Papel dos receptores Toll-Like na atividade dos neutrófilos humanos desafiados com a cepa de alta virulência do Paracoccidioides brasiliensis

Na paracoccidioidomicose, os estudos sobre os mecanismos de defesa do hospedeiro destacam o papel das células fagocitárias que participam ativamente dos mecanismos efetores diretos contra o fungo e da modulação da resposta inflamatória. Dentre essas células, nos últimos anos, os estudos têm se voltado para os neutrófilos, que exercem um importante papel efetor e modulador principalmente durante os estágios iniciais da infecção. As funções dessas células, incluindo as antimicrobicidas, podem ser reguladas por citocinas pró e anti-inflamatórias. Apesar dos mecanismos celulares e moleculares desse processo não estarem completamente entendidos, trabalhos nos últimos anos têm mostrado de forma bastante consistente o envolvimento dos “Toll Like Receptors” (TLRs). Neste contexto, os objetivos do presente trabalho foram: 1- Avaliar a expressão de TLR2 e TLR4 por neutrófilos humanos pré-ativados com GM-CSF, IL-15, TNF-a ou IFN-g e desafiados com cepa virulenta de Paracoccidioides brasiliensis cepa 18 (Pb18). 2- Avaliar a participação desses receptores na atividade fungicida, produção de H2O2 e das citocinas: IL-6, IL-8 e TNF-a e IL-10 por neutrófilos humanos pré-ativados com GM-CSF, IL-15, TNF-a, IFN-g e desafiados com Pb18. Nos ensaios referentes ao primeiro objetivo, as células foram tratadas por 18 horas com cada uma das citocinas ou LPS, posteriormente desafiadas com Pb 18 por 4 horas e avaliadas quanto a expressão de TLR2 e TLR4 por citometria de fluxo. Nos referentes ao segundo objetivo, as células foram ativadas com os mesmos estímulos, submetidas ao bloqueio de TLR2 e TLR4, através da incubação das células com anticorpos monoclonais específicos, desafiadas com Pb18 por 4 horas e analisadas quanto a atividade fungicida, produção de H2O2 e das citocinas IL-6, IL-8 e TNF-a e IL-10, por Elisa. Os resultados...In paracoccidioidomycosis, phagocytic cells appear to provide one of the major lines of host defense. In this context, in last years, various studies have focused on the role of neutrophils that are involved in primary response to the fungus. Neutrophil functions are regulated by pro and anti-inflammatory cytokines. The molecular and cellular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of “Toll Like Receptors” (TLRs). In this context, we aimed to evaluate TLR2 and TLR4 expression on human neutrophils primed with the cytokines GM-CSF, IL- 15, TNF-a or IFN-g and challenged with a virulent strain of Paracoccidioides brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H2O2 and IL-6, IL-8, TNF-a and IL-10 production, by primed and challenged cells. In first assays, neutrophils were incubated with each of cytokines or LPS by 18 h, challenged with Pb18 by 4 h and evaluated by TLR2 and TLR4 expression by flow cytometry. In other assays, cells were incubated with each of cytokines or LPS by 18h, submitted to TLR2 and TLR4 blocking by specific monoclonal antibodies, challenged with Pb18 for 4 h and evaluated for fungicidal activity, H2O2 and IL-6, IL-8, TNF-a and IL-10 production, by ELISA. All cytokines increased TLR2 and TLR4 expression. Pb18 increased TLR2 expression inducing an additional effect to that of cytokines. On the contrary, it inhibited TLR4 expression. All cytokines increased neutrophils fungicidal activity, but this process was not associated with TLR2 or TLR4 expression. All cytokines and Pb18 increased H2O2 production, but in the same manner to fungicidal activity, the process was not associated to TLR2 or TLR4 expression. Neutrophils activation with GM-CSF and TNF-a resulted in a significative increase on IL-8... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Paracoccidioides brasiliensis Uses Endogenous and Exogenous Arachidonic Acid for PGE(x) Production

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E-x (PGE(x)). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE(x) levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-gamma, TNF-alpha or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P brasiliensis is not correlated with NO concentrations produced. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a deep mycosis endemic in Latin America. Studies to elucidate the host-parasite relationship in this mycosis have demonstrated that non-activated phagocytes fail to kill the etiologic agent. Investigations of human monocytes have shown that the lack of fungicidal activity is partially associated with the capacity of a high-virulence strain to induce PGE(2) release by these cells. This eicosanoid inhibits production of TNF-alpha, the cytokine involved in cell activation for release of H2O2, the fungicidal metabolite. Cell priming with IFN-gamma was shown to partially reverse this inhibitory effect. In this study, we asked whether monocyte challenge with a low-virulence strain of this fungus would also result in PGE(2) release and consequently inhibition of antifungal activities. We also assessed whether PGE(2), besides inhibiting production of TNF-alpha, a monocyte-activating cytokine, also affects IL-10. The latter, in contrast to TNF-alpha, is a monocyte-suppressing cytokine. Finally, we evaluated whether priming cells with other cytokines, namely TNF-alpha and GM-CSF, could be more effective than IFN-gamma in reversing the PGE(2) inhibitory effect. The results revealed that the less virulent P. brasiliensis strain also induces human monocytes to release PGE(2). However, the inhibitory effect of PGE(2) was less pronounced when cells were challenged with this strain than with the more virulent one. It was also demonstrated that PGE(2), while inhibits TNF-alpha production, tends to increase IL-10 levels. Priming with GM-CSF or TNF-alpha was more effective than IFN-gamma in compensating for the inhibitory PGE(2) effect, since these cytokines induce cells to produce higher H2O2 and TNF-alpha levels.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for IS h increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism. (c) 2007 Elsevier Ltd. All rights reserved

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PNINs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H2O2, the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PNINs killing activity and H2O2 release were inhibited. Together, our results show the importance of PNINs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors

Background: Cardiovascular diseases remain leaders as the major causes of mortality in Western society. Restoration of the circulation through construction of bypass surgical treatment is regarded as the gold standard treatment of peripheral vascular diseases, and grafts are necessary for this purpose. The great saphenous vein is often not available and synthetic grafts have their limitations. Therefore, new techniques to produce alternative grafts have been developed and, in this sense, tissue engineering is a promising alternative to provide biocompatible grafts. This study objective was to reconstruct the endothelium layer of decellularized vein scaffolds, using mesenchymal stem cells (MSCs) and growth factors obtained from platelets. Methods: Fifteen nonpregnant female adult rabbits were used for all experiments. Adipose tissue and vena cava were obtained and subjected to MSCs isolation and tissue decellularization, respectively. MSCs were subjected to differentiation using endothelial inductor growth factor (EIGF) obtained from human platelet lysates. Immunofluorescence, histological and immunohistochemical analyses were employed for the final characterization of the obtained blood vessel substitute. Results: The scaffolds were successfully decellularized with sodium dodecyl sulfate. MSCs actively adhered at the scaffolds, and through stimulation with EIGF were differentiated into functional endothelial cells, secreting significantly higher quantities of von Willebrand factor (0.85 μg/mL; P < .05) than cells cultivated under the same conditions, without EIGF (0.085 μg/mL). Cells with evident morphologic characteristics of endothelium were seen at the lumen of the scaffolds. These cells also stained positive for fascin protein, which is highly expressed by differentiated endothelial cells. Conclusions: Taken together, the use of decellularized bioscaffold and subcutaneous MSCs seems to be a potential approach to obtain bioengineered blood vessels, in the presence of EIGF supplementation. © 2013 Society for Vascular Surgery