Violacein treatment modulates acute and chronic inflammation through the suppression of cytokine production and induction of regulatory T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOInflammation 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 mu g 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 naive 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.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 bacteriumChromobacterium vio105116FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [2011/17965-3]CNPq [471066/2012-5]FAPESP [2014/02631-0, 2011/23664-6, 2012/01892-0]2011/17965-3; 471066/2012-5; 2014/02631-0; 2011/23664-6; 2012/01892-0sem informaçã

Uncovering the molecular mechanisms of russet skin formation in Niagara grapevine (Vitis vinifera × Vitis labrusca)

Abstract Grape breeding programs are mostly focused on developing new varieties with high production volume, sugar contents, and phenolic compound diversity combined with resistance and tolerance to the main pathogens under culture and adverse environmental conditions. The ‘Niagara’ variety (Vitis labrusca $$\times$$ × Vitis vinifera) is one of the most widely produced and commercialized table grapes in Brazil. In this work, we selected three Niagara somatic variants with contrasting berry phenotypes and performed morphological and transcriptomic analyses of their berries. Histological sections of the berries were also performed to understand anatomical and chemical composition differences of the berry skin between the genotypes. An RNA-Seq pipeline was implemented, followed by global coexpression network modeling. ‘Niagara Steck’, an intensified russet mutant with the most extreme phenotype, showed the largest difference in expression and showed selection of coexpressed network modules involved in the development of its russet-like characteristics. Enrichment analysis of differently expressed genes and hub network modules revealed differences in transcription regulation, auxin signaling and cell wall and plasmatic membrane biogenesis. Cutin- and suberin-related genes were also differently expressed, supporting the anatomical differences observed with microscopy

Adoptive transfer of regulatory T cells elicited after violacein treatment reduces EAE through the modulation of CNS inflammation and inhibition infiltration of cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). 5x10⁵ cells were adoptively transferred into EAE-bearing mice, at the 10^th day after immunization, the clinical course was evaluated daily. At the 20^th day after immunization, mice were killed and lumbar spinal cords were removed and submitted to mRNA extraction protocols. A) The expression of FOXP3, IDO, IL-17, IL-10, IFN-γ and TNF-α was evaluated. Results show that adoptive transfer of viola-elicited Treg cells changed the CNS gene expression profile of EAE-bearing mice. B) The infiltration of cells in the CNS was evaluated as well. For that, spinal cords were collected and enriched in T cells, which were counted in hemocytometer. Results show that the treatment inhibited the infiltration of cells in the CNS of EAE-bearing mice. Representative data from two independent experiments. All values are represented as means ± standard error mean. *p<0.05.</p

Treatment with violacein reduces the severity of Experimental Autoimmune Encephalomyelitis through the modulation of CNS inflammation and infiltration of cells.

<p>C57BL/6 mice (n = 6 mice/group) were immunized with MOG_35–55 peptide emulsified in Complete Freund’s Adjuvant (CFA) and intraperitoneally injected with Pertussis toxin (200ηg) at 0 and 48h from immunization. Ten days after immunization, when the first clinical signs started to appear, mice were treated with viola (3.5 mg/Kg) for three consecutive days. At the 20^th day after immunization, mice were killed and lumbar spinal cords were removed and submitted to mRNA extraction protocols. A) The expression of FOXP3, IDO, iNOS, IL-17, IL-10, TGF-β, IFN-γ and TNF-α was evaluated and show that viola treatment changed the CNS gene expression profile of EAE-bearing mice. B) The infiltration of cells in the CNS was evaluated as well. For that, spinal cords were collected and enriched in T cells, which were counted in hemocytometer. Results show that the treatment reduced the infiltration of cells in the CNS of EAE-bearing mice. Representative data from two independent experiments. All values are represented as means ± standard error mean. *p<0.05 and **p<0.01.</p

Effect of viola treatment and viola-elicited Treg cells adoptive transfer on the severity of EAE.

