S1). In our next experiments, we used live FITC-conjugated S. aureus (strain SH1000) to investigate

the effect of PAR2-cAP alone or together with IFN-γ on the phagocytic activity of human monocytes and neutrophils APO866 against viable bacteria. We found that PAR2-cAP (1 × 10−4 m) or IFN-γ (100 ng/ml) alone enhanced phagocytic activity (Fig. 1a–d; a,b for neutrophils and c,d for monocytes). Although IFN-γ already appeared to stimulate phagocytic activity of monocytes at a concentration of 10 ng/ml, these effects were not statistically significant (Fig. 1c,d). Interferon-γ at a higher concentration (100 ng/ml) also enhanced phagocytic activity of human monocytes and neutrophils. The effects of IFN-γ at a concentration of 100 ng/ml reached statistical significance MK0683 (Fig. 1a–d). Stimulation with IFN-γ increased the number of FITC-positive human monocytes (49 ± 13% of change compared with untreated cells) and FITC-positive human neutrophils (41 ± 7% of change compared with untreated cells). The MFI also increased in IFN-γ-treated human monocytes (increased by 53 ± 14%) and neutrophils (increased by 80 ± 18%) compared with untreated controls. PAR2-cAP led to an increase in the amount of FITC-positive monocytes (increased by 35 ± 7%) and FITC-positive

neutrophils (increased by 24 ± 4%) compared with untreated samples. The MFI also increased in monocytes treated with PAR2-cAP (increased by 38 ± 8%) and in neutrophils (increased by 38 ± 4%) compared with untreated control samples.

The combined action of PAR2-cAP and IFN-γ using the same concentrations did not enhance the phagocytic activity of neutrophils or monocytes beyond that triggered by either agonist acting alone (Fig. 1a–d). Interferon-γ is a well-known endogenous modulator of phagocytic bacteria killing and secretory activity of human neutrophils and human monocytes.25,26 As an exogenous activator, LPS also affects phagocytic activity of both cell types. We wondered whether PAR2-cAP stimulation might interfere with LPS-modulated phagocytic activity of human neutrophils and monocytes. However, PAR2-cAP stimulation of human neutrophils as well MycoClean Mycoplasma Removal Kit as monocytes did not enhance the LPS-induced phagocytic activity against S. aureus (see supplementary material, Fig. S2). Hence, despite the fact that PAR2-cAP alone up-regulates the phagocytic activity of human neutrophils and monocytes against S. aureus, this agonist failed to enhance IFN-γ-induced and LPS-induced phagocytic activity. We next investigated whether treatment of isolated human neutrophils with PAR2-cAP alone or in combination with IFN-γ affects the bactericidal activity of these phagocytes. In accordance with biosafety limitations, we used live E. coli bacteria in our experiments to estimate neutrophil killing activity.