Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain

Abstract

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5-7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia.

Figure 1.

Phenotype of Flt3L-responsive CD45^hiCD11c⁺MHC II⁺ cells in the steady-state mouse brain. (A and B) Flow cytometric dot plots of EYFP expression in brain leukocytes (A) and the splenocytes (B) of untreated (top) versus Flt3L-treated (bottom) CD11c-EYFP transgenic mice. Numbers indicate percentage of gated cells. Data are representative of three independent experiments. (C) Flow cytometric dot plots of CD45⁺ mononuclear cells in the brain of untreated (top) versus Flt3L-treated (bottom) nontransgenic C57BL/6 mice. Histograms show MHC II staining on the surface of DCs and microglia (MG) from the indicated gates. Data are representative of more than three independent experiments. (D) Bar graph shows quantification of m/chDCs and microglia 14 d after Flt3L treatment in B6 mice. Bars represent data from two pooled experiments, each with two brains. Error bars represent the mean ± SEM (n = 4; ***, P < 0.0001).

Figure 2.

Flt3L-responsive cells with dendritic morphology within meninges and choroid plexus. (A) Two-photon microscopy showing fluorescent cells in coronal brain sections of untreated CD11c-EYFP mice. EYFP⁺ cells were detected in choroid plexus (C.P.) and parenchyma (top left) and meninges and parenchyma (top right). Small panels at the bottom show the morphology of individual EYFP⁺ cells from choroid plexus, meninges (en face view), and parenchyma. (B) En face two-photon view of the brains of untreated CD11c-EYFP mouse brains. Blood vessels (red) were labeled by perfusion with DiI. EYFP⁺ (green) cells were detected in the meninges. Panels show a major blood vessel in the dura mater (top) and capillary blood vessels in the pia mater (bottom). (C) Two-photon microscopy coronal sections from the brain of untreated (top) and Flt3L-treated (bottom) CD11c-EYFP mice (green, EYFP). (D) Flow cytometric analysis of DCs in meningeal isolates. Dot plots show gated CD45^hiCD11c^hi DCs in CD45⁺ leukocytes of meningeal isolates and whole brain preparation in untreated or Flt3-treated B6 mice. Numbers indicate percentage of each cell type within total CD45⁺ cells. Bar graphs summarize the percentage of m/chDCs and microglia (MG) among CD45⁺ brain leukocytes in untreated versus Flt3L-treated mice in bulk brain and meninges (gating shown in Fig. S1 B). Bars show data from one representative experiment (n = 5 mice per group). Error bars represent the mean ± SD (n = 5). (E) Two-photon microscopy and flow cytometry of EGFP⁺ cells in the brain of I-A^b–EGFP transgenic mice. (microscopy) Observation in meninges 0–30 µm (left) and parenchyma 35–90 µm (middle) from the upper limit of the brain and in choroid plexus (right) from coronal sections of untreated I-A^b–EGFP mice (yellow, EGFP; red, collagen fiber; white, autofluorescence). Flow cytometry histograms show overlay of EGFP expression on microglia and m/chDCs, gated as in Fig. 1 B, from WT B6 (shaded area) and I-A^b–EGFP (line) mice. Data are representative of two independent experiments. Bars: (A, C, and E) 50 µm; (B) 100 µm.

Figure 3.

