Astrocyte CCL2 sustains immune cell infiltration in chronic experimental autoimmune encephalomyelitis

Abstract

Chemokine (C-C motif) ligand 2 (CCL2), initially identified as monocyte chemoattractant protein-1 (MCP-1), recruits immune cells to the central nervous system (CNS) during autoimmune inflammation. CCL2 can be expressed by multiple cell types, but which cells are responsible for CCL2 function during acute and chronic phases of autoimmune disease is not known. We determined the role of CCL2 in astrocytes in vivo during experimental autoimmune encephalomyelitis (EAE) by using Cre-loxP gene deletion. Mice with a conditional gene deletion of CCL2 from astrocytes had less severe EAE late in disease while having a similar incidence and severity of disease at onset as compared to wild type (WT) control littermates. EAE mice devoid of CCL2 in astrocytes had less macrophage and T cell inflammation in the white matter of the spinal cord and less diffuse activation of astrocytes and microglia in both white and gray matter as well as less axonal loss and demyelination, compared to WT littermates. These findings demonstrate that CCL2 in astrocytes plays an important role in the continued recruitment of immune cells and activation of glial cells in the CNS during chronic EAE, thereby suggesting a novel cell specific target for neuroprotective treatments of chronic neuroinflammatory diseases.

Keywords: Astrocytes; CCL2; EAE; Macrophage; Microglia; Multiple Sclerosis; Neuroprotection; T lymphocyte.

Fig. 1

Verification of extent and selectivity of Cre targeting to astrocytes. Immunofluorescence shows complete GFAP (green) with tdTomato reporter protein (red) in healthy, untreated young adult mice mGFAP-Cre-tdTomato reporter mice. Merged images (GFAP+ tdTomato) show co-localization in yellow. Survey images (top row) show dorsal column white matter. Scale bar =100 µm. Boxed areas indicate areas shown in detail below. Scale bar = 25 µm. Arrows indicate double-labeled astrocytes. All detectable astrocytes express both GFAP and tdTomato.

Fig. 2

Verification of CCL2 deletion specificity in GFAP+ astrocytes during EAE. A, Immunofluorescence shows co-localization of CCL2 (green) with GFAP (red) in C57BL/6 wildtype (top row) and CCL2 wildtype littermate controls (CCL2 WT bottom row), but not in mice with astrocyte CCL2 conditional gene deletion (astro-CCL2-CKO middle row) during EAE. Merged images (GFAP + CCL2) show co-localization in yellow. Scale bar, 20 µm. B, Immunofluorescence shows significantly reduced CCL2 expression in the spinal cord dorsal column of astro-CCL2-CKO mice compared to CCL2 WT mice during EAE. Scale bar, 100 µm. C, Quantification shows reduced % CCL2 expression area in the spinal cord dorsal column of astro-CCL2-CKO mice. *p < 0.05 versus CCL2 WT (mean ± SEM).

Fig. 3

Astrocyte CCL2 CKO, as compared to WT littermates, has less severe EAE late in disease. A, Astro-CCL2-CKO mice had significantly better clinical EAE scores late (after EAE day 30) compared with CCL2 WT mice. *p < 0.05 (repeated-measures ANOVA with post hoc Bonferroni pairwise analysis). Graphical representation of experiment #2 from Table. B, Table showing clinical assessments in three separate experiments. Statistical analysis shows significant decrease in the cumulative disease score index after 30 days of EAE induction in astro-CCL2-CKO compared to CCL2 WT. ^aIncidence is the number of mice that developed EAE signs from the total number of mice in each group. ^bMean day (±SEM) of onset was assessed when mice showed first clinical disease signs (score of >1) after EAE induction. ^cMean peak clinical score (±SEM) was assessed when each mouse reached its maximum clinical score. ^dCumulative disease index (±SEM) was calculated by summing the daily clinical scores of each mouse and averaging them within each group. “All days” signifies day 0 of EAE induction to day of sacrifice, while “After Day 30” is from day 30 of EAE to day of sacrifice. *p < 0.05, †p < 0.08 versus CCL2 WT (mean ± SEM).

