Microglia and meningeal macrophages depletion delays the onset of experimental autoimmune encephalomyelitis

Abstract

In multiple sclerosis and the experimental autoimmune encephalomyelitis (EAE) model, both resident microglia and infiltrating macrophages contribute to demyelination as well as spontaneous remyelination. Nevertheless, the specific roles of microglia versus macrophages are unknown. We investigated the influence of microglia in EAE using the colony stimulating factor 1 receptor (CSF-1R) inhibitor, PLX5622, to deplete microglial population and Ccr2^RFP/+ fms^EGFP/+ mice, to distinguish blood-derived macrophages from microglia. PLX5622 treatment depleted microglia and meningeal macrophages, and provoked a massive infiltration of CCR2⁺ macrophages into demyelinating lesions and spinal cord parenchyma, albeit it did not alter EAE chronic phase. In contrast, microglia and meningeal macrophages depletion reduced the expression of major histocompatibility complex II and CD80 co-stimulatory molecule in dendritic cells, macrophages and microglia. In addition, it diminished T cell reactivation and proliferation in the spinal cord parenchyma, inducing a significant delay in EAE onset. Altogether, these data point to a specific role of CNS microglia and meningeal macrophages in antigen presentation and T cell reactivation at initial stages of EAE.

Fig. 1. PLX5622 microglial depletion provokes a delay in the onset of EAE and massive infiltration of macrophages.

A (Up) Scheme showing the paradigm of microglial depletion with PLX5622. (Down) Representative images showing microglial (Iba1⁺ cells) depletion in both white and gray matter of healthy spinal cord. Histogram shows the loss of microglia after PLX5622 treatment, in comparison to control mice (n = 5). Scale bar = 25 µm. B Representative images of Iba1⁺ MNR⁺ meningeal macrophages in the spinal cord of control mice and mice after complete PLX5622 treatment. Histogram shows the loss of meningeal macrophages after PLX5622 treatment, in comparison to control mice (n = 4). Scale bar = 25 µm. C (Left) Neurological score of control and PLX5622-treated mice, (n = 20–25 mice from three independent EAE experiments). (Right) Histograms showing the onset day of clinical signs of every mouse (other clinical parameters are shown in Supplementary Fig. 1) D Plots depicting the strategy to distinguish resident microglia (CD11b⁺/CD45^low), invading macrophages (CD11b⁺/CD45^hi) and lymphocytes (CD11b^-/CD45^hi) in EAE spinal cords from control and PLX5622-treated mice. Histograms show the quantification of all the populations, in relation to the total number of analyzed cells in the samples (n = 3 mice per group) E Representative images of fms⁺ microglia (green, arrows) and Ccr2⁺ macrophages (red) in the gray and white matter of control and PLX5622-treated Ccr2^RFP/+fms^EGFP/+ mice at EAE chronic phase (dpi 30–35). Dashed line delineates white matter lesions. Histograms at the right show the quantification of microglia (fms⁺ CCR2⁻ cells) and macrophages (CCR2⁺ cells) in each region (n = 4). Scale bar = 20 µm. F Representative images of fms⁺ MNR⁺ meningeal macrophages (arrows) in the spinal cord of control and PLX5622-treated EAE mice. Scale bar = 25 µm. Histogram shows the number of meningeal macrophages in relation to the area of the meninges analyzed (n = 5). Data are presented as means ± SEM. *p < 0.05, ***p < 0.001.

Fig. 2. PLX5622 microglial depletion does not alter EAE chronic phase.

A Representative images of EAE lesions in the lumbar spinal cord of control and PLX5622-treated mice (dpi 30–35). The histogram shows the percentage of lesioned white matter versus total white matter (n = 5). Scale bar = 50 µm. B Representative images showing the accumulation of CD3⁺ T cells and B220⁺ B cells in EAE lesions in control and PLX5622-treated at EAE chronic phase (dpi 30–35). Scale bar = 25 µm. Histograms show the number of T cells and B cells normalized to total white matter (n = 4). C Representative images of GFAP astrocyte immunoreactivity, indicative of astrogliosis, in lesioned and non-lesioned white matter of control and PLX5622-treated mice at EAE chronic phase (dpi 30–35; n = 3). Dashed lines delineate white matter lesions in all images. Insets show higher magnification of the indicated boxes. Data are presented as means ± SEM.

Fig. 3. PLX5622 does not alter peripheral immune priming after immunization.

