Rodolfo Thomé

Chloroquine: Modes of action of an undervalued drug

For more than two decades, chloroquine (CQ) was largely and deliberately used as first choice drug for malaria treatment. However, worldwide increasing cases of resistant strains of Plasmodium have hampered its use. Nevertheless, CQ has recently been tested as adjunct therapy in several inflammatory situations, such as rheumatoid arthritis and transplantation procedures, presenting intriguing and promising results. In this review, we discuss recent findings and CQ mechanisms of action vis-à-vis its use as a broad adjunct therapy.

Yacon (Smallanthus sonchifolius)-derived fructooligosaccharides improves the immune parameters in the mouse

Owing to its high contents of fructooligosaccharides (FOSs), the yacon (Smallanthus sonchifolius) root is used in traditional Andean medicine as a substitute for cane sugar in diabetes and for obesity prevention. This study was designed to test the hypothesis that regular consumption of yacon works to improve the immune system. BALB/c mice were fed with the AIN-93 diet supplemented with 5% commercial FOS or either 3% or 5% yacon FOS for 30 consecutive days. Animals in the control group were fed with nonsupplemented ration. Food intake; weight gain; serum levels of IgA, IgM, and IgG; levels of fecal IgA, production of nitric oxide by peritoneal macrophages, frequencies of T and B lymphocytes in the spleen and peripheral blood, T-cell proliferation, and cytokine production were evaluated in all groups. No significant differences were observed in food intake and weight gain when the experimental and control groups were compared. Also, serum levels of IgA, IgM, and IgG; nitric oxide production in peritoneal macrophages; frequencies of T and B lymphocytes in the spleen and peripheral blood; T-cell proliferation; and production of interleukin (IL)-4, interferon-γ, IL-10, and tumor necrosis factor α did not differ in the different groups. The intake of FOS, however, led to a significant reduction of the proinflammatory cytokine IL-1β in macrophage cultures and elevation of the levels of fecal IgA. Together, these results indicate that the daily consumption of yacon does not exert negative effects on the immune system, helps to preserve an anti-inflammatory state in phagocytic cells, and improves mucosal immunity, possibly preventing the risks associated with autoimmune and metabolic diseases.

Chloroquine Treatment Enhances Regulatory T Cells and Reduces the Severity of Experimental Autoimmune Encephalomyelitis

Background The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well. Methodology/Principal Findings EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG35–55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG35–55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset. Conclusion We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE.

Dendritic cells treated with chloroquine modulate experimental autoimmune encephalomyelitis

Chloroquine (CQ), an antimalarial drug, has been shown to modulate the immune system and reduce the severity of experimental autoimmune encephalomyelitis (EAE). The mechanisms of disease suppression are dependent on regulatory T cell induction, although Tregs‐independent mechanisms exist. We aimed to evaluate whether CQ is capable to modulate bone marrow‐derived dendritic cells (DCs) both phenotypically and functionally as well as whether transfer of CQ‐modulated DCs reduces EAE course. Our results show that CQ‐treated DCs presented altered ultrastructure morphology and lower expression of molecules involved in antigen presentation. Consequently, T cell proliferation was diminished in coculture experiments. When transferred into EAE mice, DC‐CQ was able to reduce the clinical manifestation of the disease through the modulation of the immune response against neuroantigens. The data presented herein indicate that chloroquine‐mediated modulation of the immune system is achieved by a direct effect on DCs and that DC‐CQ adoptive transfer may be a promising approach for avoiding drug toxicity.

Oral tolerance and OVA-induced tolerogenic dendritic cells reduce the severity of collagen/ovalbumin-induced arthritis in mice

Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3(+) and IL-10(+) cells were higher, whereas IFNγ(+) cells and IL-17(+) cells were lower, among gated CD4(+) spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.

Phosphodiesterase-5 inhibition promotes remyelination by MCP-1/CCR-2 and MMP-9 regulation in a cuprizone-induced demyelination model

