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. 2015 Sep 10;162(6):1338-52.
doi: 10.1016/j.cell.2015.08.025.

Melatonin Contributes to the Seasonality of Multiple Sclerosis Relapses

Mauricio F Farez  1 , Ivan D Mascanfroni  2 , Santiago P Méndez-Huergo  3 , Ada Yeste  2 , Gopal Murugaiyan  2 , Lucien P Garo  2 , María E Balbuena Aguirre  4 , Bonny Patel  2 , María C Ysrraelit  5 , Chen Zhu  6 , Vijay K Kuchroo  6 , Gabriel A Rabinovich  7 , Francisco J Quintana  8 , Jorge Correale  5
Affiliations

Melatonin Contributes to the Seasonality of Multiple Sclerosis Relapses

Mauricio F Farez et al. Cell. .

Abstract

Seasonal changes in disease activity have been observed in multiple sclerosis, an autoimmune disorder that affects the CNS. These epidemiological observations suggest that environmental factors influence the disease course. Here, we report that melatonin levels, whose production is modulated by seasonal variations in night length, negatively correlate with multiple sclerosis activity in humans. Treatment with melatonin ameliorates disease in an experimental model of multiple sclerosis and directly interferes with the differentiation of human and mouse T cells. Melatonin induces the expression of the repressor transcription factor Nfil3, blocking the differentiation of pathogenic Th17 cells and boosts the generation of protective Tr1 cells via Erk1/2 and the transactivation of the IL-10 promoter by ROR-α. These results suggest that melatonin is another example of how environmental-driven cues can impact T cell differentiation and have implications for autoimmune disorders such as multiple sclerosis.

