Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand

Abstract

The central nervous system (CNS) is endowed with several immune-related mechanisms that contribute to its protection and maintenance in homeostasis and under pathology. Here, we discovered an additional mechanism that controls inflammatory responses within the CNS milieu under injurious conditions, involving CD200 ligand (CD200L) expressed by newly formed endothelial cells. We observed that CD200L is constitutively expressed in the mouse healthy CNS by endothelial cells of the blood-cerebrospinal fluid barrier and of the spinal cord meninges, but not by the endothelium of the blood-spinal cord barrier. Following spinal cord injury (SCI), newly formed endothelial cells, located only at the epicenter of the lesion site, expressed CD200L. Moreover, in the absence of CD200L expression by CNS-resident cells, functional recovery of mice following SCI was impaired. High throughput single-cell flow cytometry image analysis following SCI revealed CD200L-dependent direct interaction between endothelial and local CD200R⁺ myeloid cells, including activated microglia and infiltrating monocyte-derived macrophages (mo-MΦ). Absence of CD200L signaling, both in vitro and in vivo, resulted in a higher inflammatory response of the encountering macrophages, manifested by elevation in mRNA expression of Tnfα and Il1β, increased intracellular TNFα immunoreactivity, and reduced expression levels of macrophage factors that are associated with resolution of inflammation, Dectin-1, CD206 (mannose receptor), and IL-4R. Collectively, our results highlight the importance of CD200-mediated immune dialogue between endothelial cells and the local resident microglia and infiltrating mo-MΦ within the lesion area, as a mechanism that contributes to regulation of inflammation following acute CNS injury.

Significance statement: This manuscript focuses on a novel mechanism of inflammation-regulation following spinal cord injury (SCI), orchestrated by CD200-ligand (CD200L) expressed by newly formed endothelial cells within the lesion site. Our study reveals that, in homeostasis, CD200L is expressed by endothelial cells of the mouse blood-cerebrospinal fluid barrier and of the blood-leptomeningeal barrier, but not by endothelial cells of the blood-spinal cord barrier. Following SCI, newly formed endothelial cells located within the epicenter of the lesion site were found to express CD200L at time points that were shown to be critical for repair. Our results reveal a direct interaction between CD200L⁺ endothelial cells and CD200R⁺ microglia and macrophages, resulting in attenuated inflammation, biasing macrophage phenotype toward inflammation-resolving cells, and promotion of functional recovery following SCI.

Keywords: CD200; endothelium; inflammation; microglia; monocyte-derived macrophages; spinal cord injury.

Figure 1.

Spatial distribution of CD200L in the CNS compartments in homeostasis. A, Immunostaining for βIII-tubulin, NeuN, and CD200L of neurons located in the hippocampus, cerebellum, and cortex of brains derived from noninjured WT mice. Scale bar, 500 μm. Data are representative of two different experiments; n = 2 mice in each group, on each experiment. B, Immunostaining for βIII-tubulin and NeuN of neurons expressing CD200L in the DRGs, but not in the spinal cord parenchyma of noninjured WT mice. Scale bar, 100 μm. C, Whole-mount immunostaining for CD31 and CD200L in the choroid plexus derived from noninjured WT mice. D, Immunostaining for CD200L and vimentin⁺ ependymal cells in longitudinal sections of the central canal in the spinal cord of noninjured WT mice. Inset, Immunostaining of coronal section of the central canal. E, F, Whole-mount immunostaining for CD31 and CD200L in the dura (E) and pia (F) mater of meninges derived from noninjured WT mice. G, Immunostaining for CD31 and CD200L in spinal cord parenchyma of noninjured WT mice. H, Whole-mount immunostaining for CD31 and CD200L in the choroid plexus derived from noninjured CD200L^−/− mice. C–H, Scale bar, 50 μm. Data are representative of 2–5 independent experiments; n = 3–5 mice in each group.

Figure 2.

