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Identification of a unique TGF-β–dependent molecular and functional signature in microglia

A Corrigendum to this article was published on 26 August 2014

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Abstract

Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1–deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

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Figure 1: Identification of a microglia signature by gene expression and quantitative mass spectrometry.
Figure 2: MG400 profile in microglia versus astrocytes, oligodendrocytes and neurons.
Figure 3: Identification of a miRNA microglia signature.
Figure 4: Surface expression of microglial molecules and molecular signature of recruited monocytes during EAE.
Figure 5: Microglia signature is not present in microglial cell lines.
Figure 6: Role of TGF-β in the development of microglia in vitro. (a,b) MG400 analysis of upregulated (a) and down regulated (b) microglial genes in cultured adult microglia in the presence of MCSF and TGF-β1 compared with adult microglia cultured in the presence of GM-CSF or MCSF alone.
Figure 7: Loss of microglia in CNS Tgfb1−/− mice.
Figure 8: Molecular microglial signature in CNS Tgfb1−/− mice.

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Change history

  • 19 December 2013

    In the version of this article initially published, the x-axis labels for the sets of graphs in Figure 2f corresponding to astrocyte, oligodendrocyte and neuron molecules consisted of six items, even though there were only five bars. “Red pulp macrophages” was included in error. Also, the Cleveland Clinic affiliation gave the section as the Department of Immunology; the correct affiliation is Neuroinflammation Research Center. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank D. Julius (University of California, San Francisco) for providing polyclonal antibody to P2ry12 and N. Kassam for support in antibody generation, A. Krichevsky and A. Wong Hon-Kit (Brigham and Women's Hospital, Harvard Medical School) for providing neurons, and L. Spangler for technical assistance for oligodendrocyte isolation. We thank D. Kozoriz for the FACS sorting. This work was supported by US National Institutes grant AG027437, US National Institutes Transformative Grant AG-043975, a grant from the Amyotrophic Lateral Sclerosis Association (1V78RI, ALSA 2087), a grant from Department of Defense ALS Research Program (AL120029), a Thome Foundation AMD grant, and philanthropic support. We thank Prize4Life for providing SOD1 mice.

Author information

Authors and Affiliations

Authors

Contributions

O.B. and H.L.W. conceived the study, designed the experiments and wrote the paper. O.B., A.J.L., G.G., T.K., B.D., R.C., P.M.W., C.E.D. and Z.F. performed experiments. M.P.J. and S.P.G. performed mass spectrometry experiments. C.S.M. and J.P.A. performed human microglia studies. Z.C., J.D.R., L.L. and R.M.R. performed CNS cell isolation studies. All authors discussed the results and conclusions and reviewed the manuscript.

Corresponding authors

Correspondence to Oleg Butovsky or Howard L Weiner.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of protein signature in microglia and Ly6C monocyte subsets.

(a) Schematic representation of the workflow for TMT-based quantitative proteomic analysis of microglia and splenic Ly6C monocytes. (b) Protein expression scatter plot of microglia versus monocytes subsets. (c) Venn diagram of unique and common proteins in microglia vs. Ly6CHi and Ly6CLow monocytes. (d) Heatmap of 103 enriched microglia proteins (>5-fold).

Supplementary Figure 2 Top biological functions in microglia.

Microglia and monocyte gene signature identified by AffyExon1 arrays (see Fig. 1a) were analyzed by Ingenuity pathway analysis (IPA). Bars indicate molecules present in dataset for each function.

Supplementary Figure 3 Nervous system development and function in microglia.

The subcellular localization of top microglial functions genes from Supplementary Figure 2 (Nervous system development and function, hereditary disorders, neurological disease genes) are illustrated. For each molecule in the dataset the expression fold change as compared to monocytes, P value and normalized expression level are presented.

Supplementary Figure 4 Top microglial interactions by protein function.

Illustration of microglial PU.1 network.

Supplementary Figure 5 Canonical pathways in microglia and monocytes.

Microglial and monocytes gene signatures identified with AffyExon1 arrays were analyzed by IPA for canonical pathways. Bars indicate −log P value (ratio of molecules present in the dataset out of all the function related molecules).

