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. 2009 Apr 28:6:14.
doi: 10.1186/1742-2094-6-14.

Functional interleukin-17 receptor A is expressed in central nervous system glia and upregulated in experimental autoimmune encephalomyelitis

Jayasri Das Sarma  1 , Bogoljub Ciric, Ryan Marek, Sanjoy Sadhukhan, Michael L Caruso, Jasmine Shafagh, Denise C Fitzgerald, Kenneth S Shindler, Am Rostami
Affiliations

Functional interleukin-17 receptor A is expressed in central nervous system glia and upregulated in experimental autoimmune encephalomyelitis

Jayasri Das Sarma et al. J Neuroinflammation. .

Abstract

Background: Interleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood.

Methods: EAE was induced in C57Bl/6 mice by immunization with myelin oligodendroglial glycoprotein. IL-17RA expression in the CNS was compared between control and EAE mice using RT-PCR, in situ hybridization, and immunohistochemistry. Cell-type specific expression was examined in isolated astrocytic and microglial cell cultures. Cytokine and chemokine production was measured in IL-17A treated cultures to evaluate the functional status of IL-17RA.

Results: Here we report increased IL-17RA expression in the CNS of mice with EAE, and constitutive expression of functional IL-17RA in mouse CNS tissue. Specifically, astrocytes and microglia express IL-17RA in vitro, and IL-17A treatment induces biological responses in these cells, including significant upregulation of MCP-1, MCP-5, MIP-2 and KC chemokine secretion. Exogenous IL-17A does not significantly alter the expression of IL-17RA in glial cells, suggesting that upregulation of chemokines by glial cells is due to IL-17A signaling through constitutively expressed IL-17RA.

Conclusion: IL-17RA expression is significantly increased in the CNS of mice with EAE compared to healthy mice, suggesting that IL-17RA signaling in glial cells can play an important role in autoimmune inflammation of the CNS and may be a potential pathway to target for therapeutic interventions.

