposomes. c) Flow cytometry quantification of CAMs, microglia, activated microglia or macrophages and neutrophils from young and aged mice brain treated with vehicle or clodronate-liposomes. n=4 mice/group. Two-sided Mann-Whitney U test. d) Representative immunofluorescence images of CD206+ CAMs (red), P2Y12R+ or Iba1+ microglia (green) and collagen IV in the basal lamina of blood vessels (gray), in vehicle or clodronate-liposomes treated mice cortex. Scale bar: 100 µm. e) Immunohistological quantification of CD206+ CAMs and Iba1+ microglia in vehicle or clodronate-liposomes treated mice cortex. n=5, Two-sided Mann-Whitney U-test. Data are presented as mean values +/− SEM (c, e).

Source data

Extended Data Fig. 5 Immunohistochemical analysis of P-selectin expression and fibrinogen extravasation.

a) Representative immunofluorescence images of Psel+ blood vessels in mice brain cortex at 5 days after stroke in vehicle or clodronate-treated young male mice. Scale bar: 50 µm. b) Representative immunofluorescence images of Psel+ blood vessels in mice brain cortex at 5 days after stroke in vehicle or clodronate-treated aged mice. Scale bar: 50 µm. c) Immunohistological quantification of the number of Psel+ blood vessels in mice brain cortex at 5 days after stroke. n=5 mice/group. d) Immunohistological quantification of the Psel+ area in mice brain cortex at 5 days after stroke. n=5 mice/group. e) Immunofluorescence images of Psel+ (magenta) blood vessels (gray) and Iba1+ microglia (green) in mice brain cortex at 5 days after stroke in vehicle or clodronate-treated aged mice. Scale bar: 50 µm. f) Representative immunofluorescence images of fibrinogen extravasation in mice brain cortex 1 day after stroke in young and aged mice treated with PBS (Vehicle) or clodronate (CLO). Scale bar 100µm. g) Quantification of the fibrinogen extravasation area. n=6 vehicle, young / n=6 CLO, young / n=5 vehicle, aged / n=5 CLO, aged. Multiple Mann-Whitney U-test with two-stage step up method for false discovery rate (c, d, g). Data are presented as mean values +/− SEM (c, d, g).

Source data

Extended Data Fig. 6 CAMs and microglia characterization after stroke in young and aged mice.

a) Representative flow cytometry dot-plots and gating strategy used for quantification of CAMs, microglia, macrophages and neutrophils 2 days after stroke in vehicle or clodronate-treated young and aged male mice brain. b-c) Flow cytometry quantification of CAMs, microglia, macrophages and neutrophils 2 days after stroke in vehicle or clodronate-treated young and aged mice brain. n=7 mice/group. d) Representative immunofluorescence images of CD206+ CAMs and Iba1+ microglia in perilesional area at 5 days after stroke in vehicle or CLO treated young and aged mice. Scale bar: 100 µm. e) Immunohistological quantification of CD206+ CAMs 1 day (left) and 5 days (right) after stroke. n=5 mice/group. f) Representative immunofluorescence images of Iba1+/CD68+ phagocytic microglia in perilesional area 1 day after stroke in vehicle or clodronate-treated young or aged mice. Scale bar: 100 µm. g) Immunohistological quantification of Iba1+ microglia 1 day and 5 days after stroke. n=5 mice/group. h) Immunohistological quantification of Iba1+/CD68+ phagocytic microglia 1 day and 5 days after stroke. n=5 mice/group. Multiple Mann-Whitney U-test with two-stage step up method for false discovery rate (b, c, e, g, h). Data are presented as mean values +/− SEM (b, c, e, g, h).

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Levard, D., Seillier, C., Bellemain-Sagnard, M. et al. Central nervous system-associated macrophages modulate the immune response following stroke in aged mice. Nat Neurosci 27, 1721–1733 (2024). https://doi.org/10.1038/s41593-024-01695-3

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