Supplementary Figure 6 TGF-β pathway in microglia

For each molecule in the dataset the expression fold change as compared to monocytes, P value and normalized expression level are presented.

Supplementary Figure 7 miRNA profile in microglia vs. astrocytes, oligodendrocytes and neurons.

(a) Heatmap of biological duplicates for FCRLS+ adult microglia (n = 5 mice), Glt-EGFP+ adult astrocytes (n = 9 mice), adult oligodendrocytes (n = 5 mice) and primary postnatal hippocampal and cortical neurons. Top 25 enriched miRNAs in microglia are presented (full miRNA list, Source data – Supplementary Figure 7). Each lane represents the average expression value of two biological duplicates per cell type. (b) qPCR analysis of microglia enriched miRNAs (miR-342-3p, miR-99a and miR-125b-5p) in CNS cell type. miRNA expression level was normalized against U6 miRNA using ΔCt. Bars show mean ±SEM. Shown is one of two individual experiments.

Source data

Supplementary Figure 8 Specificity of P2ry12 and FCRLS antibodies in resident microglia.

(a) Immonohistochemical analysis of mouse spinal cord, spleen red and pulp, lung, kidney, liver and skin (ear) stained with anti-P2ry12 (microglia; red), anti-Iba-1 (myeloid cells; green) and DAPI (nucleus; blue). Representative images of 3-5 mice. (b) FACS analysis of FCRLS surface expression in splenic CX3CR1GFP/+ monocytes and CD11b/CD45+ brain microglia. (c and d) Confocal images of mouse brain stained with Iba-1 and polyclonal FCRLS antibody in (c) naïve and (d) chimeric CX3CR1GFP/+ mice. Anti-FCRLS signal co-localized with Iba-1+ microglia and does not stain recruited monocytes in the brain of CX3CR1GFP chimeric mouse transplanted with bone marrow derived cells from CX3CR1GFP/+ transgenic mice.

Supplementary Figure 9 Microglia signature during development.

(a) MG400 expression profile of microglia from mice bran at E10.5, E12.5, P3, P21, P30 and 2 months of age (full gene list, Source data – Supplementary Figure 9). Results were log-transformed, normalized (to the mean expression of zero across samples) and centered, and populations and genes were clustered by pairwise centroid linkage with the Pearson correlation. Data are pooled of 2 different experiments (n = 5-10 pooled mice per group). (b) Top microglial molecules grouped according to cell localization and function. (c) qPCR analysis of 6 selected microglia genes in MCSF+TGF-β1 cultured microglia. Gene expression level was normalized against Gapdh using ΔCt.

Source data

Supplementary Figure 10 M0, M1 and M2 microglial phenotypes as measured by MG400 chip.

(a) Heatmap of significantly affected MG400 genes in M0, M1 and M2 polarized microglia. One representative of three individual experiments is shown. (b) Top 40 affected genes in M0, M1 and M2 microglia. Bars represent fold change as compared to the other two phenotypes. (c and d) IPA analysis of (c) top bio functions and (d) Top upstream regulators in M0-, M1- and M2-polarized microglia.

Supplementary Figure 11 Microglia loss in CNS-TGFβ1−/− mice.

(a) Representative FACS analysis of isolated brain- and spinal cord-derived mononuclear cells stained with CD45 and CD11b antibodies at 160d of age in IL2TGFβ1-Tg-TGF-β1+/− (n = 6) and IL2TGFβ1-Tg-TGF-β1−/− (n = 6) mice. (b and c) Increased apoptosis of CD39+CD11b+ cells in the brain of CNS-TGFβ1−/− mice. (b) Representative FACS analysis of isolated brain-derived mononuclear cells for apoptosis as measured by AnnexinV and 7AA-D in CD39+CD11b+-gated cells at 160d of age in IL2TGFβ1-Tg-TGF-β1+/− (left) and IL2TGFβ1-Tg-TGF-β1−/− (right) mice. (c) Quantitative analysis of AnnexinV+7AAD+ cell as percentage of CD39+CD11b+ cells in TGF-β1−/−, IL2TGF-β1-Tg-TGF-β1+/− and IL2TGF-β1-Tg-TGF-β1−/− mice at 20, 90 and 160 days of age. Data represent mean ±SEM (n = 3 mice per group). **P<0.01, 1-Way ANOVA followed by Dunnett's multiple-comparison post-hoc test for comparison at 20d and **P<0.01, ***P<0.001, Student's t test, 2-tailed for comparison at 90d and 160d.

