Participação das NETs (neutrophil extracellular traps) na atividade fungicida de neutrófilos humanos contra o Paracoccidioides brasiliensis

A paracoccidioidomicose é uma micose sistêmica causada pelo fungo dimófico Paracoccidioides brasiliensis, que é endêmico na América Latina. As células fagocitárias desempenham um papel importante durante a resposta imune inata contra o Pb, e se tornaram alvo de estudos neste sentido. Em especial, os neutrófilos são capazes de destruir microrganismos através de três mecanismos: fagocitose, seguida pela morte em fagolisossoma, secreção de agentes microbicidas e através do mais recente mecanismo descoberto, a netose, que consiste na liberação de redes extracelulares a partir dos neutrófilos (NETs), constituídas por proteínas granulares associadas a cromatina. Neste estudo demonstramos pela primeira vez que leveduras de P. brasiliensis induzem NETs in vitro principalmente através da ligação pelo receptor dectina-1 presente nos neutrófilos. Estas estruturas foram demonstrados através de microscopia eletrônica de varredura, e os componentes específicos das NETs: histona, elastase e DNA foram evidenciados a partir da microscopia eletrônica de imunofluorescência confocal. As leveduras foram capturadas pelas NETs, o que revelou um importante papel destas esrtruturas: impedir a disseminação do fungo. Além disso, o tratamento das co-culturas com DNAse, que degradou as NETs evidenciou aumento da sobrevivência do fungo, o que denota sua capacidade microbicidaParacoccidiodomycosis is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, which is endemic in Latin America. Since phagocytic cells play an important role during innate immune response against this fungus, we have studied the relationship between human neutrophils (PMNs) and P. brasiliensis, focusing on the effector mechanisms of these cells. Neutrophils can destroy microrganisms using at least 3 distint mechanisms: phagocytosis followed by destruction in a phagolysossome, secretion of antimicrobial molecules, and the more recently identified netosis, involving the release of neutrophil extracellular traps ( NETs) that are constituted by chromatin associated with different granule proteins with antimicrobial activities. Here, we showed for the first time, that yeast cells from P. brasiliensis strain 18 are able to induce the release of NETs in vitro, by binding to dectin-1 receptor on human neutrophils. These structures were evidenced by scanning electron microscopy, and specific NETs compounds such as histone and elastase were shown by confocal microscopy. Fungi were ensnared by NETs, denotting the role of these structures in confining infection, avoiding dissemination. In addition, disruption of NETs by treatment of cocultures with DNAse increased the fungi survival, evidencing their killing capacityFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Lemongrass and citral effect on cytokines production by murine macrophages

Ethnopharmacological relevance: Cymbopogon citratus (DC) Stapf (Poaceae-Gramineae), an herb commonly known as lemongrass (LG), is an important source of ethnomedicines as well as citral, the major constituent of Cymbopogon citratus, used in perfumery, cosmetic and pharmaceutical industries for controlling pathogens. Thus, the goal of this work was to analyze the effect of LG and citral on cytokines production (IL-1 beta, IL-6 and IL-10) in vitro, as well as before or after LPS incubation.Materials and methods: Peritoneal macrophages from BALB/c mice were treated with LG or citral in different concentrations for 24 h. The concentrations that inhibited cytokines production were tested before or after macrophages challenge with LPS, in order to evaluate a possible anti-inflammatory action. Supernatants of cell cultures were used for cytokines determination by ELISA.Results: As to IL-1 beta. only citral inhibited its release, exerting an efficient action before LPS challenge. LG and citral inhibited IL-6 release. Cymbopogon citratus showed inhibitory effects only after LPS challenge, whereas citral prevented efficiently LPS effects before and after LPS addition. Citral inhibited IL-10 production and although LG did not inhibit its production, the concentration of 100 mu g/well was tested in the LPS-challenge protocol, because it inhibited IL-6 production. LG inhibited LPS action after macrophages incubation with LPS, while citral counteracted LPS action when added before or after LPS incubation.Conclusion: LG exerted an anti-inflammatory action and citral may be involved in its inhibitory effects on cytokines production. We suggest that a possible mechanism involved in such results could be the inhibition of the transcription factor NF-kappa B. (C) 2011 Elsevier B.V. All rights reserved

The Effects of Propolis and its Isolated Compounds on Cytokine Production by Murine Macrophages

Since propolis and phenolic compounds, such as cinnamic and coumaric acids, have several biological properties, their immunomodulatory effect on cytokine production (IL-1 beta, IL-6 and IL-10) was investigated. Peritoneal macrophages from BALB/c mice were incubated with propolis, coumaric and cinnamic acids in different concentrations and the concentrations that inhibited cytokine production were tested before or after macrophage challenge with LPS, to evaluate a possible immunomodulatory action. Propolis and the acids stimulated IL-1 beta production, while IL-6 production was significantly inhibited after incubation with propolis (5, 50 and 100 mu g/well), coumaric and cinnamic acids (50 and 100 mu g/well). In LPS-challenge protocols, inhibitory concentrations of cinnamic and coumaric acids after LPS incubation prevented efficiently its effects on IL-6 production, whereas propolis inhibited LPS effects both before and after its addition. Propolis, coumaric and cinnamic acids (50 and 100 mu g/well) inhibited IL-10 production as well. Both acids showed a similar inhibitory activity on IL-10 production when added after LPS challenge, while propolis counteracted LPS action when added before and after LPS incubation. Propolis modulated the immune/inflammatory response, depending on the concentration. Its efficiency may occur due to the synergistic effect of its compounds, and cinnamic and coumaric acids may be involved in the action of propolis on cytokine production. Copyright (C) 2012 John Wiley & Sons, Ltd.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Propolis effect on Th1/Th2 cytokines production by acutely stressed mice