CD200L is involved in the functional recovery following SCI and is upregulated by newly formed endothelial cells within the lesion site. A, Immunostaining for GFAP, to detect reactive astrocytes that surround the lesion site, together with CD200L on days 3, 7, and 14 following SCI. Scale bar, 500 μm. Day 7: Enlarged image of CD200L⁺ Hoechst⁺ cells. Scale bar, 100 μm. B, C, Immunostaining for CD200L and CD31 at the epicenter of the lesion site 7 d following SCI in WT (B) and CD200L^−/− (C) mice. D, Immunostaining for CD200L and CD31 distal to the lesion site 7 d following SCI in WT mice. E, Immunostaining for βIII-tubulin, NeuN, and CD200L showing neurons that are located adjacent to the lesion site 7 d following SCI. Scale bar, 100 μm. F, Immunostaining for CD200L⁺CD31⁺ endothelium positive for Ki67 at the epicenter of the lesion site 7 d following SCI in WT mice. B–D, F, Scale bar, 50 μm. A–F, Data are representative of 2–5 independent experiments; n = 3–5 mice in each group. G, Hindlimb motor function assessed according to the BMS for WT (filled black circles) and CD200L^−/− (hollowed red circles) mice. Left, Motor score as a function of time. F_Interaction(1,152) = 10.2; ***p < 0.001 (repeated-measures two-way ANOVA). Right, Scores of individual mice (d20). ***p < 0.001 (Student's t test). H, Hindlimb motor function, assessed according to the BMS for [WT > CD200L^−/−] (hollowed orange squares) and [CD200L^−/− > WT] (filled gray squares) BM chimeras. Left, Motor score as a function of time. F_Interaction(1,60) = 4.94; ***p < 0.001 (repeated-measures two-way ANOVA). Right, Scores of individual mice (d20). *p = 0.0252 (Student's t test). G, H, Data are representative of two different experiments; n = 10 – 11 mice in each group. *p < 0.05. ***p < 0.001. Data are mean ± SEM.

Figure 3.

Myeloid cell subpopulations expressing CD200R following SCI. A, B, Immunostaining for CD200R together with (A) GFAP, to detect reactive astrocytes, and (B) with isolectin B4 (IB4), to detect the activated myeloid cells (microglia and mo-MΦ), on days 3, 7, and 14 following SCI. Scale bar, 50 μm. A, B, Data are representative of two different experiments; n = 5 mice in each group, on each experiment. C, Flow cytometry histograms and quantification (percentages of CD200R-expressing population), comparing differential CD200R expression at different time points following SCI, between CD11b^lowCD45.2^low resident microglia (orange) and CD11b^highCD45.2^high mo-MΦ (blue), compared with negative control (CD45.2⁻CD11b⁻ population (filled gray); F_{between microglia and mo-MΦ}(1,19) = 126.25; p < 0.001 (repeated-measures ANOVA). n = 2–6 mice per time point. D, Flow cytometry dot-plots and histograms showing CD200R⁺ and CD200R⁻ gates in the CD45.2⁻CD11b⁻ population. E, Left, Flow cytometry gates of CD11b^highCD45.2^high mo-MΦ (blue), CD11b^lowCD45.2^low microglia (orange), and CD45.2⁻CD11b⁻ population (gray). Right, Flow cytometry gates of the proinflammatory mo-MΦ population (CX₃CR1^lowLy6C⁺; green) and of anti-inflammatory mo-MΦ population (CX₃CR1^highLy6C⁻; red). F, Flow cytometry dot-plots showing CD200R⁺ and CD200R⁻ gates in the nonexpressing population, CD45.2⁻CD11b⁻, and in proinflammatory and anti-inflammatory mo-MΦ on day 3 following SCI. G, Percentages of CD200R⁺ cells of the proinflammatory and of the anti-inflammatory mo-MΦ on days 1, 3, 7, and 17 following SCI. F_{between groups (days 3 and 7)}(1,4) = 21.47; **p = 0.0036 (repeated-measures ANOVA). n = 2–6 mice per time point. C–G, Data are merged from two different experiments. *p < 0.05. Data are mean ± SEM.

Figure 4.

Endothelial cells form conjugates with CD200R⁺ myeloid cells via CD200L following SCI. A, Immunostaining for CD31⁺ endothelium, CD200L, and Iba1⁺ activated microglia and infiltrating mo-MΦ at the lesion site 7 d following SCI in WT and CD200L^−/− mice, to examine the spatial distribution of myeloid cells relative to CD31⁺CD200L⁺ endothelium. Scale bar, 100 μm. Data are representative of three different experiments; n = 3–5 mice in each group. B, Image of conjugate between CD11b⁺CD45.2⁺CD200R⁺ myeloid cell and CD11b⁻CD31⁺CD200L⁺ endothelial cell derived from the lesion site 7 d following SCI, acquired by high-throughput single-cell flow cytometry image analysis (ImageStream). Data are representative of two different experiments; n = 5 mice. C, CD45.2⁺CD11b⁺CD31⁺ reads were first gated, then analyzed for distances between cell centers (see Materials and Methods), and finally CD11b⁻CD31⁺ and CD45.2⁺CD11b⁺CD31⁻ cells with definite contact were classified as conjugates (purple gate). D, E, ImageStream quantification of CD11b⁻CD31⁺ and CD45.2⁺CD11b⁺CD31⁻ conjugates incubated ex vivo with PBS (control) compared with (D) cells incubated with CD200L inhibitor peptide (Student's t test_{(one tailed)}, *p = 0.042) or (E) cells incubated with OVA peptide. F, ImageStream images of conjugation between (left) single or (right) clusters of CD11b⁻CD31⁺ endothelial cells and CD45.2⁺CD11b⁺CD31⁻ myeloid cells. D, E, Data are pooled from two different experiments; n = 5 mice in each group. *p < 0.05. Data are mean ± SEM.

