Thiago Alves da Costa

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.

Growth direction and Si alloying affecting directionally solidified structures of Al–Cu–Si alloys

Abstract The roles of growth direction and Si content on the columnar/equiaxed transition and on dendritic spacings of Al–Cu–Si alloys still remain as an open field to be studied. In the present investigation, Al–6 wt-%Cu–4 wt-%Si and Al–6 wt-%Cu alloys were directionally solidified upwards and horizontally under transient heat flow conditions. The experimental results include tip growth rate and cooling rates, optical microscopy, scanning electron microscopy energy dispersive spectrometry and dendrite arm spacings. It was found that silicon alloying contributes to significant refinement of primary/secondary dendritic spacings for the upward configuration as compared with corresponding results of the horizontal growth. Experimental growth laws are proposed, and the effects of the presence/absence of solutal convection in both growth directions are discussed.

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.

Cooling thermal parameters, microstructural spacing and mechanical properties in a directionally solidified hypereutectic Al–Si alloy

Abstract Transient directional solidification experiments have been carried out with an Al-15wt. %Si alloy under cooling rates () from 0.2 to 54 K/s. A mixture of globular and fibre-like Si particles within the eutectic is shown to occur for cooling rates () > 9 K/s. The presence of refined globular Si particles arranging the microstructure either as primary particles or within the eutectic mixture seems to contribute for a combination of high tensile strength (σu) and elongation (δ). It is shown by correlations between eutectic (λΕ) and secondary dendrite (λ2) spacings and σu and δ, given by Hall–Petch type formulae that the tensile properties increase significantly with the decrease in these spacings. However, it is shown that for higher ranges of spacings, i.e.: λΕ−1/2 < 0.65 and λ2−1/2 < 0.18, both tensile strength and elongation remain unaltered.

Characterization of Dendritic Microstructure, Intermetallic Phases, and Hardness of Directionally Solidified Al-Mg and Al-Mg-Si Alloys

THE authors regret that numerical errors were identified in Figures 10(a, right graph) and (b, right graph) and 12. In the calculation of the growth rate of the Al-3.0 wt pct Ni alloy, a mistake has been found. The corrected figures and their respective legends are reproduced below. The error does not change the main discussions and conclusions of the work. In addition, a sentence containing the discussion associated with Figure 12 has been rewritten as follows. As can be observed in Figure 12, the BKmodel roughly matches the experimental tendency of evolution of the secondary dendrite arm spacing with the growth rate.

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.

Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells

Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.

Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as a Function of Microenvironmental Glucose Level

Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high‐glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257–2265, 2016.