Role of TLR2 and TLR4 in human neutrophil functions against Paracoccidioides brasiliensis.

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), 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 mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLR). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated with GM-CSF, IL-15, TNF-alpha or IFN-gamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNF-alpha and IL-10 production by activated and challenged cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression inducing an additional effect to that of cytokines. On the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production, but this process was not associated with TLR2 or TLR4. Neutrophils activation with GM-CSF and TNF-alpha resulted in a significative increase in IL-8 production, while IL-15 and IFN-gamma have no effect. Pb18 alone also increased IL-8 production. None of the cytokines activated neutrophils for IL-10 release. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. Our data suggest that Pb18 uses TLR4 to gain access to human neutrophils. This interaction results in IL-8 and IL-10 production that may be considered as a pathogenic mechanism in paracoccidioidomycosis.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: role of interleukin-8.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.