Work

Primaquine Treatment Suppresses Experimental Autoimmune Encephalomyelitis Severity

Year: 2014

Type: article

Abstract: Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent disease that mimics human multiple sclerosis (MS). The inflammation in the central nervous system (CNS) is a hallmark in these autoimmune inflammatory conditions 1. Therefore, the development of new drugs that target the infiltration of inflammatory cells to the CNS is desired 2, 3. In this context, accumulating evidences place antimalarial drugs as immunomodulatory agents. For instance, administration of chloroquine, a weak base that has tropism toward acidic organelles, was shown to modulate a series of inflammatory diseases, including EAE 4, 5. On the other hand, primaquine (PQ), a drug analogous to chloroquine 6, has much less toxicity and is poorly investigated in the control of inflammation. In this study, we aimed to evaluate whether PQ administration is able to reduce EAE severity. For that purpose, we induced EAE in C57BL/6 mice through subcutaneous injection of 100 μg of MOG35–55 peptide alongside with 200 ng of pertussis toxin, as previously described 7. When the clinical signs of EAE started to appear, mice were treated with PQ (3 mg/kg, intraperitoneally) for five consecutive days and disease outcome was evaluated (EAE+PQ group). Mice treated with vehicle (PBS) were used as controls (EAE group). We observed that PQ treatment significantly reduced the development of EAE (Figure 1A). Interestingly, disease amelioration correlated with lower infiltration of cells in the CNS (Figure 1B). Notwithstanding, the frequency of Iba-1+ cells (microglia, macrophages, and dendritic cells) was found reduced in the EAE+PQ group compared with untreated EAE-bearing mice, suggesting that probably the demyelination process is reduced in the CNS of EAE+PQ mice (Figure 1B). To evaluate whether glial cells are affected by PQ treatment, slices were stained for glial fibrillary acidic protein (GFAP) production. Our results showed that EAE+PQ mice presented lower expression of GFAP in comparison with the EAE group (Figure 1B). Of note, PQ treatment by itself did not induce GFAP expression. Collectively, these results show that improvement in EAE+PQ mice correlated with a reduction in cellular infiltration and gliosis. To investigate whether mediators of inflammation are involved in disease amelioration, the gene expression levels of inflammation-related cytokines and transcription factors were analyzed. Results showed that levels of FOXP3, IL-10, and TGF-β were significantly increased, while the levels of TNF-α, IL-17, IFN-γ, and GATA-3 were decreased in the CNS of EAE+PQ mice compared with the control (Figure 1C). Unexpectedly, levels of Th17 cell-related transcription factor, RORγt, were found reduced in EAE-bearing mice compared with naïve mice, showing that modulation of this transcription factor is not a key element in PQ-induced EAE amelioration. The increased expression in FOXP3, IL-10, and TGF-β in the CNS of EAE+PQ mice suggests that the treatment induces the expansion of regulatory T (Treg) cells. To confirm this hypothesis, naïve mice were treated with PQ and 3 days after the last dose of the treatment, mice were killed. Then, the frequency of splenic CD4+FOXP3+ cells was assessed. The results showed that PQ administration promoted a significant increase in the frequency of Treg cells (Figure 2A). To investigate whether these PQ-elicited regulatory T cells are involved in the modulation of EAE severity, we isolated CD4+CD25+ cells, also called Treg cells due to their co-expression of FOXP3, and adoptively transferred toward EAE-bearing mice at the onset of the disease (EAE+CD25+ group). As control, we transferred CD4+CD25− cells to EAE-induced mice (EAE+CD25− group). We accompanied the clinical progression of the disease. Data showed that mice recipient of CD4+CD25+ cells developed mild EAE compared with the control groups (Figure 2B). Disease improvement correlated with a lower cellular infiltration in the CNS of EAE+CD25+ mice in comparison with the EAE+CD25− and EAE groups (Figure 2C). In addition, the frequency of Iba-1+ and GFAP+ cells was found reduced in the CNS of EAE+CD25+ mice (Figure 2C). It is unknown the mechanisms by which primaquine modulates inflammation. Studies with chloroquine (CQ), a drug that possesses a similar mechanism of action, have demonstrated that the alkalization of lysosomes promotes the disruption of the antigen processing machinery in dendritic cells. In addition, CQ administration altered the iron and calcium metabolism in T cells and macrophages. CQ was also shown to reduce the secretion of inflammatory cytokines (reviewed in 4). The mechanism exerted by CQ is not restricted to inflammation, but also in the control of tumor growth, as human mammary cancer cells were unable to proliferate after chloroquine treatment 8. Although deficiency in glucose-6-phosphate dehydrogenase enzyme leads to exacerbation of its toxicity 9, primaquine is less toxic than chloroquine 10. Literature data indicate that primaquine might be an undervalued drug that needs further investigation regarding its use in the control of neuroinflammation. Taken together, our results show that primaquine, an antimalarial drug, presents antiinflammatory properties, which are dependent on the expansion of Treg cells. In the EAE model, disease amelioration correlated with both reduction of cellular infiltration and alterations in the expression of inflammatory mediators in the CNS. Still, further studies are needed to determine the mechanisms of Treg cells differentiation induced by primaquine. This work was supported by São Paulo Research Foundation (FAPESP, #2011/17965-3; 2013/08194-9). FZ and ITF were supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). RT, TAC, RdG were supported by FAPESP (2014/02631-0, 2012/22131-7 and 2013/01401-9, respectively). LV is fellow of the National Council of Technological and Scientific Development (CNPq). The authors declare no conflict of interest.

Source: CNS Neuroscience & Therapeutics

DOI: 10.1111/cns.12357