<p>*: p<0,05, in comparison with the EAE group;</p><p>#: p<0,05, in comparison with the EAE+CD25^- group.</p><p>Analyses were performed with One-Way ANOVA followed by Bonferroni’s posttest.</p><p>Effect of viola treatment and viola-elicited Treg cells adoptive transfer on the severity of EAE.</p

Violacein treatment up-regulates the frequency of Regulatory T cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (1.75, 3.5 and 7 mg/Kg) for three consecutive days. A) Kaplan-Meier curve of survival show that mice receiving the 7mg/Kg dose died after the second administration. B) After the last dose of viola, mice were killed and spleen cells were stained for flow cytometry analysis of CD4⁺CD25⁺FOXP3⁺ (Treg) cells. Results show an increase in the frequency of Treg cells after viola treatment. C) Expression of CTLA-4 was evaluated inside the Treg cell population by flow cytometry. D) The intracellular cytokine detection was performed in spleen cells from PBS- and viola-treated mice. The spleen cells were stimulated with PMA (50 ng/mL) and Ionomycin (500 ng/mL) in the presence of Brefeldin A (1 μg/mL) for 4h at 37°C. Later cells were surface stained with antibody against CD4. Following permeabilization, cells were incubated with antibody cocktail for the detection of IL-17, IL-10 and IFN-γ and preparations were acquired in Flow Cytometer equipment. Representative data from three independent experiments. All values in bar graphs are represented as means ± standard error mean. *p<0.05.</p

Reduction in EAE severity after adoptive transfer of violacein-elicited Treg cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). 5x10⁵ cells were adoptively transferred into EAE-bearing mice, at the 10^th day after immunization (arrow), the clinical course was evaluated daily and showed reduction in the severity of CD4⁺CD25⁺-transferred mice compared to the other groups. Representative data from two independent experiments. All values are represented as means from each group. **p<0.01.</p

Violacein-elicited regulatory T cells present an enhanced suppressive activity than “naïve” Treg cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. A) After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). As controls, cells were isolated from spleens from naïve mice. Treg cells were seeded in U-bottom 96-wells culture plate in increasing numbers. C57BL/6 mice (n = 3) were immunized with MOG peptide. After seven days, mice were killed and the spleens were collected and disrupted for the isolation of dendritic cells and total T cells with dynabeads. T cells were stained with CFSE (1,5 μM) and seeded to the plates containing Treg cells (5x10⁵ cells/well). Dendritic cells were isolated as well and seeded to the wells (5x10⁴ DCs/ well). As controls, encephalitogenic T cells were cultivated without Treg cells. The plates were incubated for 72h at 37°C and the suppressive activity of Treg cells was analyzed by flow cytometry. B) Treg cells were isolated from PBS- and viola-treated mice, seeded to 96-well plate and incubated for 48h at 37 ºC. The supernatants were collected and assayed for the detection of IL-10 by CBA. Representative data from two independent experiments. All values are represented as means ± standard error mean. *: p<0.05, **: p<0,01 and ***: p<0,005. Ns: not significant.</p

Treatment with violacein reduces the severity of Experimental Autoimmune Encephalomyelitis.

<p>C57BL/6 mice (n = 6 mice/group) were immunized with MOG_35–55 peptide emulsified in Complete Freund’s Adjuvant (CFA) and intraperitoneally injected with Pertussis toxin (200ηg) at 0 and 48h from immunization. Ten days after immunization, when the first clinical signs started to appear, mice were treated with violacein (3.5 mg/Kg) for three consecutive days. A) Results show that the treatment reduced the severity of EAE. B) Later mice were killed and the frequency of macrophages (F4/80+ cells) and dendritic cells (CD11c+MHC-II+ cells) were assessed. The expression of CD80 and CD86 on DCs was evaluated as well. Representative data from three independent experiments. **p<0,01.</p

Violacein administration diminishes acute inflammation induced by Lipopolysaccharide injection.

<p>C57BL/6 mice (n = 3 mice/group) were treated with viola (3.5 mg/Kg) and lipopolysaccharide (1μg/mouse) through the intraperitoneal route. Three hours after LPS injection, mice were killed and the frequencies of dendritic cells (in A), T lymphocytes (in B), B cells (in C) and Neutrophils (in D) were assessed. E) The serum levels of selected cytokines and CXCL1 were determined as well. Representative data from two independent experiments.</p