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Figures

Figure 1
Figure 1. Melatonin levels show an inverse correlation with MS clinical relapses
(a) Exacerbation rate for each season was estimated for the duration of the follow-up and depicted in the primary axis. 6-sulfatoxymelatonin levels measured in first morning urine in each season is depicted as mean ± s.e.m. in secondary axis. P value corresponds to Poisson regression model. Lack of correlation between exacerbation rate and Vitamin D (b), reported respiratory infections (c), and UV radiation in Buenos Aires city (d). See also Table 1.
Figure 2
Figure 2. Melatonin administration ameliorates EAE
(a) EAE development in C57/B6 treated with vehicle (0.01% DMSO) or melatonin (5mg/kg). Data are representative of three independent experiments (means and s.e.m.) (n ≥ 20 mice/group). P value corresponds for the effect of treatment in a repeated measures mixed effect model. (b) Flow cytometry analysis of IL-17+, IL10+, IFN-γ+ and FoxP3+ CD4+ cells from the spleen of vehicle or melatonin treated mice at day 7 after disease induction. At least 4 mice were analyzed per group and data is presented as mean ± SEM. * P<0.05 of unpaired T-test. (c-d) Flow cytometry analysis of IL-17+, IFN-γ+, IL-17+-IFN-γ+ (DP) and IL-17+-GM-CSF+ CD4+ T cells from the CNS of control- or melatonin-treated mice at the clinical peak of EAE * P<0.05 of unpaired T-test. (e) Proliferative responses of CD4+ T cells to MOG35-55 of vehicle or melatonin treated mice. At least 3 mice were analyzed per group and data is presented as mean ± s.e.m. * P<0.05 of one-way ANOVA. (f) Cytokine secretion by proliferating CD4+ T cells from vehicle and melatonin treated. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test. (g) Proliferative responses and cytokine profile (h) of CD4+ T cells in co-culture with dendritic cells derived from melatonin-treated or untreated mice. Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA. (i) Proliferative responses of melatonin treated 2D2 CD4+ T cells to MOG35-55 in the presence of dendritic cells. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of one-way ANOVA. (j) Proliferative responses of melatonin treated 2D2 CD4+ T cells to MOG35-55 stimulated only with anti-CD3 and anti-CD28. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of one-way ANOVA. (k) Proliferative responses of treated 2D2 CD4+ T cells to MOG35-55 stimulated melatonin-treated DCs. Data are representative of three independent experiments (means and s.e.m.). See also Fig. S1a–e
Figure 3
Figure 3. Melatonin interferes with human Th17 cell differentiation and boosts Tr1 generation
(a) Flow cytometry analysis of IL-17 expression in human Th17 differentiated CD4+ T cells (IL-1β, IL-6 and TGF-β1) in the presence or absence of melatonin (500ng/ml) and agomelatine (500ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (b). Cytokine quantification by ELISA of IL-17 in human Th17 differentiated CD4+ T cells in the presence or absence of melatonin (500ng/ml) and agomelatine (500ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (c) RT-PCR analysis of Th17 cells cultured as in a. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (d). Cytokine quantification by ELISA of IFN-γ in human Th1 differentiated CD4+ T cells in the presence or absence of melatonin (500ng/ml) and agomelatin (500ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (e) RT-PCR analysis of Th1 cells cultured as in d. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (f) Flow cytometry analysis of IL-10 expression in human Tr1 differentiated CD4+ T cell in the presence or absence of melatonin (500ng/ml)and agomelatin (500ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (g) Quantitative PCR analysis of Tr1 cells cultured as in f. Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA. See also Fig. S2
Figure 4
Figure 4. Melatonin interferes with Th17 cell differentiation via the Erk1/2-C/EBPα pathway
(a) CD4+ naïve T cells were differentiated into Th17 cells by the addition of TFG-β, IL-6 (0 h) and IL-23 (48hs) in the presence or absence of melatonin (2ng/ml) and analyzed by RT-PCR after 72hs. Displayed image is representative of five experiments. * P<0.05 of unpaired T-test (b) Cytokine secretion analysis of IL-17 and IL-10 after 72hs of culture as in a. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test (c) Cytokine secretion in Th17 differentiated CD4+ T cells in the presence or absence of melatonin (2ng/ml), agomelatine (20ng/ml, MTNR1A ligand) and CGP 52608 (20ng/ml, ROR-α ligand). Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA. (d) RT-PCR analysis of Th17 cells cultured as in c. Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA. (e) Flow cytometry analysis of IL-17 expression as in a, in wild type mice and MTNR1A-deficient mice. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of unpaired T-test. (f) Quantitative PCR analysis of wild type and MTNR1A deficient mice cultured as in e. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test. (g) Signal transduction profiling using reverse protein arrays. Data are representative of two independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test. (h) Immunoblot analysis of T- and P-Erk1/2. Data are representative of two independent experiments (means and s.e.m.). (i) Immunoblot analysis of T- and P-C/EBPα Data are representative of two independent experiments (means and s.e.m.). (j) Putative binding sites of C/EBPα in nr1d1 (left panel); chromatin immunoprecipitation with anti-C/EBPα (right panel). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (k) Luciferase activity of HEK-293 cells transfected with a luciferase reporter construct for the nr1d1 promoter. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test. (l) Flow cytometry analysis of IL-17 expression as in a, in wild type mice and C/EBPα-deficient mice. Data are representative of two independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. See also Fig. S3–S4
