P. aeruginosa Blocks IFNλ Activity
To elucidate how PAO1 inhibits induction of antiviral gene expression, we first analyzed IFN production in PAO1-CM treated cells after infection with RSV. IFNλ is the main IFN produced by airway epithelial cells upon viral infections, whereas RSV did not induce IFNα mRNA and only moderate levels of IFNβ mRNA (Figure S2). Interestingly, early IFNλ mRNA levels at 6 h p.i. were similar in control or CM-treated cells suggesting that initial virus recognition was not affected. However, at 10 and 14 h p.i. IFNλ mRNA levels decreased significantly in PAO1-CM treated cells (Figure 2A). Importantly, virtually no IFNλ protein was detectable in the cell culture supernatant of PAO1-CM treated cells at 14 h (Figure 2B) indicating that despite an early mRNA induction, positive amplification, and final production of IFNλ protein was blocked. For virus infection we were not able to detect functional IFNβ protein (data not shown). These findings may be explained by PAO1 modulating IFNλ protein production, secretion, stability, or signaling, thereby blocking the secondary positive feedback loop important for sustained and efficient IFN signaling. In line, STAT1 phosphorylation as an endogenous indicator of IFN signaling was missing in the presence of PAO1-CM but not with Boston-CM (Figure 2C). Next, we tested if recombinant exogenous IFNλ could overcome PAO1-CM effects as should be the case if production and secretion would be hampered: Surprisingly, addition of exogenous IFNλ resulted in MX1 and OAS1 induction in control cells and Boston-CM treated cells, yet in PAO1-conditioned BEASB cells no induction could be observed (Figure 2D). The results indicated that PAO1 could either block the recognition of IFNλ by its receptor or destabilize IFNλ protein.
Figure 2  P. aeruginosa is able to block IFN induction and signaling. (A) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV. Induction of IFNL1 and IFNL2/3 mRNA were analyzed by qRT-PCR after 6, 10, and 14 h. (B) BEAS2B cells were treated as in (A) and IFNλ levels of cell culture supernatant (14 h) were analyzed by ELISA. (C) BEAS2B cells were treated as in (A) and p-STAT1 levels of cells (6 h) were analyzed in cell lysates by western blot. (D) BEAS2B cells were pretreated as in (A). Cells were then stimulated with recombinant IFNλ (5,000 pg/ml) and induction of MX1 and OAS1 were analyzed after 14 h by qRT-PCR. 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.