m/chDCs express Flt3 and have an intrinsic dependence on Flt3L. (A) Histograms show cell surface expression of Flt3 receptor and M-CSF receptor (CD115; line) or isotype antibody control (shaded area) on gated microglia and m/chDCs in the brain versus CD8⁻ and CD8⁺ DC subsets in the spleen (Sp). (B) Bar graph shows percentage of m/chDCs and microglia among CD45⁺ brain leukocytes in WT versus Flt3L KO mice. Bars represent data from two pooled experiments (n = 4). Error bars represent the mean ± SEM (n = 4). (C) Lethally irradiated CD45.1⁺CD45.2⁺ F1 recipient mice were reconstituted with a mixture of 50% CD45.1⁺ Flt3 KO and 50% CD45.2⁺ WT bone marrow and analyzed 2–3 mo later. Bar graph shows the percentage of indicated cells from Flt3 KO CD45.1⁺ (open) and WT CD45.2⁺ (red) donor or CD45.1⁺CD45.2⁺ recipient (blue) origin. Bars represent data from three animals. Error bars represent the mean ± SEM (n = 3; *, P < 0.05; **, P < 0.01). Dot plots show percentage of donor and recipient cells in gated cell populations analyzed by flow cytometry, representing two independent experiments.

Figure 4.

Gene expression profiles of m/chDC versus microglia, monocytes, and spleen DCs. Bone marrow monocytes, spleen (Spl) CD8⁺ and CD8⁻ DC subsets from untreated B6 mice and brain m/chDCs, and microglia from B6 mice treated with Flt3L were purified, and mRNA was extracted for Affymetrix gene array analysis. (A–E) Graphs show normalized data comparison among the indicated cells for expression of receptors for growth factors (A), cell surface markers (B), pattern recognition molecules (C), antigen presentation and co-stimulatory molecules (D), and transcription factors (E). Error bars indicate mean ± SEM (n = 3).

Figure 5.

Origin and differentiation of brain m/chDCs. (A) The indicated numbers of purified MDPs, CDPs, pre-DCs, and monocytes from bone marrow of CD45.1⁺CD45.2⁺ mice were transferred into naive CD45.2⁺ congenic hosts. Dot plots show the phenotype of m/chDCs, and the numbers indicate percentages derived from the donor 7 d after transfer. (B) Dot plots show percentages of CD45⁺Lin⁻CD11c⁺MHC II⁻Flt3⁺SIRPα^lo pre-DCs among bulk brain and meningeal leukocytes. Numbers in the parentheses represent the mean pre-DC percentage of CD45⁺ leukocytes (red arrows, pre-DCs). Histograms show GFP expression of gated pre-DCs in CX₃CR1^gfp (line) versus WT (shaded) mice. (C) CD45.1 and CD45.2 mice were surgically joined for 60 d. Graph shows the percentage of partner-derived T cells, blood pre-DCs, spleen DCs, brain m/chDCs, and microglia. Dot plots show representative percentages of CD45.1 and CD45.2 cells among m/chDCs and microglia in CD45.1 and CD45.2 parabionts. The bar graph shows two independent experiments with more than three mice in total. (D) CD45.2 and CD45.1 B6 mice were surgically joined for 60 d together before being separated. Graphs show the percentage of parabiont-derived DCs among each DC subset in the spleen and brain at different time points after separation. Bars represent two separate parabionts at each time point. (C and D) Error bars show mean ± SEM.

Figure 6.

Antigen presentation by m/chDCs. (A) Flow cytometry histograms show Y-Ae antibody (line) or isotype (shaded area) staining of gated spleen DCs, brain m/chDCs, and microglia from B6 or BALB/c × B6 (CB1) mice. (B) CFSE-labeled BALB/c T cells were incubated with spleen DCs, brain m/chDCs, or microglia (MG) isolated from B6 mice treated with Flt3L and were analyzed with flow cytometry 4 d later. Graph shows percentage of proliferated T cells among total CD3⁺ T cells, indicated by dilution of CFSE. One of three representative experiments is shown. (C) CFSE-labeled 2D2 MOG-specific CD4⁺ transgenic T cells were incubated with spleen DCs, brain m/chDCs, or microglia isolated from B6 mice treated with Flt3L with the indicated doses of MOG peptide and were analyzed by flow cytometry 4 d later. Graph shows percentage of proliferated (CFSE^lo) T cells among total CD3⁺ T cells. One of three representative experiments is shown. (B and C) Error bars show the SD between triplicate wells.