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Distinguishing features of microglia- and monocyte-derived macrophages after stroke

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Abstract

After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of ‘intermediate states’ in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg−/− mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

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Acknowledgements

The technical assistance of Bettina Herrmann, Melanie Kroh, and Stefanie Balz is gratefully acknowledged. We also thank the Charité Core Facility ‘7T Experimental MRIs’ and the Deutsches Rheuma-Forschungszentrum Berlin ‘Flow Cytometry Core Facility’ (FCCF) for excellent support.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft (SFB TRR43 to M.E. and G.K.; GE2576/3-1 to K.G; DFG KR2956/5-1 to G.K; Exc257 to M.E.), the Bundesministerium für Bildung und Forschung (Center for Stroke Research Berlin to G.K., K.G., and M.E.), the European Union’s Seventh Framework Program (FP7/HEALTH.2013.2.4.2-1) under Grant agreement no. 602354 (Counterstroke consortium to K.G. and M.E.), the German Center for Neurodegenerative Diseases (DZNE; to M.E.), the German Center for Cardiovascular Research (DZHK; to M.E.), and the Corona Foundation (to M.E.).

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Author notes

  1. Matthias Endres and Karen Gertz contributed equally.

Authors and Affiliations

  1. Center for Stroke Research, Klinik für Neurologie, and Department of Experimental Neurology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1, 10117, Berlin, Germany

    Golo Kronenberg, Ria Uhlemann, Friederike Klempin, Stephanie Wegner, Matthias Endres & Karen Gertz

  2. Klinik für Psychiatrie und Psychotherapie, Campus Mitte, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1, 10117, Berlin, Germany

    Golo Kronenberg & Friederike Klempin

  3. Klinik und Poliklinik für Psychiatrie und Psychotherapie, Zentrum für Nervenheilkunde, Universitätsmedizin Rostock, Gehlsheimer Straße 20, 18147, Rostock, Germany

    Golo Kronenberg

  4. Max-Delbruck-Center for Molecular Medicine (MDC) in the Helmholtz Association, Robert‑Roessle‑Str. 10, 13125, Berlin, Germany

    Nadine Richter, Friederike Klempin, Lilian Staerck, Susanne Wolf, Wolfgang Uckert & Helmut Kettenmann

  5. Institut für Biologie, Humboldt-Universität Berlin, Robert‑Roessle‑Str. 10, 13125, Berlin, Germany

    Wolfgang Uckert

  6. DZNE (German Center for Neurodegenerative Diseases), Charitéplatz 1, 10117, Berlin, Germany

    Matthias Endres

Authors
  1. Golo Kronenberg
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  2. Ria Uhlemann
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  3. Nadine Richter
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  4. Friederike Klempin
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  5. Stephanie Wegner
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  6. Lilian Staerck
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  7. Susanne Wolf
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  8. Wolfgang Uckert
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  9. Helmut Kettenmann
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  10. Matthias Endres
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  11. Karen Gertz
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Corresponding author

Correspondence to Karen Gertz.

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The authors declare no competing financial interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Genes upregulated in blood-derived macrophages as compared to resident microglia (DsRed>EGFP) (XLSX 106 kb)

Genes upregulated in resident microglia as compared to blood-derived macrophages (EGFP>DsRed) (XLSX 185 kb)

Annotation enrichment analysis for the dataset DsRed>EGFP (XLS 1224 kb)

Annotation enrichment analysis for the dataset EGFP>DsRed (XLS 1001 kb)

Genes selectively upregulated in blood-derived macrophages (XLSX 38 kb)

Genes selectively upregulated in microglia (XLSX 64 kb)

401_2017_1795_MOESM7_ESM.xlsx

Comparison of previously described microglia-specific genes [7] with our dataset EGFP>DsRed. The first sheet of the Exel file represents a comparison of our candidate genes with the dataset published by Butovsky and co-workers [7]. Note that 56 transcripts occur in both datasets. The second sheet of the Exel file contains the transcripts specifically expressed in microglia (EGFP>DsRed) but not described by Butovsky and co-workers. Finally, the third sheet is identical to the second sheet except for the fact that all transcripts of unknown function were removed (XLSX 590 kb)

Comparison of previously described microglia-enriched transcripts in EAE [7] to our dataset EGFP>DsRed (XLSX 12 kb)

401_2017_1795_MOESM9_ESM.pdf

Flow cytometry of blood from WTDsRed → MacGreen BM chimeras. a Gating strategy. b Percentages (frequency of parent) of DsRed+ cells in granulocytes, monocytes, and lymphocytes. On average, 20.9% of blood monocytes express the fluorophore DsRed. N=31 mice. c Gating strategy for post-stroke analysis of white blood cells.Flow cytometry of CD11b pre-enriched brain cells post stroke. d Gating strategy. Infiltrating DsRed+ cells did not express CD3, Cd335 and Ly6G. N=7 WTDsRed → MacGreen BM chimeras (PDF 402 kb)

401_2017_1795_MOESM10_ESM.pdf

Transcriptomic analysis of CD11b+ DsRed+ CD45hi cells harvested from the ischemic brain of Selplg-KODsRed → WT chimeras and Selplg-WTDsRed → WT chimeras at 7 days after MCAo/reperfusion. The detailed protocol is accessible at GEO, GSE105011. a FACS gating strategy. b Hierarchical clustering did not reveal a distinction in the transcriptional response of cells derived from Selplg-WT and Selplg-KO BM. c Interexperiment correlation analysis. d Comparison of key M1 and M2 transcripts (PDF 139 kb)

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Kronenberg, G., Uhlemann, R., Richter, N. et al. Distinguishing features of microglia- and monocyte-derived macrophages after stroke. Acta Neuropathol 135, 551–568 (2018). https://doi.org/10.1007/s00401-017-1795-6

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  • DOI: https://doi.org/10.1007/s00401-017-1795-6

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