Figure 5.

CD200L inhibitor abrogates the ability of endothelial cells to regulate the inflammatory phenotype of macrophages in vitro. A, B, GFP⁺ BM-MΦ were cultured with or without brain endothelial cell-line cells (b.END3) for 24 h. To mimic injury-induced inflammation in vitro, 0.1 μg/ml LPS was added to the cultures for 4 h. GFP⁺ BM-MΦ were collected using FACS sorting, RNA was extracted, and gene expression was analyzed by qRT-PCR. C, GFP⁺ BM-MΦ gene expression of proinflammatory cytokines Tnfα, Il-1β, and Il-6 before and following LPS induction, with (filled black) or without (empty black) b.END3 cells. Tnfα: F_(2,14) = 119.5, ***p < 0.001 (one-way ANOVA). Il-1β: F_(2,14) = 45.28, ***p < 0.001 (one-way ANOVA). Il-6: F_(2,12) = 27.71, ***p < 0.001 (one-way ANOVA). Asterisks above bars without horizontal line indicate significance relative to the [mo-MΦ without LPS] sample. A, D, GFP⁺ BM-MΦ + CD200L inhibitor were cultured with or without b.END3 cells for 24 h. To mimic injury-induced inflammation in vitro, 0.1 μg/ml LPS was added to the cultures for 4 h. GFP⁺ BM-MΦ were collected using the FACS sorter, RNA was extracted, and gene expression was analyzed by qRT-PCR. E, GFP⁺ BM-MΦ gene expression of proinflammatory cytokines Tnfα, Il-1β, and Il-6 before and following LPS induction, with (filled red) or without (empty red) b.END3 cells. Tnfα: F_(2,14) = 78.44, ***p < 0.001 (one-way ANOVA). Il-1β: F_(2,14) = 23.71, ***p < 0.001 (one-way ANOVA). Il-6: F_(2,15) = 7.137, **p = 0.0066 (one-way ANOVA). Asterisks above bars without horizontal line indicate significance relative to the [mo-MΦ without LPS] sample. Data shown from two different experiments were combined, by normalization based on [BM-MΦ+LPS] samples; n = 5 – 6 replicates in each group. **p < 0.01. ***p < 0.001. Data are mean ± SEM.

Figure 6.

Absence of CD200L in vivo promotes a stronger mo-MΦ-derived inflammatory response and prevents anti-inflammatory activities. A, Flow cytometry gates of CD45.2⁺CD11b⁺Ly6C^high mo-MΦ, and their staining for TNFα and its isotype control expression levels at day 7 following SCI. B–D, Flow cytometry: (B) histogram, (C) percentage quantification, and (D) MFI quantification of the CD45.2⁺CD11b⁺Ly6C^high mo-MΦ population expressing TNFα in WT (black) and CD200L^−/− (red) mice at day 7 following SCI. Percentage TNFα: *p = 0.03 (Student's t test). MFI TNFα: *p = 0.03 (Student's t test). Each sample represents a pool of 2 animals; n = 2 – 3 replicates in each group. E, Flow cytometry gates of CD45.2⁺CD11b⁺ myeloid cells (microglia and mo-MΦ) and CD45.2⁺CD11b⁻ population at day 7 following SCI. F, Flow cytometry dot-plots showing gating strategy for the tested surface markers (Dectin-1, CD206, and IL-4R) in the CD45.2⁺CD11b⁻ population. G, H, Flow cytometry: (G) histograms and (H) quantification of the CD45.2⁺CD11b⁺ myeloid cell population expressing Dectin-1, CD206 (mannose receptor) and IL-4R in WT (black), and CD200L^−/− (red) mice at day 7 following SCI. Dectin-1: *p = 0.05 (Student's t test). CD206: **p = 0.01 (Student's t test). IL-4R: *p = 0.04 (Student's t test). Data are representative of two different experiments; n = 2 – 3 mice per group. *p < 0.05. **p < 0.01. Data are mean ± SEM.