Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

Abstract

Background: Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development.

Methods: We performed a genetic analysis of wild type and CD73-/- (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling.

Results: We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73-/- mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS.

Conclusions: We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.

Figure 1

Chemokine, chemokine receptor, and adhesion molecule genetic expression in the brains of wild type and CD73−/− mice following EAE induction. (A) EAE disease profile of wild type (n = 8) and CD73−/− mice (n = 10). Error bars represent the s.e.m. Significant differences are indicated as determined by two-way ANOVA. EAE data is combined from 2 separate experiments. (B) CD45 and CD4 stained frozen brain sections (cerebellum) from day 14 post-EAE induction wild type and CD73−/− mice. Positively stained cells (red) are shown against a hematoxylin counterstain (blue). Black scale bars represent 50 μm. (C) Brain gene expression from wild type and CD73−/− mice with EAE over time (n = 5 mice per group) as determined by quantitative real-time PCR. Gene levels were normalized to GAPDH levels and displayed in a heat map as a ratio of wild type to CD73−/− to determine fold differences in expression at each time point. (D) Brain gene expression from wild type mice treated with the broad spectrum adenosine receptor agonist NECA (0.25 mg/kg) or DMSO vehicle control as determined by quantitative real-time PCR 4 hours post treatment. Gene levels were normalized to GAPDH levels and displayed in a heat map as a ratio of NECA to vehicle treated to determine fold differences in expression.

Figure 2

CX3CL1 upregulation within the brain during EAE is associated with A2A adenosine receptor signaling. (A) CX3CL1 gene expression (relative to DMSO vehicle) in the brains of wild type mice following 4 hour A2A adenosine receptor agonist CGS21680 treatment as determined by quantitative real-time PCR. Error bars represent the s.e.m.; n ≥ 3 mice / treatment. Significant differences (P < 0.05, *; P < 0.001, ***) are indicated as determined by the Student’s t-test. (B) EAE disease profile in wild type mice given either the A2A adenosine receptor antagonist SCH58261 (1 mg/kg, open diamonds, n = 8; 5 mg/kg, open triangles, n = 7) or a vehicle control (n = 8). Error bars represent the s.e.m. EAE data is combined from 2 separate experiments. (C) CD45 and CD4 stained frozen brain sections (hippocampal area) from day 10 post-EAE induction SCH58261 and vehicle treated wild type mice. Positively stained cells (red) are shown against a hematoxylin counterstain (blue). Black scale bars represent 50 μm. (D) Brain CX3CL1 expression over time in wild type mice induced to develop EAE and given either SCH58261 or a vehicle control as determined by quantitative real-time PCR. Error bars represent the s.e.m. Significant differences (P < 0.001, ***) are indicated as determined by the Student’s t-test.

Figure 3

CX3CL1 expression in the brain during EAE progression. (A) Brain CX3CL1 expression of wild type mice with EAE as determined by quantitative real-time PCR. Gene levels were normalized to GAPDH levels and are displayed as levels relative to naïve mice. Error bars represent the s.e.m. (n = 5 mice per group). (B-M) Brains from naïve wild type mice were harvested and frozen for immunostaining. CX3CL1 expression (red) and DAPI nuclei staining (blue) in (B, F, J) naïve and (C, G, K) Day 10, (D, H, L) Day 14, and (E, I, M) Day 21 post-EAE induced wild type mice. CX3CL1 expression is displayed at the (B-E) choroid plexus, (F-I) in and near the hippocampus, and (J-M) cerebellum. White scale bars represent 50 μm. (N) CX3CL1 expression (relative to non-treated cells) in the CPLacZ-2 mouse choroid plexus cell line after 2 hour treatment with varying concentrations of the A2A adenosine receptor specific adenosine receptor agonist CGS21680. Error bars represent the s.e.m. (O) Lymphocyte migration across a transwell choroid plexus barrier following pretreatment with vehicle treatment alone, CGS21680, or CGS21680 and anti-CX3CL1. Total migration was normalized to the vehicle control (set to 100%). Error bars represent the s.e.m. These results are representative of two separate experiments (n ≤ 3).

Figure 4

The CX3CR1 receptor for CX3CL1 is expressed on immune cells. CX3CR1 expression was assessed on leukocytes isolated from the spleens of naïve mice that express GFP driven by the CX3CR1 promoter (CX3CR1-GFP/GFP mice). Cells were stained with antibodies against CD4, CD8, B220 (B cells), CX11b, F480 (macrophages), and CD49b (NK cells) and analyzed via flow cytometry. The antibody positive gated populations are displayed as histograms with the percentage of CX3CR1 expressing cells within that population noted above each histogram subgate. These results are representative of two separate experiments (n = 4 mice).

Figure 5

CX3CL1 antibody mediated blockade protects mice against EAE and its associated lymphocyte infiltration. Wild type mice were induced to develop EAE and starting at day 8 post induction given daily anti-CX3CL1 antibody or an isotype control treatments (i.p.). (A) EAE disease profile. Error bars represent the s.e.m. (n = 4 mice/group). Significant differences are indicated as determined by two-way ANOVA. EAE scoring data is representative of 2 separate experiments. (B) CD45, CD11b, and F480 stained brain (hippocampal and cerebellum areas) and spinal cord sections from day 28 post-EAE induced mice treated with either anti-CX3CL1 or control antibody. Positively stained cells (red) are shown against a hematoxylin counterstain (blue). Black scale bars represent 50 μm. (C) CD4 and (D) CD8 positive mean cells counts per field at 10x magnification from brain and spinal cord stained frozen brain sections from day 28 post-EAE induced mice treated with either anti-CX3CL1 or control antibody. Error bars represent the standard error of the mean (n ≤ 11). Significant differences (P < 0.05, *) are indicated as determined by the Student’s t-test.