Ry and lymphopenic conditions [56]. We did not observe this effect in the Title Loaded From File treatment of ongoing EAE. In fact, our results show that regulatory T cells raised by the CQ treatment were not converted into Title Loaded From File effector T cells, even at the 30th day after disease onset, as seen by the augmentation of Foxp3 expression and the reduction in IFN-c production. So, the treatment with chloroquine of established EAE resulted in reduction of EAE suggesting that a long-lasting immunomodulation can be achieved with this therapy. When CQ-elicited Treg cells are transferred to mice with ongoing EAE, the disease severity was reduced. The cellular response towards neuro-antigens in the periphery was contained and the pattern secretion of cytokines was altered as well. Transfer of CQ-Treg cells also reduced the infiltration of cell into the CNS, although the frequency of IL-10-producing cells was unaltered, which is distinct from the data observed with CQ treatment. The reduced dendritic cells number after CQ therapy may favor the higher suppression profile of CQ treatment over CQ-Treg cells transfer experiments. These data indicate that the amelioration of EAE after CQ treatment is a result of Treg-dependent and independent mechanisms. Other anti-malarial drugs are being tested in experimental models of inflammation. Recently, it was published thatartimisinin derivative, dihydroartemisinin (DHA), promoted suppression on EAE course [58]. The effects observed were dependent on Treg stimulation in the periphery. The authors showed that the mammalian target of rapamycin (mTOR) signaling cascade was attenuated in T cells [58], which also inhibits Th1 and Th17 differentiation [56]. The hypothesis that CQ treatment interferes with mTOR cascade was not investigated here. However, recent reports demonstrated that chloroquine targets the mTOR pathway in cancer cells inducing cell death [59]. We do not discard the possibility that chloroquine may influence the mTOR pathway promoting regulatory T cells and decreasing the frequency of Th1 and Th17 cells. Other studies must be conducted in order to define the precise mechanism by which chloroquine stimulates Treg cells expansion or differentiation. It was noticed that chronic abuse of chloroquine (cumulative ingestion of over 1000 g) in rheumatoid patients results in ocular toxic effects that may lead to blindness [60?2]. However, proper control of CQ consumption (3? mg/kg/day) reduced the incidence of retinopathy to 0,3? [63?5]. The dose used in this study is acceptable within the dose range for human treatment to produce less collateral effects. But, we highlight that the treatment period in this study was brief. So, other studies must be conducted to assess the efficacy of this treatment in human multiple sclerosis. The results presented herein indicate that chloroquine reduces the clinical course of EAE. We propose that this effect is due to the expansion of regulatory T cells in the spleen, which reduces the specific cellular response in the periphery. CQ may also affect other T cell subtypes that contribute to the reduced EAE severity. Interestingly, Treg cells also migrate to the CNS to reduce local inflammation and promote protection of the nervous system. Taken together, our data suggest that chloroquine may be a potential drug to be used as an adjunctive therapy in the treatment of multiple sclerosis.AcknowledgmentsThe authors thank Marcos Cesar Meneghetti for helping with animal care, ?Maria Eugenia Ribeiro de Ca.Ry and lymphopenic conditions [56]. We did not observe this effect in the treatment of ongoing EAE. In fact, our results show that regulatory T cells raised by the CQ treatment were not converted into effector T cells, even at the 30th day after disease onset, as seen by the augmentation of Foxp3 expression and the reduction in IFN-c production. So, the treatment with chloroquine of established EAE resulted in reduction of EAE suggesting that a long-lasting immunomodulation can be achieved with this therapy. When CQ-elicited Treg cells are transferred to mice with ongoing EAE, the disease severity was reduced. The cellular response towards neuro-antigens in the periphery was contained and the pattern secretion of cytokines was altered as well. Transfer of CQ-Treg cells also reduced the infiltration of cell into the CNS, although the frequency of IL-10-producing cells was unaltered, which is distinct from the data observed with CQ treatment. The reduced dendritic cells number after CQ therapy may favor the higher suppression profile of CQ treatment over CQ-Treg cells transfer experiments. These data indicate that the amelioration of EAE after CQ treatment is a result of Treg-dependent and independent mechanisms. Other anti-malarial drugs are being tested in experimental models of inflammation. Recently, it was published thatartimisinin derivative, dihydroartemisinin (DHA), promoted suppression on EAE course [58]. The effects observed were dependent on Treg stimulation in the periphery. The authors showed that the mammalian target of rapamycin (mTOR) signaling cascade was attenuated in T cells [58], which also inhibits Th1 and Th17 differentiation [56]. The hypothesis that CQ treatment interferes with mTOR cascade was not investigated here. However, recent reports demonstrated that chloroquine targets the mTOR pathway in cancer cells inducing cell death [59]. We do not discard the possibility that chloroquine may influence the mTOR pathway promoting regulatory T cells and decreasing the frequency of Th1 and Th17 cells. Other studies must be conducted in order to define the precise mechanism by which chloroquine stimulates Treg cells expansion or differentiation. It was noticed that chronic abuse of chloroquine (cumulative ingestion of over 1000 g) in rheumatoid patients results in ocular toxic effects that may lead to blindness [60?2]. However, proper control of CQ consumption (3? mg/kg/day) reduced the incidence of retinopathy to 0,3? [63?5]. The dose used in this study is acceptable within the dose range for human treatment to produce less collateral effects. But, we highlight that the treatment period in this study was brief. So, other studies must be conducted to assess the efficacy of this treatment in human multiple sclerosis. The results presented herein indicate that chloroquine reduces the clinical course of EAE. We propose that this effect is due to the expansion of regulatory T cells in the spleen, which reduces the specific cellular response in the periphery. CQ may also affect other T cell subtypes that contribute to the reduced EAE severity. Interestingly, Treg cells also migrate to the CNS to reduce local inflammation and promote protection of the nervous system. Taken together, our data suggest that chloroquine may be a potential drug to be used as an adjunctive therapy in the treatment of multiple sclerosis.AcknowledgmentsThe authors thank Marcos Cesar Meneghetti for helping with animal care, ?Maria Eugenia Ribeiro de Ca.