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Figures

Figure 1
Figure 1
IL-17RA expression in mouse CNS. Spleen, brain and spinal cord were harvested from 10-week-old C57BL/6 and IL-17RA-deficient mice and used for quantification of IL-17RA mRNA by RT-PCR. Absolute copy number (mRNA molecules/μg total RNA) is shown. (A) Quantification of IL-17RA mRNA in wild-type mice using a primer set from exon boundary 1–2. IL-17RA expression was detected in all samples with > 5-fold more expression in spleen than CNS. One experiment of three is shown. (B) Expression of IL-17RA assessed by RT-PCR using a primer set from exon boundary 3–4. IL-17RA expression was again observed in wild-type (WT) CNS, but not in IL-17RA-deficient mice. Mean and SEM generated from multiple animals in one experiment (of three) is shown.
Figure 2
Figure 2
IL-17RA expression in the CNS of EAE mice. (A) Clinical profile of EAE. Female C57BL/6 mice (n = 8) were immunized with MOG35–55 and scored daily. Data represent mean clinical scores ± SEM. One experiment of three is shown. (B-G). CNS inflammation and demyelination. Mice were sacrificed at day 20 p.i., spinal cords were harvested and 5 μm sections were stained with H&E (B, D, F) or LFB (myelin stain; C, E, G). Magnifications are 40× (B-E) and 100× (F, G). EAE mice had significant cellular infiltration (arrows; D, F) and demyelination (arrows; E, G). No inflammation or demyelination occurred in control mice (B, C). (H) IL-17RA expression is up-regulated in the inflamed CNS of EAE mice. EAE and control mice (n = 5) were sacrificed at day 20 p.i. and IL-17RA expression from isolated spinal cords was assessed by RT-PCR using a primer set from exon boundary 1–2. Expression of IL-17RA in EAE mice is upregulated > 5-fold (*** P < 0.0001).
Figure 3
Figure 3
Expression of IL-17RA protein in spleen tissue from adult mouse. 5 μm thick serial cross sections of adult mouse spleen were processed for immunohistochemisty and immunostained with anti-IL-17RA antibody (A, B) followed by hematoxyline counterstaining. Immunostained sections demonstrated a subpopulation of IL-17RA positive cells (appear in brown). Original magnification for A was 100× and B was 400×.
Figure 4
Figure 4
Expression of IL-17RA protein in brain, spinal cord and spleen tissue from adult mouse. 5 μm thick serial cross sections were processed for immunohistochemisty. Immunostaining with anti-IL-17RA antibody and counterstaining with hematoxyline demonstrated a subpopulation of IL-17RA positive cells in spleen cells as shown here and in Figure 3 (C, D; appear in brown). Immunostained section of brain surrounding the hippocampal region (A) and cross section of spinal cord (B) demonstrated non-specific binding of IL-17RA. Original magnification for A, B and C was 100× and D was 400×.
Figure 5
Figure 5
IL-17RA expression in astrocytes and microglia in vitro. (A-C) Phenotypic characterization of glial cells by immunofluorescence. Mixed glial cultures (A), and purified astrocytic (B) and microglial cultures (C) were established from neonatal C57BL/6 mice. Cultures were stained with anti-GFAP antibody (astrocytic marker; green) and anti-CD11b (microglial marker; red) and counterstained with nuclear stain DAPI (blue). Mixed glial cultures primarily consist of astrocytes (70–80%) and microglia (5–10%); whereas, purified astrocyte cultures consist of 80–90% GFAP-positive cells. Purified microglial cultures are 98–99% CD11b-positive. (D-F). Flow cytometry. Glial cells were immunostained for flow cytometric analysis. Mixed glial cultures (D) contain both GFAP- and CD11b-positive cells. Astrocyte cultures were free from microglia (< 0.5%) (E) and microglial cultures free of astrocytes (< 0.5%) (F). (G) IL-17RA expression in vitro. mRNA was extracted from glial cultures and IL-17RA expression was quantified by RT-PCR using a primer set from exon boundary 1–2. Data represent the mean ± SEM expression of total IL-17RA mRNA from isolated cultures from three different batches of donors. IL-17RA is expressed 4-fold higher in microglia compared to astrocytes (***p < 0.0001). Mixed glial culture confers more expression of IL-17RA mRNA in comparison to astrocyte cultures devoid of microglia (*p = 0.0329).
Figure 6
Figure 6
Detection of IL-17RA protein on the cell surface of neonatal glial cells. Purified astrocytes (A) and microglia (B) cultured on chamber slides were fluorescently labeled with anti-IL-17RA antibody and CY2 -conjugated Hamster anti-goat IgG secondary antibody. The majority of astrocytes showed punctuate surface staining (A), whereas IL-17RA staining was much weaker in microglia (B). Binding of polyclonal-anti-mouse IL-17R-fluorescein on the surface of isolated astrocytes and microglia was tested using flow cytometric analysis. Cells were double labeled with anti-GFAP (intracellular marker for astrocytes) and IL-17RA antibody (C) or anti-CD11b (for microglia) and IL-17RA antibody (D). Protein G- coupled normal goat-IgG conjugated with carboxyfluorescein was used as isotype. Cells were gated either for GFAP or for CD11b, and IL-17RA expression on gated cells shown in single parameter FACS plot against the isotype staining demonstrates a prominent population of IL-17RA positive astrocytes, but only rare positive microglia.
Figure 7
Figure 7
Exogenous IL-17A treatment induces chemokine secretion in vitro. (A-H) Isolated astrocyte and microglia cultures were treated with IL-17A (10 ng/ml). Culture supernatants from treated and non-treated cultures were collected at 12 hr and assessed for chemokine levels by a multiplex array system. In response to IL-17A, microglia and astrocytes each upregulated secretion of MCP-1, MCP-5, MIP-2 and KC. One experiment of three is shown. *p < 0.05, ***p <0.0001. Mean ± SEM was generated from three multiple wells of the single experiment.
Figure 8
Figure 8
Exogenous treatment of IL-17A does not alter IL-17RA expression in glial culture. mRNA was isolated from either non-treated resting culture or IL-17A (10 ng/ml) treated culture supernatants at 12 hr in vitro. IL-17RA gene expression was measured by RT-PCR using a primer set from exon boundary 1–2. Data represent the mean expression from three different non- treated and IL-17A- treated culture batches ± SEM. IL-17A treatment did not alter IL-17RA expression in neonatal glial cells (*p > 0.05).
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