A Flow cytometry gating strategy for analysis of immune populations in the spleen and peripheral blood of mice at EAE pre-onset (dpi 8–9). Quantification of αβ T cells (CD3⁺ γδTCR^-), γδ T cells (CD3⁺ γδTCR⁺), CD4 T cells (CD3⁺ CD4⁺) CD8 T cells (CD3⁺ CD8⁺), dendritic cells (CD11b⁺ CD11c⁺), neutrophils (CD11b⁺ Ly6G+), and macrophages/monocytes (CD11b⁺ CD11c^- Ly6G^-) populations in spleen (B) and blood (C), in relation to the total cells analyzed at this stage (n = 10). D Concentration of cytokines in blood serum from control and PLX5622-treated mice at EAE pre-onset stage (n = 8). E Relative mRNA expression of Ror, Foxp3 and Ifnγ in spleen and lymph nodes of both mice groups, at EAE pre-onset stage (dpi 9; n = 5). Data are presented as means ± SEM.

Fig. 4. PLX5622 does not alter early, CNS-related events at the pre-onset EAE.

A Flow cytometry gating strategy for analysis of immune populations from the spinal cord of mice at EAE pre-onset (dpi 8–9). B Quantification of the same populations as in the previous figure plus microglia (CD11b⁺ CD45^low) in spinal cord, in relation to the total number of analyzed cells, at this stage (n = 5). C Relative mRNA expression of Ror, Foxp3 and Ifnγ in the spinal cord of both mice groups, at EAE pre-onset stage (n = 3). D Representative images showing Evans Blue staining restricted to CD31⁺ blood vessels and lack of BBB disruption in the spinal cord of both control and PLX5622-treated mice. Scale bar = 50 µm. Data are presented as means ± SEM. ***p < 0.001.

Fig. 5. PLX5622 provokes a delay in CNS infiltration of immune cells.

A Histogram showing the neurological score in control and PLX5622-treated mice (used for FACS analysis). B Quantification of immune populations in spleen and peripheral blood, in relation to the total number of analyzed cells at this timepoint (n = 10). Gating strategy is specified in Fig. 3A. C Quantification of immune populations in spinal cord, in relation to the total number of analyzed cells, at EAE onset (dpi 13–14; n = 10). Gating strategy is specified in Fig. 4A. D Histogram showing the neurological score in control and PLX5622-treated mice at dpi 14 (used for immunohistochemistry and qPCR). E (Left) Representative images of CD3⁺ T cells, B220⁺ B cells and Ly6G⁺ neutrophils infiltration into spinal cord parenchyma. (Right) Histogram shows the number of cells per total white matter area in control and PLX5622-treated mice (n = 5). Scale bar = 30 µm. Representative images and histogram showing the quantification of fms⁺ microglia (fms⁺ Ccr2^- cells; green) and Ccr2⁺ macrophages (red) in control and PLX5622-treated Ccr2^RFP/+ fms^EGFP/+ mice at EAE onset (dpi 14; F) and at a timepoint coincident with the beginning of symptoms in both groups (dpi 14 for control mice and dpi 20 for PLX5622-treated mice: G) (n = 7). Scale bar = 50 µm. Data are presented as means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001.

Fig. 6. Microglial depletion with PLX5622 alters antigen presentation and T cell reactivation in the spinal cord.

Histograms showing MHC-II, CD80 and CD86 fluorescence intensity in different APCs (dendritic cells (DCs), macrophages/monocytes (MCs/MOs) and microglia (Mglia)) and in diverse tissues, as analyzed by flow cytometry at the pre-onset stage of EAE (A; dpi 8–9) (n = 5 spinal cord samples, n = 10 spleen and blood samples), and at the onset stage of EAE (B; dpi 13–14) (n = 10). C Representative images showing CD3⁺ lymphocytes and Ki67 proliferative-associated expression in spinal cord, at EAE onset (dpi 14) in control and PLX5622-treated mice. Histogram show the proportion of Ki67⁺ CD3⁺ T cells in relation to the total number of CD3⁺ T cells (n = 3). Scale bar = 25 µm. D Representative images showing the early Ki67⁺ CD3⁺ proliferating lymphocytes in the spinal cord at EAE onset (dpi 10, D). Scale bar = 30 µm. E Representative images and quantification of CD3⁺ lymphocytes proliferation at a timepoint coincident with the beginning of symptoms in both groups (dpi 14 for control mice and dpi 20 for PLX5622-treated mice (n = 3). Scale bar = 25 µm. Data are presented as means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001.