Adrenomedullin protects from experimental autoimmune encephalomyelitis at multiple levels

Abstract

Adrenomedullin is a neuropeptide known for its cardiovascular activities and anti-inflammatory effects. Here, we investigated the effect of adrenomedullin in a model of experimental autoimmune encephalomyelitis (EAE) that mirrors chronic progressive multiple sclerosis. A short-term systemic treatment with adrenomedullin reduced clinical severity and incidence of EAE, the appearance of inflammatory infiltrates in spinal cord and the subsequent demyelination and axonal damage. This effect was exerted at multiple levels affecting both early and late events of the disease. Adrenomedullin decreased the presence/activation of encephalitogenic Th1 and Th17 cells and down-regulated several inflammatory mediators in peripheral lymphoid organs and central nervous system. Noteworthy, adrenomedullin inhibited the production by encephalitogenic cells of osteopontin and of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), two critical cytokines in the development of EAE. At the same time, adrenomedullin increased the number of IL-10-producing regulatory T cells with suppressive effects on the progression of EAE. Furthermore, adrenomedullin generated dendritic cells with a semi-mature phenotype that impaired encephalitogenic responses in vitro and in vivo. Finally, adrenomedullin regulated glial activity and favored an active program of neuroprotection/regeneration. Therefore, the use of adrenomedullin emerges as a novel multimodal therapeutic approach to treat chronic progressive multiple sclerosis.

Keywords: Adrenomedullin; Autoimmunity; Dendritic cells; Glial cells; Multiple sclerosis; Neuropeptide; Regulatory T cells.

Figure 1. Treatment with adrenomedullin reduces severity of chronic EAE

Chronic progressive EAE was induced in C57BL/6 mice by immunization with MOG_35–55. Mice were treated i.p. with PBS (control) or adrenomedullin (1 nmol/day, black bars) for 5 consecutive days at disease onset (A, arrow indicated adrenomedullin administration started at clinical score <1) or at two points during the acute phase in the therapeutic regimen (B, arrow indicated adrenomedullin administration started at clinical score 1–2; C, at clinical score >2). Data represent the mean clinical score ± SEM (A–C) or maximum peak of disease per animal (D, mean: horizontal line). n=10 mice/group in A; n=13 mice/control group and 10 mice/adrenomedullin group in B; n=24 mice/control group and 15 mice/adrenomedullin group in C; all performed in three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. control with Mann-Whitney test (A–C) and Kruskal Wallis Test (D). ^#p<0.001 with ANOVA t-test.

Figure 2. Adrenomedullin reduces inflammatory infiltration and demyelination in CNS of mice with EAE

Mice with MOG_35–55-induced chronic EAE were treated with PBS (control) or with adrenomedullin for 5 days starting at disease onset (6/group, two independent experiments). Naïve animals were used as negative controls. (A) Transverse sections of spinal cord randomly selected at the peak of clinical disease were stained with Kluver-Barrera or immunostained for myelin (anti-MBP). Original magnification for upper images, ×40; lower, ×100 for Kluver-Barrera images and ×200 for anti-MBP images. Arrows point to areas of demyelination and inflammatory infiltration. A section of spinal cord of naïve mice stained with isotype control antibody was included to assess non-specific immunostaining (magnification, × 200). The mean number of plaques of demyelination in the lumbar and cervical regions and the incidence of occurrence of plaques (in parenthesis) was determined. (B) The phenotype of the infiltrating cells in the lumbar region of spinal cord was assayed by immunofluorescence for CD45, CD4 and Iba1. AM: adrenomedullin. Scale bars: 50 µm.

Figure 3. Adrenomedullin reduces the production of inflammatory markers in the CNS of EAE mice

Mice with MOG_35–55-induced chronic EAE were treated with PBS (control) or adrenomedullin for 5 days at the disease onset (clinical score 1–2). Levels of inflammatory cytokines, chemokines, GM-CSF, and osteopontin (OPN) were determined by ELISA in protein extracts purified from spinal cord at peak of the disease. Samples from naïve mice were used as reference (n.d.: under detection limit). n=6 mice/group, two independent experiments. *p<0.05; **p<0.01, vs. control with Mann-Whitney test.

Figure 4. Adrenomedullin ameliorates EAE severity by downregulating the peripheral encephalitogenic Th1/Th17 response

Mice with MOG_35–55-induced chronic EAE were treated with PBS (control) or adrenomedullin for 5 days at disease onset (clinical score 1–2). (A) Adrenomedullin-treated EAE mice showed reduced encephalitogenic Th1/Th17-mediated responses in lymphoid organs. Proliferation and cytokine production by DLN cells isolated at peak of the disease and stimulated with medium, the encephalitogenic antigen (MOG_35–55) or a polyclonal stimulus (Con A). We obtained similar results with spleen cells (data not shown). n=6 mice/group, two independent experiments. (B) Adrenomedullin treatment regulated the number of Th1, Th17 and Th2 cells in EAE mice. Spleen cells isolated at peak of the disease were assayed for intracellular cytokine expression by flow cytometry in the CD4 gated population. We used naïve mice as basal controls. We obtained similar results with DLNs (data not shown). n=6–8 mice/group, three independent experiments. (C) MOG-specific IgG1 and IgG2a serum levels (peak of disease). n=14 mice/group, three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs control with Mann-Whitney test (A) and t-test (B and C).

