Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides brasiliensis induce cytokine production by human monocytes.

The glycoprotein gp43 is an immunodominant antigen secreted by Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. The present study evaluated whether gp43 can interact with toll-like (TLR2, TLR4) and mannose (MR) receptors on the surface of human monocytes, and how that affects their expression and cytokine production. Monocytes were incubated with or without monoclonal antibodies anti-TLR2, anti-TLR4, or anti-MR, individually or in combination, prior to the addition of gp43. The gp43 binding to monocyte surface, as well as expression of TLR2, TLR4, and MRs were analyzed by flow cytometry, while production of TNF-α and IL-10 was monitored by ELISA. The results suggested that gp43 binds to TLR2, TLR4, and MR receptors, with TLR2 and MR having the strongest effect. All three receptors influenced the production of IL-10, while TNF-α production was associated with expression of TLR4 and MR. The modulatory effect of gp43 was demonstrated by high levels of TLR4 expression associated with increased production of TNF-α after 4 h of culture. Alternatively, high levels of TLR2 expression, and elevated production of IL-10, were detected after 18 h. We showed that interaction between gp43 and monocytes may affect the innate immune response by modulating the expression of the pattern recognition receptors TLR2, TLR4 and MR, as well as production of pro- and anti-inflammatory cytokines.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis.

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-ß1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-ß1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-ß1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Hepatoprotective and anti-inflammatory effects of silibinin on experimental preeclampsia induced by L-NAME in rats.

AIMS: Inhibition of nitric oxide synthase with N-omega-nitro-l-arginine methyl ester (l-NAME) has been employed as an experimental model of human preeclampsia. This study determined the protective effect of silibinin, a flavonoid with anti-inflammatory and hepatoprotective properties on the deleterious effects observed in experimentally induced preeclampsia in rats. MAIN METHODS: Pregnant Wistar rats were treated during gestation (days 10-20) with l-NAME (70-80mg/kg/day) in drinking water or with l-NAME plus silibinin (100mg/kg/day, orally) starting at day 0, day 7 or day 14 of pregnancy. Systolic blood pressure was recorded from gestation days 0 to 21. A control group of pregnant non-treated rats was analyzed similarly. On day 21 the rats were euthanized and the following parameters were evaluated: proteinuria, platelet count, liver histopathology and reproductive outcome. Tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1ß), IL-6, IL-10 and interferon-gamma (IFN-γ) were determined in liver homogenate by enzyme immunoassay. KEY FINDINGS: In comparison with the l-NAME group the silibinin treatment reduced the values of systolic blood pressure, proteinuria, TNF-α, IL-1ß and IFN-γ in liver, normalized the platelet count and improved fetal outcomes. Histopathological lesions in liver of the l-NAME group showed intense mononuclear inflammatory infiltrate and thickening of muscle tunica of arterial vessel, mainly in the periportal area. Silibinin treatment induced attenuation of periportal inflammatory infiltrate, showing an association between inflammatory infiltrate and TNF-α, IL-1ß and IFN-γ levels in liver homogenate. SIGNIFICANCE: Silibinin administration to l-NAME-treated rats displays anti-inflammatory and immunomodulatory actions that may contribute to its hepatoprotective effects and improve reproductive outcomes in experimental preeclampsia.

Downregulation of nuclear factor-kappa B (NF-kappaB) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis.

AIMS: Silibinin is the major active component of silymarin, a polyphenolic plant flavonoid that has anti-inflammatory effects. The modulatory effect of silibinin on monocyte function against Paracoccidioides brasiliensis (Pb18) has not yet been demonstrated. The present study investigated whether the effect of silibinin on nuclear factor-kappa B (NF-kappaB) pathways may affect the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), transforming growth factor beta (TGF-beta1), prostaglandin E(2) (PGE(2)), nitric oxide (NO) and fungicidal activity of human monocytes challenged in vitro with Pb18. MAIN METHODS: Peripheral blood monocytes from healthy individuals were treated with silibinin and challenged with Pb18 for 18h. TNF-alpha, IL-10, TGF-beta1 and PGE(2) expression were determined by immunoenzymatic assay (ELISA) and NO release was determined by the accumulation of nitrite in culture supernatants. Fungicidal activity of monocytes was analyzed after treatment with interferon-gamma plus silibinin and challenge with Pb18. NF-kappaB activation in cultured monocytes was evaluated by flow cytometry and ELISA. KEY FINDINGS: Silibinin partially inhibited p65NF-kappaB activation as the number of cells expressing this factor was reduced and the concentration of nuclear p65NF-kappaB was low, compared to untreated controls. The addition of silibinin also resulted in suppression of TNF-alpha, IL-10, TGF-beta1, PGE(2) and NO production but did not affect the fungicidal activity of monocytes against Pb18. SIGNIFICANCE: Silibinin exerts anti-inflammatory and anti-fibrotic effects on CD14+/- human monocytes challenged by Pb18 by partial inhibition of p65NF-kappaB activation.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.