Production of leukotriene B4 by Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. The production of eicosanoids during fungal infection has been associated with the biology of these microorganisms and modulation of host immune response. The aim of our study was to evaluate whether P. brasiliensis strains with high or low virulence produce leukotriene B4 (LTB4), using endogenous and/or exogenous sources of arachidonic acid (AA). Moreover, we assessed whether this fungus might use the same metabolic pathway, described for mammalian cells, that involves the lipoxygenase (LOX) enzyme. The association between the production of this eicosanoid and fungus survival and growth was also evaluated. Our results showed that P. brasiliensis, irrespective of its virulence, produces high levels of LTB4 using endogenous AA. In addition, in cultures treated with exogenous AA, LTB4 levels were significantly higher, showing that this fungus also uses exogenous sources of fatty acids. Treatment with MK886, which blocks the activity of lipoxygenase, by inhibiting five-lipoxygenase-activating protein (FLAP) or with nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, resulted in a significant reduction in LTB4 levels, indicating that the fungus produces this eicosanoid by using the LOX pathway or an enzyme with biochemically similar function. The significant reduction in viability detected in cultures treated with these inhibitors was, however, restored by adding exogenous LTB4 , confirming the role of this eicosanoid in fungus survival. Moreover, the addition of LTB4 to cultures capable of producing LTs induces fungal growth. These results provide a foundation for additional studies on the contributions of LTB4 in P. brasiliensis virulence.

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-α, the cytokine involved in cell activation for release of H(2)O(2), the fungicidal metabolite. Cell priming with IFN-γ 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-α, a monocyte-activating cytokine, also affects IL-10. The latter, in contrast to TNF-α is a monocyte-suppressing cytokine. Finally, we evaluated whether priming cells with other cytokines, namely TNF-α and GM-CSF, could be more effective than IFN-γ 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-α production, tends to increase IL-10 levels. Priming with GM-CSF or TNF-α was more effective than IFN-γ in compensating for the inhibitory PGE(2) effect, since these cytokines induce cells to produce higher H(2)O(2) and TNF-α levels.

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.

The immunomodulatory effect of propolis on receptors expression, cytokine production and fungicidal activity of human monocytes.

OBJECTIVES: Propolis is a beehive product and its immunomodulatory action has been documented; however, little is known concerning its mechanisms of action on human cells. Propolis influence on the initial events of the immune response was assessed, evaluating cell markers, cytokine production and the fungicidal activity of human monocytes. METHODS: Toll-like receptor (TLR)-2, TLR-4, human leukocyte antigen-DR and cluster of differentiation (CD)80 expression by human monocytes was assessed using a FACSCalibur flow cytometer, cytokine production (tumour necrosis factor (TNF)-α and interleukin (IL)-10) was determined by ELISA and the candidacidal activity was investigated after monocytes incubation with propolis and challenged with Candida albicans. The role of TLR-2 and TLR-4 on propolis action was assessed as well. KEY FINDINGS: Propolis upregulated TLR-4 and CD80 expression and affected TNF-α and IL-10 production, depending on concentration. Propolis also increased the fungicidal activity of monocytes. Cytokine production was decreased by blocking TLR-4, whereas the fungicidal activity was affected by blocking TLR-2. CONCLUSIONS: Propolis exerted an immunomodulatory action on cell receptors, cytokine production and fungicidal activity of human monocytes without affecting cell viability and depending on concentration. TLR-2 and TLR-4 may be involved in its mechanism of action.

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-γ, TNF-α 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.