Since P. aeruginosa is known to secret various proteases, we next tested whether CM of P. aeruginosa is able to degrade recombinant IFNλ directly (Figure 3A). CM of both strains, PAO1 and Boston, degraded recombinant IFNλ but PAO1 was much more efficient with complete destruction of added IFNλ even after 1h of incubation. To elucidate the mechanism of action, we heat treated (10 min, 95°C) or filtered (molecular weight cut-off 30 kDa) the conditioned medium from both P. aeruginosa strains (Figures 3B,C). Both treatments were able to restore MX1 and OAS1 induction by RSV in PAO1-conditioned cells indicating that the inhibitory factors in PAO1-CM might be proteins of <30 kDA. We next analyzed the general secretion of proteases by both strains using casein degradation (Figure 3D) or zymography (Figure 3E). Although both strains showed protease secretion, protease activity was much higher in PAO1 (Figure 3D). Zymography also showed that PAO1 had higher AprA and LasA/B activity (as determined by size) whereas the Boston strain only expressed low amounts of AprA (Figure 3E).