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

Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

T cell maturation takes place within the thymus, a primary lymphoid organ that is commonly targeted during infections. Previous studies showed that acute infection with Paracoccidioides brasiliensis (Pb), the causative agent of paracoccidioidomycosis (PCM), promotes thymic atrophy that is associated with the presence of yeast cells in the organ. However, as human PCM is a chronic infection, it is imperative to investigate the consequences of Pb infection over the thymic structure and function in chronic infection. In this sense, we developed a new experimental model where Pb yeast cells are injected through the intraperitoneal route and mice are evaluated over 120 days of infection. Thymuses were analyzed in chronically infected mice and we found that the thymus underwent extensive morphological alterations and severe infiltration of P. brasiliensis yeast cells. Further analyses showed an altered phenotype and function of thymocytes that are commonly found in peripheral mature T lymphocytes. We also observed activation of the NLRP3 inflammasome in the thymus. Our data provide new information on the severe changes observed in the thymic microenvironment in a model of PCM that more closely mimics the human infection

Correction: Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

[This corrects the article DOI: 10.1371/journal.pone.0164745.]

Severe changes in thymic microenvironment in a chronic experimental model of paracoccidioidomycosis

T cell maturation takes place within the thymus, a primary lymphoid organ that is commonly targeted during infections. Previous studies showed that acute infection with Paracoccidioides brasiliensis (Pb), the causative agent of paracoccidioidomycosis (PCM), promotes thymic atrophy that is associated with the presence of yeast cells in the organ. However, as human PCM is a chronic infection, it is imperative to investigate the consequences of Pb infection over the thymic structure and function in chronic infection. In this sense, we developed a new experimental model where Pb yeast cells are injected through the intraperitoneal route and mice are evaluated over 120 days of infection. Thymuses were analyzed in chronically infected mice and we found that the thymus underwent extensive morphological alterations and severe infiltration of P. brasiliensis yeast cells. Further analyses showed an altered phenotype and function of thymocytes that are commonly found in peripheral mature T lymphocytes. We also observed activation of the NLRP3 inflammasome in the thymus. Our data provide new information on the severe changes observed in the thymic microenvironment in a model of PCM that more closely mimics the human infection1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informação#2014/19492-3; 2013/08194-9; #2012/22131-7; #2013/01401-9; #2014/02631-0; #2012/03238-

Protection against Paracoccidioides brasiliensisinfection in mice treated with modulated dendritic cells relies on inhibition of interleukin-10 production by Cd8(+) T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOParacoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensisconidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are ant1463486495FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP [2013/08194-9, 2011/17965-3]FAPESP [2012/22131-7, 2013/01401-9, 2014/02631-0]2013/08194-9; 2011/17965-3; 2012/22131-7; 2013/01401-9; 2014/02631-

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

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

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO123384386FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/17965-3; 2013/08194-9; 2014/02631-

Recirculating mature T cells home to infected thymuses, leading to increased frequency of cytokine producing Th17 and T CD8⁺ IFN-γ⁺.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Frequency of CD44^hiCD24^lo T cells increased among CD4⁺ and CD8⁺ subpopulations in 120dpi compared to the naive group. B) Cytokine producing T cells, Th17 and CD8⁺IFNγ⁺ was found in 120dpi, while practically absent in the naive group. Representative data from three independent experiments with similar results. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. ***p<0.001, ****p<0.0001. Representative data from three independent experiments with similar results.</p

Increased inflammasome and caspase-1 activity in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased initiator caspase-8 gene expression on 120dpi group compared to the naive group. Increased inflammatory caspase-1 gene expression on 120dpi group compared to the naive group. Increased NLRP3 inflammasome gene expression on 120dpi group compared to the naive group. B) Increased pro-caspase-1 production and increased caspase-1 activity on 120dpi group compared to the naive group. C) Increased NLRP3 inflammasome complex assembly on 120dpi compared to the naive group. Statistical analysis was carried out with Student’s t-test. **p<0.01, ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt). Image J software (NIH, MD, USA) was used for the estimation of the pro-caspase-1, the active form of caspase-1 and NLRP3 inflammasome assembly, through a GAPDH ratio.</p