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. 2018 Jun 19;23(12):3501-3511.
doi: 10.1016/j.celrep.2018.05.048.

Sex-Specific Features of Microglia from Adult Mice

Alessandro Villa  1 , Paolo Gelosa  2 , Laura Castiglioni  3 , Mauro Cimino  4 , Nicoletta Rizzi  3 , Giovanna Pepe  1 , Federica Lolli  3 , Elena Marcello  3 , Luigi Sironi  5 , Elisabetta Vegeto  1 , Adriana Maggi  6
Affiliations

Sex-Specific Features of Microglia from Adult Mice

Alessandro Villa et al. Cell Rep. .

Abstract

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

Keywords: cell transfer; estrogens; ischemic stroke; microglia; neuroinflammation; sexual differentiation.

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Figures

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Graphical abstract
Figure 1
Figure 1
Microglia Transcriptome in the Male and Female Mouse Brain (A) Volcano plot of RNA-seq data obtained from microglia isolated from male and female adult C57BL/6 mice. Analyses were conducted on two pools of six brains each, a total of 12 males and 12 females. Blue dots represent genes for which RPKM values are significantly higher in males than in females (204 genes; p < 0.01 by Benjamini-Hochberg correction). Red dots represent genes for which RPKM values are significantly higher in females than in males (342 genes; p < 0.01). (B) Gene Ontology and ChIP enrichment analysis (ChEA) transcription factor (TF) term enrichment analysis of transcripts with higher expression in male (blue) or in female (red) microglia. (C) In vivo imaging of luciferase expression in male and female NFκB-luc2 mice. Bioluminescence is measured ventrally and whole body, and the pseudocolors represent radiance (p/s/cm2/sr). The image is representative of three independent measures on three mice per group per experiment. In the graph, each line corresponds to average radiance ± SEM. (D) Luciferase activity measured in extracts of microglia isolated from the whole brains of male (n = 3) and female (n = 3) NFκB-luc2 mice and expressed as relative luciferase units (RLUs) per microgram of proteins. Each sample was measured in triplicates. Lines represent the mean ± SEM of n = 3. ∗p < 0.05 by unpaired, two-tailed t test.
Figure 2
Figure 2
The Role of Estrogens in Preventing Microglia Inflammatory Phenotype (A) Heatmap and hierarchical clustering of expression profiles for a subset of NF-κB-regulated genes measured in microglia, isolated from adult C57BL/6 mice: males, intact females (Cyc Met), and ovariectomized females treated with vehicle (OVX) or 17β-estradiol for 3 hr (OVX + E2 3 hr) and 24 hr (OVX + E2 24 hr). The group size was n = 2 samples per condition, each sample consisting of a pool of six brains. The results were log-transformed, normalized, and centered, and populations and genes were clustered by Pearson correlation. Data were obtained from two pools of six brains each. The right panel shows general expression plots for each cluster. The purple line represents the mean expression of the clustered genes. (B) Immunocytochemistry of microglia stained with Iba1 antibody (red signal) showing different phenotypes in males or females. Upper panel: details of microglia (×100 magnification); lower panel: lower magnification images (×20) including neurons expressing EGFP (green). In the graph, each column corresponds to the percentage of cells showing either the resting or the activated phenotype. We blind-counted the cells present in 18 ×20 images (for about 200 cells/sex in total). The blind count was repeated by three different operators. ∗p < 0.05 by unpaired, two-tailed t test. Scale bar: 10 μm. (C and D) qPCR analyses of genes showing higher expression in male (C) or female (D) microglia. Data are expressed as 2−ΔΔCt using the 36B4 transcript as an internal reference standard. Each column represents the mean ± SEM of n = 6 plates measured in triplicate. ∗p < 0.05; ∗∗p < 0.01 by a one-way ANOVA and Tukey’s method for multiple comparisons versus expression in neurons + male microglia (blue column).
Figure 3
Figure 3
Female Microglia Maintain Their Characteristic Gene Expression When Transplanted in Male Brain (A) Bioluminescence-based optical imaging of brain slices of WT male mice before (left panel) and after (3 days, central panel; 5 days, right panel) transnasal administration of 400,000 bioluminescent microglial cells isolated from female G4063 mice; pseudocolors represent the intensity of light emission (p/s/cm2/sr). The images are representative of three independent measures on n = 3 individual animals/group. (B) Three-dimensional representation of the distribution of bioluminescent signals in WT mouse brain, 5 days after transnasal administration of bioluminescent microglial cells isolated from G4063 mice. Pseudocolors represent the intensity of light emission (p/s/cm2/sr), according to the color bar reported in (A). (C) RT-PCR analysis was performed using primers for Xist mRNA (amplicon: 124 bp) on microglia RNA isolated from naive male (M) or female (F) mice, vehicle-treated male mice (Veh, 5 days [5d]) or after transnasal