Role of the receptor Mas in macrophage-mediated inflammation in vivo

Abstract

Recently, an alternative renin-angiotensin system pathway has been described, which involves binding of angiotensin-(1-7) to its receptor Mas. The Mas axis may counterbalance angiotensin-II-mediated proinflammatory effects, likely by affecting macrophage function. Here we investigate the role of Mas in murine models of autoimmune neuroinflammation and atherosclerosis, which both involve macrophage-driven pathomechanisms. Mas signaling affected macrophage polarization, migration, and macrophage-mediated T-cell activation. Mas deficiency exacerbated the course of experimental autoimmune encephalomyelitis and increased macrophage infiltration as well as proinflammatory gene expression in the spleen and spinal cord. Furthermore, Mas deficiency promoted atherosclerosis by affecting macrophage infiltration and migration and led to increased oxidative stress as well as impaired endothelial function in ApoE-deficient mice. In summary, we identified the Mas axis as an important factor in macrophage function during inflammation of the central nervous and vascular system in vivo. Modulating the Mas axis may constitute an interesting therapeutic target in multiple sclerosis and/or atherosclerosis.

Keywords: EAE; atherosclerosis; inflammation; macrophages; renin–angiotensin system.

Fig. 1.

Mas is expressed on different macrophage subsets. Mas expression is detectable on the mRNA (A) and protein (B) level in mature M(unstimulated) as well as M(LPS+IFNγ) and M(IL-4+IL-13) polarized BMDM. Mas expression in the spleen, testis, and aorta did not significantly differ compared with macrophages but was higher in the heart, kidney, and hindbrain (A). Data are presented as relative expression with the respective gene/protein expression in M(unstimulated) macrophages set to 1 [n = 4–5 per group for qRT-PCR and n = 4 for Western Blot analysis, mean ± SEM, **P < 0.01, ***P < 0.001 compared with M(unstimulated)]. A receptor Mas control peptide antigen is used as negative control.

Fig. S1.

The extent of Mas expression in the brain is region-dependent. (A) Mas mRNA expression is detectable on all types of CNS cells [n = 3–4, mean ± SEM, ***P < 0.001 compared with M(unstimulated)]. (B) Mas expression is widely distributed throughout the brain in mice [n = 4, mean ± SEM, **P < 0.01, ***P < 0.001 compared with M(unstimulated)].

Fig. 2.

Mas-deficient macrophages show an increase in proinflammatory M(LPS+IFNγ) but a decrease in anti-inflammatory M(IL-4+IL-13) marker gene expression, enhanced migration, and increased T-cell activation capacities. (A) Compared with WT mice, M(LPS+IFNγ) macrophages from MasKO mice display a significantly enhanced expression of the proinflammatory cytokines ccl2 and tnfa and a trend toward higher levels of il6 and inos. (B) A significant decrease in M(IL-4+IL-13) marker gene expression, for example, in ym1, fizz, mrc1, and mgl2, is detected in Mas-deficient macrophages compared with WT controls. Data are presented as relative expression of the indicated genes (n = 4–6 per group, mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001). (C) The migration rate of Mas-deficient peritoneal macrophages is increased by around 20% compared with WT macrophages (data are pooled from a total of three independent experiments; **P < 0.01, mean ± SEM). Data are compiled from an in vitro FCS-gradient Transwell assay. (D) Coculture assays of naive T cells with in vitro-generated BMDM display a significant increase in proliferating T cells when cultured with Mas-deficient M(LPS+IFNγ) and M(IL-4+IL-13) macrophages (data are pooled from a total of three independent experiments; *P < 0.05, **P < 0.01, mean ± SEM).

Fig. S2.

The selective Mas agonist AVE 0991 counteracts the effects of Mas deficiency on macrophage polarization and function. (A) Treatment with AVE 0991 decreases the expression of the M(LPS+IFNγ) marker genes tnfa and il6 but significantly increases the expression of the anti-inflammatory M(IL-4+IL-13) marker gene ym1. Data are presented as relative expression of the indicated genes (n = 3–7, *P < 0.05, **P < 0.01, mean ± SEM). (B) Expression of the surface markers CD80, CD86, and MHCII is reduced on M(LPS+IFNγ) whereas expression of CD206 is increased on M(IL-4+IL-13) macrophages after treatment with 0.1 µM AVE 0991 (one of two independent experiments is shown; *P < 0.05, **P < 0.01, ***P < 0.001, mean ± SEM). (C) Coculture assays of naive T cells with in vitro-generated BMDM reveal a significant decrease in proliferating T cells when cultured with AVE 0991-treated M(IL-4+IL-13) but not M(LPS+IFNγ) macrophages (data are pooled from two independent experiments, **P < 0.01, mean ± SEM). (D) The disease incidence is significantly reduced in AVE 0991-treated mice (n = 13 per group; data are pooled from a total of three independent experiments, *P < 0.05, mean ± SEM). (E) Mice treated with AVE 0991 showed a slightly ameliorated clinical EAE course (n = 4 per group; one of three independent experiments is shown; mean ± SEM).

Fig. S3.

Mas has no effect on in vitro Th1 and Th17 differentiation or ex vivo T-cell proliferation. (A) Mas deficiency has no influence on Th1 (28.0% versus 31.5%) or Th17 (13.6% versus 16.7%) cell differentiation in vitro (data are pooled from two independent experiments, mean ± SEM). (B) The selective Mas agonist AVE 0991 has no effect on Th1 or Th17 differentiation in vitro (data are pooled from two independent experiments, mean ± SEM). (C) Proliferation assays with splenocytes stimulated in vitro with aCD3/aCD28 display no difference in the percentage of proliferating CD3⁺, CD4⁺, or CD8⁺ cells between the WT and the Mas-deficient group (n = 5 MasKO versus n = 4 WT).

