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.

Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis.

Paracoccidiodomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), which is endemic in Latin America. The host innate immune response against the fungus has been well characterized and several studies have shown the important role played by phagocytic cells. Our laboratory has studied the relationship between human neutrophils (PMNs)/Pb, focusing the effector mechanisms of these cells against the fungus. However, in last years, studies have shown that in addition to their phagocytic and killer functions, PMNs can modulate and instruct the immune response, since these cells have been shown to produce and release several cytokines. Thus, we evaluated whether PMNs stimulated with Pb can modulate the immune response to a Th1 phenotype through the production of IFN-γ, as well as the role of "pattern-recognition receptors" (PRRs) such as TLR2, TLR4 and Dectin-1 in this production. Furthermore, we asked whether activation of the cells with the cytokines IL-12, IL-15 and IL-18 could result in increased levels of this cytokine. Peripheral blood PMNs obtained from 20 healthy donors were nonactivated or activated with IL-12, IL-15 or IL-18 in different concentrations and challenged with strain 18 Pb (Pb18) for 2 h, 4 h, 12 h, 24 h and 48 h and evaluated for IFN-γ production, by ELISA. In other experiments, PMNs were treated with monoclonal antibodies anti-TLR2, TLR4 and Dectin-1, challenged with Pb and evaluated for IFN-γ production. We found that Pb induces human PMNs to produce IFN-γ, probably by binding to TLR4 and Dectin-1 receptors expressed by these cells. Moreover, IFN-γ levels were significantly increased when cells were activated with each of the tested cytokines or a combination of two of them, being the association IL-12 plus IL-15 the most effective. The results support our hypothesis that during infection by Pb, human PMNs modulate the adaptive immune response to a Th1 response pattern, via IFN-γ production.

Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: Role on extracellular killing.

Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.