MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway

Abstract

MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45(low), major histocompatibility complex (MHC) class II(low) phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo. These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.

Figure 1

Analysis of expression of miR-124 in CNS-resident microglia compared to peripheral macrophages. (a,b) Real-time qRT-PCR analysis of miR-124 (a) and miR-223 (b) expression in F4/80⁺CD11b⁺ mononuclear cells isolated from different tissues of healthy adult C57BL/6 mice. Mean ± s.d. of triplicate wells is shown. The data are representative of three different experiments. BM, bone marrow; Perit., peritoneal cavity; TGM, thioglycolate-elicited inflammatory macrophages. (c,d) Analysis of expression of miR-124 in populations of microglia and peripheral macrophages isolated from the CNS of chimeric mice during EAE. (c) GFP fluorescence (x axis) and staining for CD11b (y axis) are shown for mononuclear cells isolated from the CNS of chimeric mice at the peak of EAE (day 21). In d, miR-124 expression in populations of F4/80⁺CD11b⁺GFP⁻ microglia (c, left rectangle) and F4/80⁺CD11b⁺GFP⁺ peripheral macrophages (c, right rectangle) sorted from CNS of healthy chimeric mice (no disease) or chimeras with EAE at the disease onset (day 14), peak (day 21) and recovery phase (day 40). One representative of four experiments is shown.

Figure 2

Quantitative analysis of expression of miR-124 in activated microglia. (a) Analysis of expression of activation markers CD45 and MHC class II in populations of gated F4/80⁺CD11b⁺GFP⁻ microglia (upper row) and F4/80⁺CD11b⁺GFP⁺ peripheral macrophages (lower row) isolated from CNS of healthy chimeric mice (no disease) or chimeric mice with EAE at the disease onset (day 14), peak (day 21) or recovery (day 40). Four to five mice per group were used. The isotype controls are shown in upper left quadrants of contour plots. Percentages of CD45⁺ cells (right quadrants) and MHC class II⁺ cells (upper quadrants) are shown. (b) Comparison of miR-124 expression in populations of resting CD45^lowMHC class II⁻GFP⁻ and of activated CD45^int–hiMHC class II⁺GFP⁻ microglia, as well as populations of activated CD45^hiMHC class II⁺GFP⁺ and deactivated CD45^int–hiMHC class II⁻GFP⁺ peripheral macrophages. The cells were sorted by FACS from the CNS of chimeric mice with EAE at day 14, and miR-124 expression was assessed by real-time qRT-PCR. Representative results from two independent experiments are shown, with mean ± s.d. of quadruplicate wells plotted; **P < 0.01. (c) Analysis of expression of miR-124 in microglia activated in vitro by GM-CSF and IFN-γ or LPS and IFN-γ. Microglia cells were isolated from healthy mice and incubated in medium alone, with GM-CSF and IFN-γ, or with LPS and IFN-γ for 6 h, and miR-124 expression was assessed by real-time qRT-PCR. Mean ± s.d. of triplicate experiments is shown; *P < 0.05 compared to ex vivo–isolated microglia.

Figure 3

Analysis of expression of activation markers, pro- and anti-inflammatory cytokines and markers for M2 macrophages in BMDMs with ectopic overexpression of miR-124. (a,b) Flow cytometry analysis of the expression of CD45 (x axes) and either CD11b, F4/80, MHC class II or CD86 (y axes) in BMDMs transfected with miR-124 or control miRNA. Percentages of CD45^hi cells (right quadrants) and activation marker–positive cells (upper quadrants) are shown. A representative experiment is shown in a, and mean ± s.e.m. of four independent experiments is shown in b. MFI, mean fluorescence intensity. *P < 0.05. (c) Flow cytometry analysis of TNF-α production by BMDMs transfected with miR-124 or control miRNA. (d) Real-time qRT-pPCR analysis of the mRNA expression of C/EBP-α, inducible form of nitric oxide synthase (iNOS), IL-4, IL-10, TGF-β1, arginase I and FIZZ1 in BMDMs transfected with miR-124 or control miRNA. (e) Analysis of TGF-β1 protein expression. The cells were stained for intracellular TGF-β1 or for isotype control and analyzed by FACS. Open histograms show TGF-β1 staining; filled histograms show staining for isotype control. Percentages of TGF-β1⁺ cells are shown.