Fig. 3.

Mas deficiency aggravates clinical symptoms of EAE and enhances macrophage infiltration and Th1 frequencies in the CNS. Active EAE is induced in C57BL/6 and MasKO mice. (A and B) Compared with healthy control mice, mas expression is significantly down-regulated in the spleen (A) but up-regulated in the spinal cord (B) during the acute phase of EAE (d10). In the early chronic phase (day 28 p.i.), expression of mas in the spinal cord (B) returns to baseline levels. (C) Mas deficiency significantly exacerbates the course of EAE (n = 7, **P < 0.01 on day 22 p.i., mean ± SEM; one of two representative experiments is shown). (D) Mas deficiency leads to enlarged infiltrated areas and enhanced infiltration of Mac-3⁺ macrophages/microglia and CD3⁺ T cells on spinal cord cross-sections obtained at the maximum of disease. Representative images from the anterior columns of the thoracolumbar spinal cord are shown (Scale bar, 50 µm for all images.) (E) Ex vivo FACS phenotyping of the spleen and spinal cord infiltrates revealed that Mas deficiency increases CD11b⁺ antigen-presenting cell frequencies in the CNS but not in the spleen on day 14 p.i. (n = 7–8, *P < 0.05, mean ± SEM). (F) Ex vivo FACS analysis also shows enhanced Th1 frequencies in the spleen as well as spinal cord of MasKO mice on day 14 p.i. whereas Th17 and Treg cells remain unchanged (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, mean ± SEM). (G) Mas deficiency enhances CCL2 and IL-6 secretion after MOG_35–55-specific recall in total splenocytes on day 10 p.i. (n = 8, mean ± SEM).

Fig. S4.

Behavior of MasKO mice is not altered in an open field test. Compared with WT mice, MasKO mice showed the same percentage of time spent in the center (A) and rearing frequency (B) in a modified open field test (n = 4 per group).

Fig. S5.

Mas deficiency results in increased myelin and in axonal as well as neuronal loss in the spinal cord during MOG-EAE. Representative images of thoracic spinal cord cross-sections on day 22 p.i. are shown. (A) In contrast to WT mice, Luxol Fast Blue staining reveals enhanced demyelination in the spinal cord of MasKO animals during EAE. (For images of whole spinal cord cross sections, a multiple image alignment of 4–6 single images was performed via Cell^P software, Olympus). Silver impregnation studies (B) and Cresyl Violet staining (C) disclose a reduction in axonal and neuronal densities in Mas-deficient compared with WT mice. (D) CD11b⁺ cells in the spinal cord of MasKO mice display significantly reduced levels of the M(IL-4+IL-13) markers CD14 and CD206 but no significant difference in the expression of the M(LPS+IFNγ) markers CD80 and CD86. Data are presented as mean ± SEM (n = 4; *P < 0.05, **P < 0.01).

Fig. 4.

Mas knockout increases proinflammatory gene expression in the spinal cord and spleen. mRNA expression in the spinal cord (A and B) and spleen (C and D) was analyzed by qRT-PCR on day 22 p.i.. Mas deficiency increases the expression level of the proinflammatory M(LPS+IFNγ)-like macrophage markers il6, il1b, tnfa, inos, and ccl2 in the spinal cord (A) and spleen (C) of EAE mice by up to 10-fold. The expression of some M(IL-4+IL-13)-like marker genes is also altered in the spinal cord (B) and spleen (D) of MasKO mice compared with the WT controls (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, mean ± SEM).

Fig. 5.

Mas deficiency in ApoEKO mice increases oxidative stress and endothelial dysfunction. (A) Carbachol-induced vascular relaxation is significantly impaired in kidneys of ApoEKO/MasKO mice compared with ApoEKO mice (n = 5–10 per group). (B and C) In ApoEKO mice, Mas deficiency leads to increased oxidative stress measured by urinary 8-isoprostane and aortic nitrotyrosine expression levels (measurements were repeated for a total of three times; n = 6–8, *P < 0.05, mean ± SEM).

Fig. 6.

Increased atherosclerotic plaque size, macrophage infiltration, and proinflammatory cytokine expression in ApoEKO/MasKO mice. (A) Mas deficiency leads to a significant exaggeration of atherosclerosis in the aortic arch region of ApoEKO mice. (B) The relative and the specific area of atherosclerotic lesions in the aorta are significantly enlarged in Mas-deficient ApoEKO mice (n = 11, *P < 0.05). (C) Mas deficiency causes enhanced infiltration of F4/80⁺ macrophages within the atherosclerotic plaques of the aortic root (n = 6, *P < 0.05, mean ± SEM). (D) The migration rate of Mas-deficient peritoneal macrophages from ApoEKO mice, measured with an in vitro FCS-gradient Transwell assay, is significantly increased compared with ApoEKO mice (two independent experiments, each comprising two mice; *P < 0.05, mean ± SEM). (E) Mas deficiency induces proinflammatory M(LPS+IFNγ)-like cytokines, such as il6, inos, ccl2, and il12p40, in atherosclerotic aortas of ApoEKO mice (n = 6, *P < 0.05, **P < 0.01, mean ± SEM).

Fig. S6.

Mas has no effect on proinflammatory cytokine expression in the plasma or splenic immune cell frequencies. (A) Cytokine levels in the plasma of ApoEKO mice are not significantly affected by Mas deficiency (n = 6–7, mean ± SEM). (B) Ex vivo flow cytometry shows no difference in the frequency of CD11c⁺, CD11b⁺, CD3⁺, CD4⁺, and CD8⁺ cells in the spleen of MasKO/ApoEKO versus ApoEKO mice (n = 3–5, mean ± SEM).