administration of 400,000 microglial cells isolated from female C57BL/6 mice (F/M 3d: microglia isolated 3 days after transnasal administration; F/M 5d: microglia isolated 5 days after transnasal administration). Bars represent the percentage ratio of Xist and C1qa mRNA accumulation in naive female microglia (two independent measures, n = 3) or in microglia derived from males, 5 days after transnasal administration of 400,000 microglial cells isolated from female C57BL/6 mice (5d: microglia isolated 5 days after transnasal administration, two independent measures, n = 3). Data are normalized to naive female mice. (D) qPCR analyses of C1qa, Xist, Ki67, and Cdk3 mRNA accumulation in microglia isolated from naive female (F) or male (M) mice or 5 days after transnasal administration of 400,000 microglial cells isolated from female (F/M) or male (M/M) C57BL/6 mice. Data are expressed as 2−ΔΔCt using the 36B4 transcript as an internal reference standard. Columns represent the mean ± SEM of n = 6 animals measured in triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by a one-way ANOVA and Tukey’s method for multiple comparisons versus (F). #p < 0.05 by a two-way ANOVA and Tukey’s method for multiple comparisons. (E) qPCR analyses of Shank3, Fxyd1, Aqp1, and Timp3 mRNA accumulation in microglia isolated from naive female (F) or male (M) mice or fluorescence-sorted 5 days after transnasal administration of 400,000 microglial cells isolated from female (F/M) or male (M/M) CX3CR1-GFP mice. Data are expressed as 2−ΔΔCt using the 36B4 transcript as an internal reference standard. Columns represent the mean ± SEM of n = 6 animals measured in triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by a one-way ANOVA and Tukey’s method for multiple comparisons versus (F). #p < 0.05 by a two-way ANOVA and Tukey’s method for multiple comparisons. (F) qPCR analyses of Akt1s1, Trem1, S100a9, and Cxcl2 mRNA accumulation in microglia isolated from naive female (F) or male (M) mice or fluorescence-sorted 5 days after transnasal administration of 400,000 microglial cells isolated from female (F/M) or male (M/M) CX3CR1-GFP mice. Data are expressed as 2−ΔΔCt using the 36B4 transcript as an internal reference standard. Columns represent the mean ± SEM of n = 6 animals measured in triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by a one-way ANOVA and Tukey’s method for multiple comparisons versus microglia isolated from naive female mice. #p < 0.05; ###p < 0.001 by a two-way ANOVA and Tukey’s method for multiple comparisons. (G) qPCR analyses of Akt1s1, Trem1, S100a9, and Cxcl2 mRNA accumulation in microglia isolated from naive male (♂), female (♀), or masculinized female (⚥) C57BL/6 mice. Data are expressed as 2−ΔΔCt using the 36B4 transcript as an internal reference standard. Columns represent the mean ± SEM of n = 6 animals measured in triplicates. ∗p < 0.05 by a one-way ANOVA and Tukey’s method for multiple comparisons versus ♂.
Figure 4
Figure 4
Sex of Microglia Influence the Progression of Ischemic Stroke (A) Time-dependent evolution of infarct volume in permanent middle cerebral artery occlusion (pMCAO) mice. Diffusion-weighted imaging (DWI) representative of male mice transnasally administered with male (M/M) or female (F/M) microglia, taken at 2, 24, and 48 hr after pMCAO. The ischemic lesion is detectable as a hypointense area in the right cerebral hemisphere (delineated by the yellow dotted line). The image is representative of n = 8 animals. (B) Scatterplot showing the quantitative analysis of the brain damage volume determined by DWI measurements and expressed as percent change relative to the initial 2 hr value set to 100%. Solid lines represent mean ± SEM for M/M (n = 8) and F/M mice (n = 8). ∗∗p < 0.01; ∗∗∗p < 0.001 by two-way ANOVA and Sidak’s method for multiple comparisons. (C) Representative, low-magnification (×20) immunofluorescence analysis of coronal sections of brains excised from adult mice that had undergone pMCAO, stained for Iba1 (white), CD16/32 (green), Ym1 (red), and Hoechst 33258 (blue). M/M: male recipient transplanted with male microglia; F/M: male recipient transplanted with female microglia. Images are representative of n = 3 mice per experimental group and show the fluorescence images acquired in the areas indicated by red squares in the upper schematics. Scale bar: 10 μm. (D) Representative high-magnification (×100) confocal z stack projections of cells stained for Iba1 (white), CD16/32 (green), Ym1 (red), and Hoechst 33258 (blue) in mice that had undergone pMCAO. The pictures show the colocalization of pro- (CD16/32) and anti-inflammatory markers with Iba1-stained microglia. M/M: male recipient transplanted with male microglia; F/M: male recipient transplanted with female microglia. Images are representative of n = 3 mice per experimental group. Scale bar: 10 μm. (E) Graph columns show the mean ± SEM of fluorescence brightness for Ym1 (red channel) in Iba1-positive cells, measured in a double-blind manner. ∗p < 0.05 by unpaired, two-tailed t test.
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