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Origin, fate and dynamics of macrophages at central nervous system interfaces

Abstract

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

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Figure 1: Molecular census of non-parenchymal macrophages, microglia and monocytes.
Figure 2: Development of CNS macrophages.
Figure 3: Ontogeny of brain macrophages at brain interfaces.
Figure 4: Maintenance of non-parenchymal macrophages in adulthood.
Figure 5: In vivo dynamics of CNS macrophages.
Figure 6: Turnover of CNS macrophages.

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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).

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Authors

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 to Marco Prinz.

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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 Cx3cr1CreERRosa26-YFP animals.

b) Scheme and timeline for labelling and analyses of pvMΦ, mMΦ and cpMΦ in adulthood using TAM injection in adult Cx3cr1CreERRosa26-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 Cx3cr1CreERRosa26-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 Figure 5 Model for experimental findings on the origin, fate and dynamics of microglia and macrophages at CNS boundaries.

Traditional and proposed view on the origin, fate and turnover of non-parenchymal CNS macrophages and microglia.

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 Cx3cr1CreER: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 Cx3cr1CreER: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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