New tools for studying microglia in the mouse and human CNS

Abstract

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Keywords: RNAseq; developmental neuroscience; glia; macrophage; microglia.

Fig. 1.

Tmem119 is specifically expressed by parenchymal myeloid cells in the CNS. (A) In situ hybridization of mouse brain revealed widespread Tmem119 expression by myeloid (C1q⁺) cells. Composited from two adjacent imaging fields. (B) Sagittal brain schematic of panel locations. (C) Higher power showing C1q⁺ Tmem119⁺ and a rare parenchymal C1q⁺Tmem119⁻ cell (arrow). C1q⁺ cells in the choroid plexus (D) and meninges (E) were Tmem119⁻ (arrows). (F) Tmem119 mRNA was not detected in Cd163⁺ perivascular macrophages (arrows; dotted line highlights one vessel). (G) qPCR analysis of Tmem119, Csf1r, and Cx3cr1 expression by whole tissues and Tmem119 by CD45⁺ cells compared with average expression of each gene in whole brain. Data presented as mean ddCT ± SEM; *P < 0.01 compared with whole brain Tmem119; **P < 0.02 by ANOVA with post hoc Tukey HSD for dCT values. n = 4–9 except BM, CD45⁺ liver, CD45⁺ thymus where n = 2. x, no sample for CD45⁺ BM. (Scale bars: 100 μm in A, D, and E; 40 μm in C and F.) See also SI Appendix, Fig. S1.

Fig. 2.

Monoclonal antibodies reveal microglia-specific Tmem119 IR. (A) ICD Tmem119 mAbs stained all Cx3cr1-GFP⁺ cells except in meninges (arrow and Inset) in Tmem119 WT but not KO brain. [Scale bars, 100 μm in A (50 μm in Inset).] Tmem119 protein localized to microglia cell surface, by epifluorescence (B) and confocal microscopy (C). (Scale bars, 10 μm in B, 7 μm in C.) (D) Tmem119 IR is limited to myeloid cells in CNS parenchyma and was not detected in choroid plexus (outlined with dotted line) Iba1⁺ macrophages or liver (E). (Scale bars, 50 μm in D and E.) (F) FACS plot schema showing cell populations revealed by CD11b and CD45 expression at P60 (Left). Tmem119 (TM⁺) is restricted to CD45^loCD11b⁺ microglia (blue cell population, Right). (G) ECD mAbs are specific, as revealed by staining in WT but not KO brains (G). See also SI Appendix, Fig. S2.

Fig. 3.

Tmem119 is a developmentally regulated but stable microglia marker. (A) Schematic showing timing of Tmem119 IR in microglia. (Scale bars, 50 μm.) (B) Tmem119 and Iba1 IR in brain sections from different ages (maximum intensity projection, MIP). (C) Percent Tmem119⁺ cells by age and post-LPS treatment, using ECD mAb. Bars show mean± SEM *P < 0.01 P14–P60; **P < 0.01 all ages; n.s., no significant differences by ANOVA with Tukey HSD. n = 2 experiments of two animals (P10 and older) or one litter (under P10) for each age/condition, except for P7 (n = 3), P21 (n = 3), P60 (n = 8, 3 PBS injected pooled with naïve). (D) Tmem119 and Iba1 IHC 3 d post-PBS or LPS reveals no gross reduction in Tmem119 IR (MIP). (Scale bars, 20 μm.) (E) Representative MIP of optic nerve crush site in 120 d CCR2-RFP^+/− mouse. Tmem119 (T) IR (white arrows) and CCR2-RFP (C, yellow arrows) detected at the crush site. There are no Tmem119⁺RFP⁺ cells. Autofluorescence channel (auto) is digitally subtracted from merge (T/C/D) to differentiate microglia (green) from infiltrating cells (red) in debris-filled crush site. (Scale bars, 25 μm.) (F) GFP⁺ donor BM cells in the CNS 6 mo posttransplant are Tmem119⁻ in the parenchyma (yellow) and out (white) despite the presence of Tmem119⁺ microglia. (Scale bars, 20 μm.) See also SI Appendix, Fig. S3.

Fig. 4.

Isolation and RNAseq profiling of microglia using Tmem119 IR. (A) Schematic showing streamlined microglia isolation procedure for minimizing baseline microglia reactivity and death. Abbreviations: abs, antibodies; CV, column volumes; TM, Tmem119. (B) Heatmap of Spearman correlation between individual microglia, myeloid, and whole-tissue RNAseq replicates for genes expressed FPKM > 5. (C) Four-way diagram demonstrating top 100 expressed genes by microglia (Tmem119⁺ except for E17) at each age. (D) Venn diagram showing number of differentially expressed genes between P7 Tmem119⁺ and mature microglia (P14, P21, P60). Black numbers outside diagram represent the few differentially expressed genes between these time points. Diagrams adapted from jVenn (58). See also SI Appendix, Fig. S4 and Dataset S1.

Fig. 5.

Developmental changes in microglial gene expression. Bar plots of RNAseq data showing FPKM expression of selected microglia-enriched (A) and proliferation marker genes (B) by developing microglia and myeloid cells. Error bars depict mean ± SEM **P < 0.01, *P < 0.05 for values compared with each P60, P21, and P14 microglia; ^#P < 0.01 compared with P60. (C) Representative Ki67, Tmem119, and Iba1 epifluorescence image quantified in D, showing Iba1⁺Ki67⁺ (white arrowheads), Iba1⁺only (cyan), and Ki67⁺only (yellow) cells. Auto: autofluorescence. (D) Box-and-whisker plots depicting percentage of Iba1⁺ cells that were Ki67⁺ (Left) and Tmem119⁺ (Right). n.d., no difference; *P < 0.01 compared each with P6, P12, and P60; **P < 0.01 compared with P6; ***P < 0.01 compared with P6 and P12; n = 2 mice each, quantified four to eight fields from two to three sections. Box/whiskers represent standard values. All P values calculated by one-way ANOVA with Tukey HSD and verified by pairwise t test with Bonferroni correction. (E) In situ of S100a8 at E17 and P60, the latter 24 h after LPS injection, revealing bright, S100a8-expressing cells not detected in naïve P60 brain (not shown). (Scale bars, 50 μm.) See also SI Appendix, Fig. S5 and Tables S2–S8.

Fig. 6.

Tmem119 is also highly enriched in and specific to human microglia. (A) Relative expression of TMEM119, CD11B, and CX3CR1 mRNA in human peripheral blood leukocytes (PBL), whole brain (WB), and CD11B⁺ brain cells *P < 0.01 by ANOVA with Tukey HSD. Error bars show SEM, for n = 2 independent samples per tissue type. (B) Representative confocal images of 51-y-old normal appearing cortical tissue showing localization of TMEM119 (green) to highly ramified, Iba1⁺ cells (red) at low (Upper) and high (Lower) magnification. Autofluorescence channel (red) overlays green to highlight nonspecific fluorescence from lipofuscin (arrows), which are absent in Lower because of Sudan Black treatment. (Scale bars, 30 μm.)