Origin, fate and dynamics of macrophages at central nervous system interfaces (original) (raw)

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Acknowledgements

We thank M. Oberle, M. Ditter and T. el Gaz for excellent technical assistance and T. Leng Tay for critical reading of the manuscript. Supported by the DFG (SFB 992, SFB 1160, PR 577/8-1, Reinhart Koselleck Grant for M.P.; SFB 1160, ZE872/3-1 for R.Z.; and FOR1336 for J.P., I.B., M.P. and S.J.), the Fritz-Thyssen Foundation (M.P.), the European Union's Seventh Framework Program FP7 under Grant agreement 607962 (nEUROinflammation for M.P.), the Gemeinnützige Hertie Foundation (GHST for M.P.), the Sobek Foundation (M.P.) and the BMBF-funded Competence Network on Multiple Sclerosis (KKNMS for M.P. and M. Kerschensteiner).

Author information

Author notes

1. Tobias Goldmann, Peter Wieghofer and Marta Joana Costa Jordão: These authors contributed equally to this work.

Authors and Affiliations

1. Institute of Neuropathology, Freiburg University Medical Centre, Freiburg, Germany
   Tobias Goldmann, Peter Wieghofer, Marta Joana Costa Jordão, Fabiola Prutek, Nora Hagemeyer, Kathrin Frenzel, Lukas Amann, Ori Staszewski, Katrin Kierdorf & Marco Prinz
2. Faculty of Biology, University of Freiburg, Freiburg, Germany
   Peter Wieghofer, Marta Joana Costa Jordão, Kathrin Frenzel & Lukas Amann
3. Institute of Anatomy, University of Leipzig, Leipzig, Germany
   Martin Krueger & Ingo Bechmann
4. Institut für Klinische Neuroimmunologie, Ludwig-Maximilians Universität München, Munich, Germany
   Giuseppe Locatelli & Martin Kerschensteiner
5. Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
   Hannah Hochgerner & Sten Linnarsson
6. Department of Hematology and Oncology, Freiburg University Medical Centre, Freiburg, Germany
   Robert Zeiser
7. BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany
   Robert Zeiser & Marco Prinz
8. Peter Munk Cardiac Centre, University Health Network Toronto, Ontario, Canada
   Slava Epelman
9. Centre for Molecular and Cellular Biology of Inflammation, King's College London, London, UK
   Frederic Geissmann
10. Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité – Universitätsmedizin Berlin, Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité – Universitätsmedizin Berlin,
    Josef Priller
11. Cluster of Excellence NeuroCure, DZNE & BIH, Berlin, Germany
    Josef Priller
12. The Biomedical Research Centre, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
    Fabio M V Rossi
13. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
    Martin Kerschensteiner
14. Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
    Steffen Jung

Authors

1. Tobias Goldmann
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2. Peter Wieghofer
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3. Marta Joana Costa Jordão
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4. Fabiola Prutek
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5. Nora Hagemeyer
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6. Kathrin Frenzel
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7. Lukas Amann
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8. Ori Staszewski
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9. Katrin Kierdorf
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10. Martin Krueger
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11. Giuseppe Locatelli
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12. Hannah Hochgerner
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13. Robert Zeiser
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14. Slava Epelman
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15. Frederic Geissmann
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16. Josef Priller
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17. Fabio M V Rossi
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18. Ingo Bechmann
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19. Martin Kerschensteiner
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20. Steffen Jung
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21. Marco Prinz
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Contributions

T.G., P.W., M.J.C.J., F.P., N.H., K.F., O.S., K.K., L.A., M. Krueger, G.L. and H.H. conducted the experiments and analyzed the data. R.Z., S.E., F.G., J.P., F.M.V.R., I.B., S.L., M. Kerschensteiner and S.J. analyzed the data, contributed to the in vivo studies and provided mice or reagents. T.G. and M.P. supervised the project and wrote the manuscript.

Corresponding author

Correspondence toMarco Prinz.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Single-cell RNA sequencing of pvMΦ, microglia and monocytes.

(a) Work flow for obtaining and analyzing single-cell RNA-seq from mouse cortical cells and monocytes from dissection to single-cell RNA-seq and unbiased biclustering.

(b) Representative image of single cells captured in the chip. All cells were checked individually by light microscopy and chambers with no or damaged cells (red squares) were omitted from subsequent analysis.

(c-f) Bar graphs for commonly expressed genes (c), markers selectively expressed by pvMΦ (d), selectively expressed by cortical microglia (e) or just by monocytes (f) evaluated by single cell RNA-seq.

Picture from cell capture for RNAseq is representative of one independent experiment (b). Single cell RNAseq data (c-e) is representative of two independent experiments with 167 ptMΦ, 246 monocytes, 33 microglia and 65 pvMΦ. Data is represented as mean ± s.e.m.

