Expression of RAS components during MOG-EAE (day 31 p.i.). Induction of MOG-EAE leads to an (A) up to 2-fold increase in AT1aR expression and (B) up to 6-fold increase in AT1bR in the spinal cord, as well as to a (C) small increase in AT1aR expression and (D) significant increase in AT1bR expression in the spleen. In comparison to naive control mice, AT1aR (E) and AT1bR (F) on peritoneal macrophages derived from EAE diseased mice are both massively up-regulated, and can be down-regulated by losartan. Data are presented as relative expression with the respective gene expression in naive control mice or EAE mice set to 1 (n = 4–8 per group). n.d. = not detectable. In myeloid DCs prepared from murine bone marrow, expression of AT1aR (G) and AT1bR (H) is significantly up-regulated during differentiation in vitro from day 0 (d0), day 4 (d4), and day 7 (d7) until day 9 (d9).

Pharmacologic blockade of AT1R in MOG-EAE targets antigen presenting cells. (A) Losartan treatment does not alter T-cell proliferation in response to MOG35–55 or ConA. Data are pooled from 3 independent experiments (n = 10 vs. 13 mice). (B) FACS analysis of CD25⁺ FoxP3⁺ T cells on day 10 p.i. reveals similar frequencies of CD4⁺ regulatory T cells in the mesenterial (mLN), inguinal lymph nodes (iLN), spleen, or spinal cord (SC) on day 10 of MOG-EAE or after immunization with CFA only. Data are pooled from 2 representative out of 4 experiments (n = 4 mice per group). (C) Upon FACS analyses of APC frequencies in the spleen on day 10 p.i., losartan treatment leads to about a 50% reduction of different APC subsets (n = 5 per group). (D) Reduction of CD11c⁺ DCs in the spleen and CD11b⁺ APC as well as F4/80⁺ macrophages on spinal cord cross-sections on day 18 p.i. after preventive losartan application. Representative images from the spleen and the anterior columns of the thoracolumbar spinal cord are shown. (Scale bar, 50 μm for all images.) (E) In comparison to baseline, injection of ovalbumin and CFA leads to increased foot swelling on day 7 and 14 p.i, which is significantly reduced after losartan treatment starting on day –3 p.i. (n = 8 per group).

Functional role of the RAS during MOG-EAE. In (A) to (E), the duration of treatment is indicated by a black bar on top of the respective graphs. (A–C) Preventive inhibition of RAS components. (A) Treatment with aliskiren (n = 22 vs. 20 mice per group, P < 0.05 on day 28 p.i., data are pooled from a total of 3 experiments), (B) enalapril (n = 7 vs. 8 mice; P < 0.05 on day 26 p.i., a representative experiment is shown), and (C) losartan (n = 19 vs. 21 mice; P < 0.01 on day 31 p.i., data are pooled from 3 independent experiments) lead to an ameliorated course of MOG-EAE. (D) Therapeutic losartan application after onset of EAE results in an ameliorated disease course. Data are pooled from 2 independent experiments (n = 11 vs. 12 mice; P < 0.05 on day 38 p.i.). (E) Agtr1^–/– mice display a more severe disease course of MOG-EAE (n = 7 wild-type and n = 6 agtr1^–/– mice, P < 0.05 on day 27 p.i.). Upon treatment with losartan, there is a therapeutic effect both in wild-type and agtr1^–/– mice. (F) Therapeutic efficacy of losartan in MOG-EAE is independent from the blood pressure. Application of losartan or dihydralazine in MOG-EAE leads to a similar lowered blood pressure as in agtr1^–/– mice, where the effect can only slightly be extended by AT1R blockade. In contrast, immunization with MOG or CFA does not alter the blood pressure in comparison to naive control mice. Data are pooled from 4 independent experiments (n = 7 for naive mice, n = 15 for the EAE control, n = 9 for the losartan treated and n = 5 for the hydralazine treated group, n = 5 for the complete Freund's adjuvant (CFA) control, n = 4 for agtr1^–/– mice).

AT1R blockade in MOG-EAE affects chemokine-induced APC migration. (A) After losartan treatment, CCL2 and CXCL10 are significantly reduced in the spleen and CCL2, CCL3, and CXCL10 are reduced in the spinal cord on day 31 p.i. of MOG-EAE. Data are presented as relative expression with the respective chemokine expression in the vehicle-treated mice set to 1 (n = 5 per group). (B) Preventive AT1R blockade until day 16 p.i. impairs CCL2 and CCL3 production in macrophages on the protein level (n = 4 vs. 3 mice per group). (C) In an ex vivo migration assay, application of CCL2 induces migration of macrophages in comparison to medium, which can be blocked by addition of losartan. Data are pooled from 2 independent experiments (total of 5 mice per group).