Figure 5
Figure 5. Melatonin interferes with Th17 cell differentiation by limiting NFIL3 expression
(a) Schematic diagram of the proposed mechanisms mediating the effects of melatonin on Th17 cell differentiation. (b) RT-PCR analysis of nr1d1 expression in CD4+ T cells activated under Th0, Th17 and Tr1 polarizing conditions for 3 days. Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of unpaired T-test. (c) RT-PCR analysis of nr1d1 (left panel) and nfil3 (right panel) expression in CD4+ T cells activated under Th17 polarizing conditions for 3 days treated with vehicle, melatonin (2ng/ml) or agomelatine 20ng/ml). Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of unpaired T-test. NFIL3 expression was further confirmed by western blot (d) Data are representative of two independent experiments (means and s.e.m.). (e) RT-PCR analysis of nfil3 expression in CD4+ T cells activated under Th17 polarizing conditions for 3 days in the presence of melatonin (2ng/ml) and/or UO126. Data are representative of five independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA. (f,g) Flow cytometry analysis of IL-17 expression (f) and rorc expression (g) in CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml) and/or UO126. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (h,i) Flow cytometry analysis of IL-17 expression (h) and rorc and il17 expression (i) in CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml), following infecting with a control or an nr1d1-encoding retrovirus. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (j,k) Flow cytometry analysis of IL-17 expression (j) and rorc and il17 expression (k) ) in wild type and REV-ERBα deficient CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (l) Putative binding sites of Nfil3 in rorc and rora (left panel); ChIP analysis of the interaction of NFIL3 with its putative binding sites in CD4+ T cells activated under Th17 polarizing conditions (right panel). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (m) RT-PCR analysis of rorc and rora expression in CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of unpaired T-test. (n,o) Flow cytometry analysis of IL-17 expression (n) and rorc and il17 expression (o) in CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml) and transduced with a control or nfil3-encoding retrovirus. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (p,q) Flow cytometry analysis of IL-17 expression (p) and rorc and il17 expression (q) in wild type mice and NFIL3-deficient in CD4+ T cells activated under Th17 polarizing conditions in the presence of melatonin (2ng/ml). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (r) Flow cytometry analysis of IL-17 and IFN-γ expression in CD4+ T cells from RAG-1 deficient mice reconstituted with wild type, MTNR1A-REV-ERBα or NFIL3-deficient CD4+ T cells, immunized with MOG35-55 in CFA and treated with vehicle or melatonin (5mg/kg). *P<0.05 of unpaired T-test. See also Fig. S5
Figure 6
Figure 6. Melatonin boosts Tr1 cell differentiation
(a) RT-PCR analysis of il10, ahr and maf expression in Tr1 differentiated CD4+ T cells in the presence or absence of melatonin (2ng/ml). Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of one-way ANOVA (b) In vitro suppression assay, treated or untreated differentiated Tr1 cells as in a, were co-cultured after 72hs with CD4+ T cells previously labeled with CSFE, and proliferation cycles (CSFE dilution) were measured after 48hs by flow cytometry. Data are representative of two independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (c) Flow cytometry analysis of IL-10 expression in Tr1 differentiated CD4+ T cells in the presence or absence of melatonin (2ng/ml), agomelatine (20ng/ml, MTNR1A ligand) and CGP 52608 (20ng/ml, ROR-α ligand). Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (d) RT-PCR analysis of Tr1 cells cultured as in c. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (e) RT-PCR analysis of il10 expression as in c, in wild type mice and MTNR1A deficient mice. Data are representative of three independent experiments (means and s.e.m.) * P<0.05 of one-way ANOVA. (f) RT-PCR expression of il10 in melatonin treated Tr1 cells with or without the addition of UO126. Data are representative of five independent experiments (means and s.e.m.). * P<0.05 of unpaired T-test vs vehicle and signaling inhibitor control condition. ** P<0.05 vs vehicle of UO126-treated condition. (g) Flow cytometry analysis of IL-10 expression as in c, in wild type mice and ROR-α deficient mice. (h) ROR-α putative binding site present in the il10 promoter (lower panel), and chromatin immunoprecipitation with anti-ROR-α (upper panel) Data are representative of three independent experiments (means and s.e.m.). * P<0.05 of unpaired T-test. (i) Luciferase activity of HEK-293 cells transfected with a luciferase reporter construct for the il10 promoter. Data are representative of three independent experiments (means and s.e.m.)* P<0.05 of unpaired T-test. (j) Schematic diagram depicting the effects of melatonin in Tr1 cells.
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