Fig. 4

Astrocyte CCL2 CKO mice with EAE have a reduction in macrophage and T cell infiltrates in both dorsal and ventral white matter of the spinal cord during EAE. Representative immunofluorescence images of A, Iba-1 (green) globoid macrophage (arrowhead) and B, CD3 (red) T cell (arrow) infiltrates, each reduced in the dorsal (top rows) and ventralateral (bottom rows) white matter of spinal cords during EAE. Scale bar, 100 µm. Quantitative analysis show that astro-CCL2-CKO mice have a significant reduction in C, Iba-1 globoid macrophages and D, CD3 T cells in the dorsal and ventralateral white matter of the spinal cord during EAE. *p < 0.05 versus CCL2 WT (mean ± SEM).

Fig. 5

Selective deletion of CCL2 from astrocytes results in less activation of astrocytes diffusively in the spinal cords during EAE. A, Representative immunofluorescence images show that astro-CCL2-CKOmice have less reactive GFAP⁺ astrocytes (red) in the gray matter (GM) and white matter(WM) of the spinal cord compared to CCL2 WT during EAE. Scale bar, 100 µm. B, Quantitative analysis shows that astro-CCL2-CKO mice have a significant reduction of GFAP expression in both white and gray matter of the spinal cord compared to CCL2 WT. *p < 0.05 versus CCL2 WT (mean ± SEM).

Fig. 6

Selective deletion of CCL2 from astrocytes results in less microglial activation diffusively in the spinal cords during EAE. Representative immunofluorescence image of A, ramified/activated and B, resting microglial cell using Iba-1 staining (green). Scale bar, 10 µm. C and D, astro-CCL2-CKO mice show a reduced number of ramified/activated microglial cells in white matter (WM) and gray matter (GM) of the spinal cord compared to CCL2 WT mice during EAE. Scale bar, 100 µm. E, Quantitative analysis confirms that astro-CCL2-CKO mice have reduced activated microglial cells in the white and gray matter of the spinal cord during EAE. *p < 0.05 versus CCL2 WT (mean ± SEM).

Fig. 7

Astrocyte CCL2 CKO, as compared to WT littermates, has less demyelination and axonal loss during EAE. A and B, Immunofluorescence images of MBP⁺ myelin (green) and NF200⁺ axon (red) in the spinal cord dorsal column of astro-CCL2-CKO and CCL2 WT mice induced with EAE. Left images show entire dorsal column of the spinal cord, with images in boxes shown in higher magnification. Astro-CCL2-CKO, compared to CCL2 WT, has more myelin and axons in the dorsal column shown in individual (left) and merged (right) images. Scale bars; yellow, 100 µm: white, 20 µm. Quantitative analysis shows that astro-CCL2-CKO mice have more C, myelin and D, axons in the dorsal column of the spinal cord compared to CCL2 WT during EAE. *p < 0.05 versus CCL2 WT (mean ± SEM).

Fig. 8

No differences were found between astro-CCL2-CKO mice as compared to WT littermates during the early phase of EAE. A, Astro-CCL2-CKO mice have similar clinical disease scores as WT mice early during EAE. B, Representative immunofluorescence images and quantitative analysis show that astro-CCL2-CKO and WT have similar levels of Iba-1 (green) globoid macrophage (arrowhead) and CD3 (red) T cell (arrow) infiltrates in the spinal cord dorsal column. Scale bar, 100 µm. Quantitative analyses confirm that astro-CCL2-CKO mice and CCL2 WT mice also have similar levels of diffuse activation of C, reactive astrocytes and D, activated microglial cells in the white and gray matter of the spinal cord early during. E, Quantitative analyses show that astro-CCL2-CKO mice and CCL2 WT mice have similar levels of myelin and axons in the dorsal column of the spinal cord.