Aim of the study: Propolis has gained special attention due to its biological properties, however, little is known about its immunomodulatory effects in stress conditions. The purpose of this study was to investigate propolis effect on Th1/Th2 cytokines production by spleen cells of acutely stressed mice. Serum corticosterone concentration was determined as a stress indicator.Materials and methods: Male BALB/c mice were submitted to restraint stress and treated with propolis (200 mg/kg) for 3 days. Supernatants of splenocytes cultures were assessed for Th1/Th2 cytokines determination.Results: Regarding Th1 cytokines production, no alterations were seen in IL-2 production; however, IFN-gamma production was inhibited in stressed mice, even when treated with propolis. As to Th2 cytokines, IL-4 was inhibited in stressed mice, but normal levels were seen when these animals were treated with propolis. No significant differences were found in IL-10 production between the experimental groups. Stressed groups (treated or not with propolis) showed higher corticosterone concentrations in comparison to control group.Conclusions: Data suggest that propolis treatment was not able to counteract the stress-induced immunosuppressive effect on IFN-gamma production; however, propolis showed an immunorestorative role, increasing IL-4 production in stressed mice, favoring humoral immune response during stress. Since the exact mechanisms of this natural product on immune system are still unclear, further studies are still required for a better comprehension of propolis use as a therapeutic alternative against the stress-induced negative effects that could lead to the development of various diseases. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Clove and eugenol in noncytotoxic concentrations exert immunomodulatory/anti-inflammatory action on cytokine production by murine macrophages

Objectives The extract and essential oil of clove (Syzygium aromaticum) are widely used because of their medicinal properties. Eugenol is the most important component of clove, showing several biological properties. Herein we have analysed the immunomodulatory/anti-inflammatory effect of clove and eugenol on cytokine production (interleukin (IL)-1 beta, IL-6 and IL-10) in vitro. Methods Macrophages were incubated with clove or eugenol (5, 10, 25, 50 or 100 mg/well) for 24 h. Concentrations that inhibited the production of cytokines were used before or after incubation with lipopolysaccharide (LPS), to verify a preventive or therapeutic effect. Culture supernatants were harvested for measurement of cytokines by enzyme-linked immunosorbent assay. Key findings Clove (100 mg/well) inhibited IL-1 beta, IL-6 and IL-10 production and exerted an efficient action either before or after LPS challenge for all cytokines. Eugenol did not affect IL-1 beta production but inhibited IL-6 and IL-10 production. The action of eugenol (50 or 100 mg/well) on IL-6 production prevented efficiently effects of LPS either before or after its addition, whereas on IL-10 production it counteracted significantly LPS action when added after LPS incubation. Conclusions Clove exerted immunomodulatory/anti-inflammatory effects by inhibiting LPS action. A possible mechanism of action probably involved the suppression of the nuclear factor-kB pathway by eugenol, since it was the major compound found in cloveFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2008/06120-0

Propolis Immunomodulatory Action In Vivo on Toll-Like Receptors 2 and 4 Expression and on Pro-Inflammatory Cytokines Production in Mice

Propolis is a bee product and its immunomodulatory action has been the subject of intense investigation lately. The recent discovery and characterization of the family of Toll-like receptors (TLR) have triggered a great deal of interest in the field of innate immunity due to their crucial role in microbial recognition and development of the adaptive immune response. This work aimed to evaluate propolis's effect on TLR-2 and TLR-4 expression and on the production of pro-inflammatory cytokines (IL-1 beta and IL-6). Male BALB/c mice were treated with propolis (200 mg/kg) for three consecutive days, and TLR-2 and TLR-4 expression as well as IL-1 beta and IL-6 production were assessed in peritoneal macrophages and spleen cells. Basal IL-1 beta production and TLR-2 and TLR-4 expression were increased in peritoneal macrophages of propolis-treated mice. TLR-2 and TLR-4 expression and IL-1 beta and IL-6 production were also upregulated in the spleen cells of propolis-treated mice. One may conclude that propolis activated the initial steps of the immune response by upregulating TLRs expression and the production of pro-inflammatory cytokines in mice, modulating the mechanisms of the innate immunity. Copyright (C) 2009 John Wiley & Sons, Ltd.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Th1/Th2 cytokine production by clove-treated mice

Although clove possesses several biological and therapeutic properties, its immunomodulatory action has not been fully investigated. The goal of this work was to investigate the effect of administration of the water extract of clove over a short-term to BALB/c mice on Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokine production. After treatment, spleen cells were aseptically removed and cells were stimulated with concanavalin A. Supernatants of cell cultures were used for cytokine determination by ELISA. The chemical composition of the extract was also carried out, revealing that eugenol(4-allyl-2-methoxyphenol) was the major component in our sample. Although the anti-inflammatory action of clove has been mentioned, our data showed that clove administration to mice did not influence the Th1/Th2 cytokine balance. Further studies dealing with cytokine expression and production will provide a better understanding of clove's immunomodulatory and anti-inflammatory actions, using different extract concentrations and different intake periods

Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice

Introduction: Propolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1 beta and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells.Methods: BALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator.Results: Stressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1 beta and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1 beta and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress.Conclusion: Propolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES