Expression of AT1R in MS plaques. Immunohistochemical analysis of AT1R in human CNS tissue. No AT1R expression is detected in normal spinal cord (A) or white matter from a patient with Alzheimer disease (B). Strong AT1R expression is detected in perivascular cuffs of a chronic active MS plaque (C–F), particularly in foamy macrophages (F, arrows). CD3 staining (E) of a neighbouring section of D shows presence of T cells. AT1R is also detectable in endothelial cells (G, arrow), astrocytes (H, arrows), and axons (I) within a chronic inactive MS plaque. AT1R is also strongly expressed in axons during viral encephalitis (J), suggesting that inflammation itself may drive neuronal AT1R expression. Magnifications: 20× (J), 40× (A and I), 60× (C, F–H), and 90× (D and E).

Molecular mechanisms of tolerance induction by suppressing signaling through AT1R. (A, C, and D) Western blot analyses of STAT protein expression (A), SOCS protein expression (C) and expression of proteins of the NF-κB signaling pathway (D) in CD4⁺ T cells from naive SJL/J mice or SJL/J mice 10 days after PLP immunization with or without treatment with lisinopril. (B) IFN-γ release from CD4⁺ T cells from untreated SJL/J mice (black circles) or SJL/J mice treated with lisinopril for 10 days (open circles) after stimulation with IL-12 in vitro. (E) In vitro DNA binding studies of proteins from the NF-κB signaling pathway in CD4⁺ T cells from naïve untreated (black bars), naïve lisinopril-treated (white bars), PLP-immunized untreated (light gray bars) and PLP-immunized lisinopril-treated SJL/J mice (dark gray bars). Values represent mean ± SEM of arbitrary units of colorimetric analysis of DNA-binding activity. *, P < 0.05; **, P < 0.01.

Suppression of myelin-specific TH1 and TH17 cells by blocking AII production. (A) Analysis of AII in serum and in the supernatant of CD11b+ monoctyes and CD4⁺ T cells of naive (white bars) or SJL/J mice 10 days after immunization with PLP p139–151 (black bars) by ELISA. Cells were purified to 95–99% using CD4 MACS beads and were then incubated for 12 h with full stimulation media and supernatents were harvested. *, P < 0.05; **, P < 0.01. (B) Intracellular cytokine analysis using flow cytometry in CD4⁺ LNC from naive SJL/J mice or SJL/J mice 10 days after immunization with PLP p139–151 treated with vehicle (control) or lisinopril (LIS) at 10 mg/kg/day (n = 3). LNC were isolated and restimulated with αCD3/αCD28 and pulsed with PMA, lonomycin, and golgi stop. Numbers indicate percent positive cells. (C) Adoptive transfer of CD4⁺ LNC from SJL/J mice immunized with PLP p139–151 and treated with vehicle (filled circles) or lisinopril at 10 mg/kg/day (open circles) for 12 days. Cells were transferred into naive SJL/J recipient mice. Data represent clinical scores as described in the Materials and Methods section.

Expression of AT1R during autoimmune neuroinflammation and MS. (A) Analysis of mRNA expression of ATR1R in LNC from naive SJL/J mice (open bars) and SJL/J mice 10 days after immunization with PLP p139–151 (PLP, closed bars) (n = 3) using real-time PCR. Values represent mean arbitrary expression levels of triplicates and SEM normalized to expression of β-actin. *, P < 0.05; **, P < 0.01. (B) Western blot analysis of AT1R protein expression in splenic CD4⁺ T cells from naive SJL/J mice and SJL/J mice 10 days after immunization with PLP p139–151 (PLP). (C) Binding of FITC-labeled AII to myelin-specific CD4⁺ T cells as measured by flow cytometry. Splenocytes were isolated from SJL/J mice 5 days after immunization with PLP p139–151 (red histogram) or naive mice (blue histogram). Splenoytes were restimulated ex vivo with PLP p139–151 and CD4⁺ T cells were gated. Unlabelled AII served as a control (gray histograms). Numbers indicate percent AII binding cells. (D) Analysis of mRNA expression of ATR1R in CNS tissue from naive SJL/J mice (open bars) and SJL/J mice 14 days after immunization with PLP p139–151 (PLP, closed bars) (n = 5) using real-time PCR. Values represent mean arbitrary expression levels of triplicates and SEM normalized to expression of β-actin. *, P < 0.05. (E and F) Immunohistochemical analysis of AT1R expression in meningeal infiltrates of spinal cords from naive SJL/J mice (E) and SJL/J mice 14 days after PLP-immunization (F). Magnifications: 40× (E) and 60× (F).

