Figure 3  P. aeruginosa secretes a protease degrading directly IFNλ. (A) Recombinant IFNλ was incubated in conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium at 37°C for 1, 2, or 4 h. Subsequently, levels of IFNλ were analyzed by western blot. (B) Conditioned media was heated for 10 min at 95°C. Subsequently BEAS2B cells were pretreated with heat treated or non-treated conditioned medium of P. aeruginosa (PAO1 and Boston) or with control medium and subsequently infected with RSV. Induction of MX1 mRNA was analyzed by qRT-PCR after 14 h post-infection. (C) Conditioned media was filtered using a cutoff of 30 kDa. Subsequently BEAS2B cells were pretreated with the fraction larger than 30 kDa, smaller than 30 kDa, non-filtered conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV. Induction of MX1 mRNA was analyzed by qRT-PCR after 14 h post-infection. (D) Protease activity in conditioned medium of P. aeruginosa (PAO1 and Boston) was assessed using skim-milk agar. (E) Elastase activity in conditioned medium of P. aeruginosa (PAO1 and Boston) was assessed by zymography. All experiments n = 3–4, ANOVA and Bonferroni post-test was used for statistical analysis. Significant differences were considered at *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the control condition. n.s., not significant.