While it has recently been shown that sildenafil (Viagra®) has a protective effect on myelination/remyelination, the mechanism of this protection is still unknown. In general, cytokines, chemokines and metalloproteinases have a pro-inflammatory action, but can also exert a role in modulating glial cell activation, contributing to the balance of cell response. Investigating these molecules can contribute to clarifying the mechanisms of sildenafil neuroprotection. In addition, it is not known whether sildenafil is able to restore an already installed neurodegenerative process or if the treatment period is critical for its action. The aim of the present study was to evaluate, in a cuprizone (CPZ)-induced demyelination model, the effects and mechanisms of time-dependent treatment with sildenafil (beginning 15 days after neurodegeneration and continuing for 15 days, or starting concomitantly with neurodegeneration and continuing for 30 days) on neuroinflammation and remyelination. Neuroinflammation and demyelination induced by CPZ in rodents has been widely used as a model of multiple sclerosis (MS). In the present study, five male C57BL/6 mice aged 7-10 weeks were used per group. For four weeks, the groups received either cuprizone (CPZ) 0.2% mixed in feed or CPZ combined with the administration of sildenafil (Viagra®, Pfizer, 25 mg/kg) orally in drinking water, starting concurrently with (sild-T0) or 15 days (sild-T15) after the start of CPZ treatment. Control animals received pure food and water. The cerebella were dissected and processed for immunohistochemistry, immunofluorescence (frozen), Western blotting, Luxol fast blue staining and transmission electron microscopy. Magnetic resonance was performed for live animals, after the same treatment, using CPZ 0.3%. CPZ induced an increase in the expression of IL-1β and a decrease in MCP-1, CCR-2, MBP and GST-pi, as well as promoting damage in the structure and ultra-structure of the myelin sheath. Interestingly, the administering of sild-T0 promoted a further increase of MMP-9, MCP-1, and CCR-2, possibly contributing to changes in the microglia phenotype, which becomes more phagocytic, cleaning myelin debris. It was also observed that, after sild-T0 treatment, the expression of GST-pi and MBP increased and the myelin structure was improved. However, sild-T15 was not efficient in all aspects, probably due to the short treatment period and to starting after the installation of the degenerative process. Therefore, the present study shows that sildenafil modulates inflammation, with the involvement of MMP-9, MCP-1, and CCR-2, and also contributes to myelin repair. These protective effects were dependent on the therapeutic strategy used. This clarification can strengthen research proposals into the mechanism of action of sildenafil and contribute to the control of neurodegenerative diseases such as MS.

Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy

Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.

Dendritic cells treated with crude Plasmodium berghei extracts acquire immune-modulatory properties and suppress the development of autoimmune neuroinflammation.

Dendritic cells (DCs) are professional antigen‐presenting cells specifically targeted during Plasmodium infection. Upon infection, DCs show impaired antigen presentation and T‐cell activation abilities. In this study, we aimed to evaluate whether cellular extracts obtained from Plasmodium berghei‐infected erythrocytes (PbX) modulate DCs phenotypically and functionally and the potential therapeutic usage of PbX‐modulated DCs in the control of experimental autoimmune encephalomyelitis (EAE, the mouse model for human multiple sclerosis). We found that PbX‐treated DCs have impaired maturation and stimulated the generation of regulatory T cells when cultured with naive T lymphocytes in vitro. When adoptively transferred to C57BL/6 mice the EAE severity was reduced. Disease amelioration correlated with a diminished infiltration of cytokine‐producing T cells in the central nervous system as well as the suppression of encephalitogenic T cells. Our study shows that extracts obtained from P. berghei‐infected erythrocytes modulate DCs towards an immunosuppressive phenotype. In addition, the adoptive transfer of PbX‐modulated DCs was able to ameliorate EAE development through the suppression of specific cellular immune responses towards neuro‐antigens. To our knowledge, this is the first study to present evidence that DCs treated with P. berghei extracts are able to control autoimmune neuroinflammation.

Nitric oxide plays a key role in the suppressive activity of tolerogenic dendritic cells.

Tolerogenic dendritic cells (DCs) are widely studied for their possible use in the treatment of inflammatory disorders, such as autoimmune diseases. One of the obstacles for the use of this cell-based therapy is the characterization of drugs that are able to modulate DCs. We have previously shown that chloroquine (CQ), an antimalarial agent, has the ability to modulate DCs towards a tolerogenic phenotype.1 These tolerogenic DCs are able to suppress the development of experimental autoimmune encephalomyelitis (EAE), a T cell-driven mouse model of human multiple sclerosis. In addition, several studies have proposed that nitric oxide (NO) plays a major role in the differentiation of regulatory T cells (Tregs) and the suppression of Th1/Th17 cells.2,3 However, little is known about the role of DC-derived NO in the modulation of inflammatory autoimmune responses. Thus, we aimed to evaluate whether NO plays a role in the tolerogenic activity of CQ-treated DCs (CQ-DCs). We found that CQ induces DC production of NO and expression of indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (iNOS). In addition, CQ-DCs stimulated the differentiation of Tregs at the expense of Th1/Th17 cells. On the other hand, iNOS−/− DCs did not acquire a tolerogenic phenotype following CQ treatment. Rather, CQ-DCsiNOS−/− stimulated the differentiation of Th1/Th17 cells as well as Tregs. In a therapeutic approach, CQ-DCsiNOS−/− were unable to suppress the development of EAE. Gene expression analyses of central nervous system (CNS) tissue from mice that received CQ-DCsiNOS−/− showed an increased expression of inflammatory modulators compared with mice that received CQ-DCsWT. In this work, we show that NO is an important factor in the modulatory activity of tolerogenic dendritic cells.

Exacerbation of Autoimmune Neuro-Inflammation in Mice Cured from Blood-Stage Plasmodium berghei Infection

The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P.berghei NK65 and treated with chloroquine (CQ), with MOG35–55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35–55-immunized mice after adoptive transfer of P.berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.