As observed in cell cultures from CD25+ transferred- mice when compared to control group (Figure 6D).Figure 5. Chloroquine administration after the onset of EAE reduces the clinical signs of the disease. (A) CQ 1317923 was administrated ten days after immunization with MOG35?5. (B) and (C) Animals were accompanied for weight changes and clinical score. (D) The spinal cords were removed at day 30 and 10 mm slices were stained with HE for detection of infiltrating cells in the CNS. Figures are representative of at least six mice. Bar: 500 mm. (E) Gene expression of IL-17, IFNc, Foxp3 and RORc in the CNS was evaluated. (F) The infiltrating cells in the CNS were collected and stained for flow cytometric characterization of IL-10, IL-17 and IFN-c production. (G) Spleen cells from EAE mice, CQ- and PBS-treated mice, were removed and CFSE-stained cells were cultivated (56105/well) in the presence of MOG35?5 peptide (20 mg/mL) for 96 h. The dye decay and cytokine production were measured by flow cytometry. Results are representative of three independent experiments and are expressed as mean 6 SEM for at least five animals. * p,0.05. doi:10.1371/journal.pone.0065913.gChloroquine Supresses EAE?Figure 6. Transfer of CQ-elicited Treg cells reduces the severity of ongoing EAE. (A) Naive C57BL/6 mice were treated with chloroquine (5 mg/kg/day) for five consecutive days. Three days after the last dose of the treatment, splenic CD4+CD25+ cells were isolated using magnetic beads and cells (56105 cells 1317923 was administrated ten days after immunization with MOG35?5. (B) and (C) Animals were accompanied for weight changes and clinical score. (D) The spinal cords were removed at day 30 and 10 mm slices were stained with HE for detection of infiltrating cells in the CNS. Figures are representative of at least six mice. Bar: 500 mm. (E) Gene expression of IL-17, IFNc, Foxp3 and RORc in the CNS was evaluated. (F) The infiltrating cells in the CNS were collected and stained for flow cytometric characterization of IL-10, IL-17 and IFN-c production. (G) Spleen cells from EAE mice, CQ- and PBS-treated mice, were removed and CFSE-stained cells were cultivated (56105/well) in the presence of MOG35?5 peptide (20 mg/mL) for 96 h. The dye decay and cytokine production were measured by flow cytometry. Results are representative of three independent experiments and are expressed as mean 6 SEM for at least five animals. * p,0.05. doi:10.1371/journal.pone.0065913.gChloroquine Supresses EAE?Figure 6. Transfer of CQ-elicited Treg cells reduces the severity of ongoing EAE. (A) Naive C57BL/6 mice were treated with chloroquine (5 mg/kg/day) for five consecutive days. Three days after the last dose of the treatment, splenic CD4+CD25+ cells were isolated using magnetic beads and cells (56105 cells 1315463 per mouse) were transferred into mice with ongoing EAE (10 days after immunization). As controls, mice received the same number of CD4+CD252 cells. (B) The clinical course of the disease was evaluated routinely. (C) The brains and spinal cords were collected and the enriched infiltrating cells were counted. The frequency of IL-17-, IL-10- and IFN-c-producing cells was analyzed by flow cytometry as well. (D) The spleens were collected and CFSE-stained cells were cultivated in the presence of MOG35?5 peptide for 96 h. Dye decay and cytokine production were analyzed by flow cytometry. Results are expressed as mean 6 SEM for at least five animals. p,0,05 (*), p,0,01 (**) and p,001 (***). doi:10.1371/journal.pone.0065913.gDiscussionAutoimmune diseases develop in deregulated immune systems that fail to control chronic inflammation. Although the events that trigger disease development are unknown, multiple sclerosis is an immune mediated syndrome with characteristics of acute and chronic inflammation [35,36]. Therapies that focus on reestablishing homeostasis and immunomodulation are of great value. Regulatory T cells play an important role in the control of inflammation and suppression of auto-reactive cells [3,8,37]. In this context, we found that chloroquine administration provokes an increase in Treg cells frequency in the spleen of normal mice. When administrated, both prophylactic and therapeutically, CQ modulated the course of EAE, an animal model for multiple sclerosis. The transfer of CQ-elicited Treg cells into mice with ongoing EAE promoted a reduction in disease severity as well. Chloroquine, an anti-malarial agent, was shown to have antiinflammatory properties. The administration of the drug resulted in impaired iron metabolism and TNF-a production by macrophages [20,38], as well as altered cytokine secretion profile [19,39,40]. It was also shown that chloroquine affects T cell priming to minor MHC complexes and may be used to modulate graft-versus-host dise.
