TLR9 stimulation induces increase in fungicidal activity of human dendritic cells challenged with Paracoccidioides brasiliensis.

Microorganisms killing by dendritic cells (DCs) is an important effector mechanism during innate immune response, as it can avoid dissemination of infection during migration of these cells toward draining lymph nodes. However, this function depends on pattern recognition receptors (PRRs) to which the microorganism will bind in these cells. Regarding this, TLR9 activation, by stimulating the oxidative metabolism, induces increase in microbicidal activity of these cells. Accordingly, we showed that DCs treatment with a TLR9 agonist results in an increase in fungicidal activity of these cells against the fungus Paracoccidioides brasiliensis (Pb), which however, was not associated to higher H2O2 levels.

Interleukin-18 increases TLR4 and mannose receptor expression and modulates cytokine production in human monocytes.

Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs) that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10.

Interleukin-10 but not Transforming Growth Factor beta inhibits murine activated macrophages Paracoccidioides brasiliensis killing: effect on H2O2 and NO production.

Paracoccidioidomycosis is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). Most often, this mycosis runs as a chronic progressive course affecting preferentially the lungs. In vitro fungicidal activity against a high virulent strain of P. brasiliensis by murine peritoneal macrophages preactivated with IFN-gamma or TNF-alpha is high and correlates with increased NO and H2O2 production. Within this context, the purpose of this work was to study the role of suppressor cytokines, such as IL-10 and TGF-beta, in this process. Incubation of either IFN-gamma or TNF-alpha with IL-10 inhibits fungicidal activity of these cells. However, TGF-beta had no effect on fungicidal activity of IFN-gamma or TNF-alpha-activated macrophages. The suppression of fungicidal activity by IL-10 correlated with the inhibition of NO and H2O2 production supporting the involvement of these metabolites in P. brasiliensis killing. These results suggest that IL-10 production in vivo could represent an evasion mechanism of the fungus to avoid host immune response.

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.

Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis.

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H(2)O(2), NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H(2)O(2) production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.

Prostaglandin E(2) production by high and low virulent strains of Paracoccidioides brasiliensis.

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E(2) (PGE(2)); however another potential source of PGE(2) is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE(2) in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE(2) and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE(2) is produced by P. brasiliensis by a cyclooxygenase-dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.