Modulation of EAE by suppressing AII production or blocking AT1R. (A) Prevention of EAE after PLP immunization by lisinopril at 1 mg/kg/day (gray circles) or 10 mg/kg/day (open circles) compared with vehicle-treated controls (black circles), n = 12 per group. Treatment was initiated 2 days before immunization. Values are displayed as mean clinical scores as in Fig. 3. (B) Treatment of EAE after PLP immunization with lisinopril at 10 mg/kg/day (open circles) with vehicle controls (black circles), n = 15 per group. Treatment was initiated at the peak of first clinical disease activity (day 15 after immunization). Values are displayed as mean clinical scores. (C) Treatment of EAE after PLP immunization with lisinopril at 10 mg/kg/day (open circles) or candesartan at 1 mg/kg/day (gray circles) compared with vehicle controls (black circles), n = 15 per group. Treatment was initiated at the peak of first clinical disease activity (day 15 after immunization). Values are displayed as mean clinical scores. (D) H&E stained spinal cord sections of SJL/J mice with EAE with (EAE + LIS) or without (vehicle) treatment with lisinopril. Arrows indicate meningeal inflammatory infiltrate. (Magnification: D, 20×.) (E) Number of inflammatory foci (±SEM) in brains and spinal cords of SJL/J mice treated with vehicle (black bars), lisinopril (white bars), or candesartan (gray bars). Lesions were counted by a neuropathologist blinded to the type of treatment, n = 6 per group. *, P < 0.05. (F) Flow cytometric analysis of FoxP3 and CD25 in CD4⁺ CNS-infiltrating T cells from SJL/J mice with EAE 14 days after immunization with PLP139–151 treated with vehicle (Top) or lisinopril at 10 mg/kg/day (LIS, Bottom) (n = 3). Numbers indicate percent positive cells.

Induction of myelin-specific FoxP3+ regulatory T cells by blocking AII production. (A) Flow cytometric analysis of FoxP3 and CD25 in CD4⁺ LNC from naive SJL/J mice (Left) or SJL/J mice 10 days after immunization with PLP p139–151 treated with vehicle (Center, control) or lisinopril (Right, LIS) at 10 mg/kg/day (n = 3). Numbers indicate percent positive cells. (B) Western blot analysis of SMAD 2/3 phosphorylation in CD3+ LNC from SJL/J mice 10 days after immunization with PLP p139–151 treated with vehicle (co) or lisinopril (LIS) at 10 mg/kg/day (n = 3). (C) Adoptive transfer of CD4⁺ LNC from SJL/J mice (n = 5) immunized with PLP p139–151 to induce EAE and treated with vehicle (filled circles) or lisinopril at 10 mg/kg/day (open circles) for 12 days. Cells were transferred into SJL/J recipient mice (n = 10 per group). Recipient mice were immunized with PLP p139–151 24 h after the adoptive transfer. Data represent clinical scores as described in the Materials and Methods section. (D) Intracellular expression of IFN-γ (black bars) and IL-17 (white bars) using flow cytometry in CD4⁺ T cells from naive 2D2 mice, transfected with AT1R siRNA at different concentrations using nucleofection and activated with MOG35–55 for 48 h. Histograms represent AT1R cell surface expression, numbers represent percent AT1R-positive cells. Values in the bar graphs represent percent IFN-γ+ or IL17+ cells.