Before Pb18 challenge, neutrophils were pre-activated with the cytokines GM-CSF, IL-15, TNF-α or IFN-γ or LPS and evaluated by TLR2 and TLR4 expression, using flow cytometry. LPS was used as positive TGF-beta inhibitor control for TLR2 and TLR4 expression by neutrophils. Cells treated only with CTCM expressed very low levels of TLR2 that increased after activation with cytokines or LPS. After Pb18 challenge, all cultures expressed higher TLR2 levels when compared to their respective non-challenged cultures (Fig. 1A). All cytokines and LPS

increased TLR4 expression. However, after Pb18 challenge, a decrease in this expression was detected when compared to that detected in non-challenged cells (Fig. 1B). Together, the results showed that neutrophil activation with all cytokines resulted in an increase in TLR2 and TLR4 expression. However Pb18 modulation was different for TLR2 or TLR4. While this fungus increased GSK2118436 manufacturer TLR2 expression inducing an additional effect

to that of cytokines, it decreased TLR4 expression. As all cytokines increased TLR2 and TLR4 expression, we performed experiments to assess the role of these receptors on antifungal activities by activated neutrophils, such as fungicidal activity, H2O2 release and IL-6, IL-8, TNF-α and IL-10 production. For this, before fungus challenge, neutrophils were treated with anti-TLR2 or anti-TLR4 monoclonal antibodies, for TLR2 and TLR4 blockade. Parallel experiments confirmed inhibition of TLR2 and TLR4 expression after blockade (data not presented). Figure 2 shows the results on fungicidal activity. Non-activated cells presented a very low fungicidal activity. However, this activity was significatively increased after cells activation with all cytokines. Interestingly, Niclosamide this response profile was not significatively altered after TLR2 or TLR4 blockade, leading us to suggest that these receptors were not involved in this activity. Figure 3A–D show the results concerning TLR2 and TLR4 role

on H2O2 production by neutrophil activated with GM-CSF, IL-15, TNF-α or IFN-γ, respectively. A similar response profile was detected for all assays, because H2O2 levels were significatively increased after activation with the four cytokines, but differences among them not being detected. Moreover, there was a tendency towards Pb18 to increase metabolite release and to induce an additional effect to that of cytokines (data not statistically significant). However, as detected for fungicidal activity, H2O2 release was not significatively altered with TLR2 or TLR4 blockade showing the non-involvement of these receptors on this neutrophil activity. We also studied the possible role of TLR2 and TLR4 on IL-6, IL-8, TNF-α and IL-10 production by human neutrophils activated with the different cytokines and Pb18 challenged. IL-6 and IL-8 were not detected in neutrophil supernatants. Then, Figs.