Figure 5. Treatment with adrenomedullin impairs the encephalitogenic response in the CNS of mice with EAE

Mice with MOG_35–55-induced chronic EAE were treated with PBS (control) or adrenomedullin for 5 days at disease onset (clinical score 1–2). (A) Phenotypic analysis by flow cytometry of brain infiltrating mononuclear cells isolated at the peak of disease. Results show total numbers of CD4 T cells, CD4⁺IFNγ⁺ cells or CD4⁺IL-17⁺ cells per brain. n=6 mice/group, two independent experiments. (B) Proliferation (depicted by the formation of cell clusters of activation; original magnification, ×100) and (C) cytokine production by mononuclear cells isolated from brains and spinal cords at peak of the disease and restimulated with MOG_35–55 or with a polyclonal stimulus (ConA). n=6–8 mice/group, two independent experiments. AM: adrenomedullin. *p<0.05; **p<0.01 vs control with Mann-Whitney test.

Figure 6. Treatment with adrenomedullin increases the number of Treg cells which suppress EAE progression

Mice with MOG_35–55-induced chronic EAE were treated with PBS (control) or adrenomedullin for 5 days at disease onset (clinical score 1–2). (A) Adrenomedullin induces the emergence of peripheral Treg cells in EAE mice. Percentage and number of CD4⁺CD25⁺FoxP3⁺IL-10⁺ Treg cells in spleens isolated at peak of the disease. n=18 mice/group, three independent experiments. (B) Adrenomedullin increases the percentages of Treg cells in the CNS inflammatory infiltrates. Spinal cord and brain mononuclear cells isolated at peak of the disease were analyzed for the presence of CD4⁺CD25⁺FoxP3⁺ Treg cells by flow cytometry and expressed as percentage of FoxP3⁺ cells in the CD4 population. n=10 mice/group, two independent experiments. (C) Treatment (arrow) of EAE mice with CD4 T cells or CD25-depleted CD4 T cells isolated from spleen/DLNs of EAE mice that were previously treated with PBS (control) or adrenomedullin. Untreated EAE mice were used as reference. n=7–9 mice/group, two independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs control with Mann-Whitney test (A and B) and Kruskal-Wallis test (C). ^#p<0.01; NS: not significant vs control with Kruskal-Wallis test.

Figure 7. Adrenomedullin generates dendritic cells (DCs) with the capacity to deactivate the encephalitogenic response in vitro and in vivo

(A) DCs matured with LPS and pulsed with MOG_35–55 in the absence (DC-control) or presence of adrenomedullin (DC-adrenomedullin) were co-incubated with DLN cells isolated from mice suffering EAE and stimulated with MOG_35–55 or Concanavalin A. Cell proliferation (evidenced by the presence of clusters of activation in the culture; original magnification, ×100) and the production of cytokines were determined 48 h later. n=5, in duplicate. (B) DCs matured with LPS and pulsed with MOG_35–55 in the absence (DC-control) or presence of adrenomedullin (DC-adrenomedullin) were administered i.p. to EAE mice at disease onset (arrow). Untreated EAE mice were used as reference. Numbers in parenthesis represent the mortality rate in each group. n=6–8 mice/group, two independent experiments. **p<0.01 vs control with Mann-Whitney test; ^#p<0.001 vs control with Kruskal-Wallis test.

Figure 8. Adrenomedullin promotes neuroprotective responses in EAE mice

(A) Adrenomedullin downregulates the inflammatory response of CNS resident cells. Microglia, astrocytes and mixed neuron-glia isolated from newborn mice were cultured in medium or stimulated with LPS in the absence or presence of adrenomedullin (100 nM) and cytokine contents in culture supernatants were determined 24 h later. n=4, in duplicates. (B) Adrenomedullin protects oligodendrocytes from oxidative-induced cell death. Cell survival of precursor and mature oligodendrocytes cultured for 24 h with 200 µM H₂O₂ in the absence or presence of 100 nM adrenomedullin. n=3, in duplicates. (C) Adrenomedullin increases neurotrophic factors. BDNF and ADNP gene expression (relative to β-actin) in spinal cords isolated from EAE mice treated with PBS (control) or adrenomedullin at disease onset. Dashed line corresponds to expression in naïve mice normalized to 1. n=8 mice/group, three independent experiments. *p<0.05; **p<0.01; ***p<0.001vs control with Mann-Whitney test.

Figure 9. EAE mice show decreased CNS adrenomedullin content

Levels of adrenomedullin in culture supernatants of LPS-activated astrocytes and microglia (24 h) (A, n=5, in duplicates) and in the brains and spinal cords of naïve and EAE mice (B, n=5–8 mice/group, in duplicates). *p<0.05 vs medium with Mann-Whitney test; *p<0.05; **p<0.01 vs naïve with t-test.