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.

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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 (CQDCs). 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 2/2 DCs did not acquire a tolerogenic phenotype following CQ treatment. Rather, CQDCs iNOS2/2 stimulated the differentiation of Th1/Th17 cells as well as Tregs. In a therapeutic approach, CQ-DCs iNOS2/2 were unable to suppress the development of EAE. Gene expression analyses of central nervous system (CNS) tissue from mice that received CQ-DCs iNOS2/2 showed an increased expression of inflammatory modulators compared with mice that received CQ-DCs WT . In this work, we show that NO is an important factor in the modulatory activity of tolerogenic dendritic cells.DCs are antigen-presenting cells that can dictate the course of the immune response via the modulation and activation of naive T cells. DC modulation is a possible approach to address the immunosuppression that is often caused by tumors 4 and the exacerbated immune response observed in autoimmune diseases. 5 Multiple sclerosis, one such autoimmune disease, is a debilitating condition that affects the CNS. Studies in EAE, an experimental mouse model of multiple sclerosis, have found that much of the immunological etiology of the disease development is due to the activity of Th1/Th17 cells, and these studies have found that NO plays a major role in disease progression. 2 To verify whether NO is involved in the modulatory activity of tolerogenic DCs, we generated DCs from bone marrow precursors obtained from wild-type (DCs WT ) and iNOS 2/2 (DCs iNOS2/2 ) mice and treated these DCs with CQ or vehicle (PBS-DCs WT ). All protocols involving laboratory animals were approved by the institutional committee (protocol no. 2687-1). NO measurements revealed that CQ treatment induced DCs WT to produce large amounts of NO in an iNOS-dependent manner (Figure 1a). It has been demonstrated that CQ administration results in NO production by the endot...

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MS and EAE are T cell-driven autoimmune diseases of the CNS where IL-17-producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ-induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ-treated DCs. Opposed to its WT counterparts, CQ-treated STAT1 BMDCs were unable to suppress Th17 cells and increased EAE severity. Our findings show that STAT1 is a major signaling pathway in CQ-induced tolDCs and may shed light on new therapeutic avenues for the induction of tolDCs in autoimmune diseases such as MS.

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

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