Supplementary Figure 12 Macrophages, dendritic cells and Langerhans cells are not affected in peripheral organs of CNS-TGFβ1−/− mice.

(a-d) Nonlymphoid and lymphoid tissue myeloid cells isolated from IL2TGFβ1-Tg-TGF-β1+/− (left) and IL2TGFβ1-Tg-TGF-β1−/− (right) mice at 160d were analyzed by flow cytometry. FACS representative dot plots show the percentage and absolute numbers of (a) kidney, liver, lung, spleen and skin derived CD11b+F4/80+ macrophages and (b) Langerhans cell (LC) stained with MHCII+CD11b+ and Langerin+CD11b+ in the ear skin cell suspension among DAPICD45+ cells of IL2TGFβ1-Tg-TGF-β1+/− (n = 6) and IL2TGFβ1-Tg-TGF-β1−/− (n = 6) mice. (c) Plots show percentage of CD11c+ DCs among gated DAPICD3CD19NK1.1+ cells. (d) Dot plots show percentage of CD103+ and CD11b+ DCs among gated DAPI-CD45+CD11c+I-A+ cells. Bars represent data from 2 pooled experiments. Errors bars represent ±SEM.

Supplementary Figure 13 Loss of microglia during development in CNS-TGF-β1 deficient mice

FACS analysis of CD39 and CD11b expression among CD45+ cells in the brain during development and aging. Data represent mean ±SEM (n = 4-6 mice per group).

Supplementary Figure 14 Microglia loss in the spinal cord of CNS-TGFβ1−/− mice.

(a) Representative FACS analysis of isolated spinal cords-derived mononuclear cells stained for FCRLS, F4/80, CD39 and CD11b among hematopoietic (CD45+) cells at 20d of age in in IL2TGF-β1-Tg-TGF-β1+/− and IL2TGF-β1-Tg-TGF-β1−/− mice). Immonohistochemical analysis of mouse spinal cord axial section of lumbar level stained with (b) anti-P2ry12 (microglia) and anti-NeuN (neurons) and (c) anti-P2ry12 (microglia), Iba-1 (myeloid cells) and anti-NeuN (neurons) at 20 days of age (IL2TGF-β1-Tg-TGF-β1+/− and IL2TGF-β1-Tg-TGF-β1−/−) mice. Representative images of 3-5 mice. Scale bar represents 500μm (top panel) and 250μm (zoomed are indicated, bottom panel). (d) Quantitative analysis of NeuN+, Iba-1+ and P2ry12+/Iba-1+ cells in IL2TGF-β1-Tg-TGF-β1+/− and IL2TGF-β1-Tg-TGF-β1−/− mice at 20, 90 and 160 days of age. Data represent mean ±SEM (n = 5). **P<0.01, ***P<0.001, Student's t test, 2-tailed.

Supplementary Figure 15 TGF-β pathway and downstream microglial molecules are suppressed in CNS-TGFβ1−/− mice.

For each molecule in the dataset the expression fold change as compared to microglia from IL2TGF-β1-Tg-TGF-β1+/− mice, P value and normalized expression level are presented.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1–4 (PDF 11887 kb)

Supplementary Table 2

Top unique microglial genes and shared genes between microglia, neurons, astrocytes and oligodendrocytes. List of 152 identified genes which are enriched in adult microglia and not in adult astrocytes, oligodendrocytes and neurons. (XLSX 25 kb)

Rotorod performance of CNS-TGFβ1−/− mice

IL2TGFβ1-Tg-TGF-β1−/− (left) and IL2TGFβ1-Tg-TGF-β1+/− (right) mice at the age of 140 days of age. (MOV 8329 kb)

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Butovsky, O., Jedrychowski, M., Moore, C. et al. Identification of a unique TGF-β–dependent molecular and functional signature in microglia. Nat Neurosci 17, 131–143 (2014). https://doi.org/10.1038/nn.3599

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