Fig. 4. Pf phage-mediated immune inhibition is TRIF- and TLR3-dependent and associated with production of phage RNA.

(A) TNF production by WT and Trif^−/− BMDCs stimulated with alginate and Pf4 for 48 hours. (B) E. coli -pHrodo-positive cells in WT and Trif^−/− Pf-stimulated BMDCs. (C) TNF production by WT and Myd88^−/− BMDCs stimulated with alginate and Pf4 for 48 hours. (D) TNF production by WT and T/r3^−/− BMDCs stimulated with alginate and Pf4 for 24 hours. (E) E. coli-pHrodo-positive cells in WT or Tlr3^−/− BMDMs stimulated with Pf4. (F) Type I IFN production by BMDCs stimulated with LPS and Pf4 for 24 hours. (A) to (F) are each representative of n ≥ 3 experiments and depict mean with SEM of n ≥ 3 replicates. Statistics: two-tailed Student’s t test. (G) Wound infection rate 3 days after infection in Tlr3^−/− and WT mice inoculated with 7.5 ± 2.5 × 10² CFU/ml PAO1 or PAOlΔPf4. Summary of n = 2 experiments, n = 30 to 34 wounds/group. Statistics: two-tailed Fisher’s exact test. (H and I) Tlr3-reporter signal in response to (H) whole Pfl and Pf4 phage; (I) RNA from Pf4 genes. (J) RNA detected in human monocytes stimulated with whole Pfl or Pf4 phage for 24 hours. Summary of n = 2 (I and J) or n ≥ 3 (H) experiments with n > 3 replicates; depicted are means and SEM.