Supplementary Figure 2 Flow cytometry of cortical pvMΦ and microglia.

(a) Representative gating strategy for the flow cytometry-based isolation of bone marrow monocytes for subsequent single-cell RNA-sequencing as shown in Fig. 1d-g.

(b) Representative gating strategy for the isolation of cortical CD11b+ CD45lo microglia and CD11b+ CD45hi cells from the cortex (meninges and choroid plexus were removed) as shown in Fig. 1j-k.

(c) CD11b+ CD45hi pvMΦ can be further separated from CD11b+CD45lo microglia by gating them as CD45hi CD11b+ Ly6C− Ly6G− CD206+ F4/80+ cells. Representative histograms for CD206, CD36 and F4/80 staining on pvMΦ and microglia (black line) are shown over isotype controls (filled gray).

d) Representative gating strategy for the flow cytometry-based characterization of Ly6C cells as shown in Fig. 6a-b. Peripheral blood leukocytes were gated according to physical parameters and further subdivided in myeloid cells by the expression of CD45 and CD11b. CD45+ CD11b+ SSClo CD115+ monocytes are then further discriminated into Ly6Chi inflammatory monocytes and Ly6Clo patrolling monocytes.

Gating strategies are representative of six mice from two independent experiments (a, d) or from three biological replicates from two independent experiments (b, c).

Supplementary Figure 3 Irradiation induces engraftment of bone-marrow-derived CNS macrophages and microglia.

a) Scheme for the induction of recombination (injection of tamoxifen [TAM]) and subsequent analysis in Cx3cr1_CreER_Rosa26-YFP animals.

b) Scheme and timeline for labelling and analyses of pvMΦ, mMΦ and cpMΦ in adulthood using TAM injection in adult Cx3cr1_CreER_Rosa26-YFP animals.

c) Direct fluorescence microscopic visualization revealed numerous GFP+ donor-derived Iba-1+ pvMΦ, mMΦ, cpMΦ and few microglia 20 weeks after transfer of bone marrow from _Acta1_-GFP mice into lethally irradiated wild-type mice. Arrows indicate double positive, asterisks single Iba-1 (red) positive cells. Scale bar = 25 μm.

d) Quantification of donor-derived GFP+ Iba-1+ cells.

Immunofluorescence pictures (c) are representative of four mice from one independent experiment. Data were obtained from five mice per group from one independent experiment (d). Each symbol represents one mouse and three tissue sections per mouse were quantified. (means ± s.e.m.)

Supplementary Figure 4 CNS macrophages do not require Batf3.

a) Localization and presence of pvMΦ, mMΦ and cpMΦ in adult wild-type (WT) and _Batf3_−/− mice evaluated using Iba-1 immunohistochemistry. Representative figure are presented (upper images) and quantification thereof.

b) Confocal pictures showing Tomato+ cells in the choroid plexus of Cx3cr1_CreER_Rosa26-Tomato mice (tomato, red) at 8 weeks after TAM expressing the macrophage marker F4/80 and to a much lesser extent CD206. Scale bar: 25 μm.

Immunofluorescence pictures are representative of of five mice from two independent experiments (b). Immunohistochemistry pictures (a) are representative of three mice per genotype from one independent experiment. Macrophage density (a) is representative of three mice per genotype from one independent experiment (Meninges: P= 0.7; perivascular space: P=0.7; choroid plexus P= 1). Mann Whitney test was applied. Each symbol represents one mouse with quantification of a minimum of three tissue sections. (error bars, s.e.m.). N.S. = not significant.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 1295 kb)

Location and morphology of pvMΦ, mMΦ and microglial cells.

3D-rendering (Imaris, Bitplane) of the confocal image stack represented in Fig. 5b that illustrates the distinct pvMΦ, mMΦ and microglial cells in the intact spinal cord of a Cx3cr1_CreER:Rosa26-Tomato_ mice (tomato, red) at 8 weeks after TAM and injected with dextran-AF647 (blood vessel, blue). (MP4 8147 kb)

Dynamics of the myeloid cells.

Confocal projection of the dorsal spinal cord as shown in Fig. 5c and 5d followed by in vivo 2-photon time-lapse imaging of the different myeloid cells in Cx3cr1_CreER:Rosa26-Tomato_ mice at 8 weeks after TAM. (MP4 979 kb)

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Goldmann, T., Wieghofer, P., Jordão, M. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces.Nat Immunol 17, 797–805 (2016). https://doi.org/10.1038/ni.3423

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• Received: 20 August 2015
• Accepted: 01 March 2016
• Published: 02 May 2016
• Issue Date: July 2016
• DOI: https://doi.org/10.1038/ni.3423