Figure 4

Validation of downstream target genes for miR-124. (a) Alignment of three predicted miR-124 binding sites to C/EBP-α 3′ UTR is shown for different species (Mus musculus, Homo sapiens, Pan troglodytes, Macaca mulatta, Rattus norvegicus and Oryctolagus cuniculus). (b) Western blot analysis of C/EBP-α expression in BMDMs transfected with miR-124 or control miRNA (c) Flow cytometry analysis of the expression levels of C/EBP-α and CD45, or PU.1 and CD45 in BMDMs transfected with miR-124 or control miRNA. Percentages of CD45^hi C/EBP-α⁺ and CD45^hiPU.1⁺ cells are shown in upper right quadrants. Populations of miR-124–transfected CD45^low cells were negative for C/EBP-α and PU.1 expression, as shown in double staining for cell-surface CD45 and intracellular C/EBP-α or PU.1 (lower left quadrants). Staining for CD45 (x axes) and either C/EBP-α, PU.1 or corresponding isotype controls (y axes) are shown. (d) The mean ± s.e.m. of percentages of CD45^hi C/EBP-α⁺ and CD45^hiPU.1⁺ cells from four independent experiments. **P < 0.01. (e) Luciferase activity in NIE115 cells transfected with reporter constructs containing either intact or mutated C/EBP-α 3′ UTR. The NIE115 cell line was co-transfected with the indicated constructs and either miR-124 or control miRNA, and normalized levels of luciferase activity are shown.

Figure 5

Flow cytometry and histology analysis of extent of inflammation and demyelination in the CNS of mice treated with miR-124 or control miRNA. (a,b) EAE disease course (scored as described in Online Methods) in mice treated with miR-124. Mice with EAE were injected intravenously (i.v.) with miR-124 or control miRNA on days 7, 11, 15 and 18 (a) or days 13, 16, 18, 20 and 22 (b) after EAE induction, as indicated by arrows. The data represent average disease scores from three experiments with four or five mice per group. (c) Flow cytometry analysis of immune cell infiltrate in the CNS of mice with EAE treated with miR-124 or control miRNA. Mononuclear cells were isolated from the CNS on day 21 after induction of EAE, stained for CD11b and CD45 and analyzed by FACS. Percentages of resting CD11b⁺CD45^low microglia (region R1), CD11b⁺CD45^hi activated microglia and peripheral macrophages (R2) and CD11b⁻CD45^hi lymphocytes (R3) are shown. (d) Quantification of absolute number of activated microglia and macrophages (CD11b⁺CD45^hi), lymphocytes and CD4 T cells in the CNS of mice treated with either miR-124 or control miRNA. Mean ± s.e.m. of three independent experiments is shown. (e) Histology analysis of extent of inflammation and demyelination in the spinal cords of mice with EAE treated with miR-124 or control miRNA. Spinal cords were harvested on day 21 after induction of EAE, and sections of spinal cord were stained for myelin or CD11b. Each panel shows a representative histopathology image (scale bar, 200 μm); three mice were analyzed. Myelin sheath is stained light blue and nucleated cells are stained dark blue (left images). The cells colored dark gray are positive for CD11b (right images).

Figure 6

Effect of miR-124 inhibitor on phenotype of macrophages cocultured with neural and astroglial cells. (a) Flow cytometry analysis of the expression of CD45 (x axis) and MHC class II (y axis) in populations of CD11b⁺GFP⁺ gated BMDMs cultured alone (left contour plot) or cocultured with an astroglial (middle contour plot) or neuronal (right contour plot) cell line. Percentages of CD45^hiMHC class II⁺ cells are shown in upper right quadrants. BMDMs were isolated from ACTB-GFP transgenic mice that ubiquitously express GFP under the actin promoter. The cells were then analyzed for the expression of GFP, CD11b, MHC class II and CD45 using four-color flow cytometry. (b) qRT-PCR analysis of miR-124 expression in microglia, astroglial and neuronal lines, in BMDMs cultured alone or in CD11b⁺GFP⁺ BMDMs sorted from the cocultures. (c–e) BMDMs isolated from ACTB-GFP transgenic mice were cocultured with either an astroglial (c) or neuronal (d) cell line in the presence of anti–miR-124 or a control antagomir. (c,d) CD11b⁺GFP⁺ gated cells were analyzed for expression of CD45 (x axis) and MHC class II (y axis). Percentages of CD45^hiMHC class II⁺ cells are shown in upper right quadrants. (e) Data from three independent experiments are summarized, which show mean ± s.e.m. of percentages of CD45^hiMHC class II⁺ macrophages. *P < 0.05; **P < 0.01.