Pseudomonas aeruginosa Inhibition of Flagellin-Activated NF-κB and Interleukin-8 by Human Airway Epithelial Cells

Pseudomonas aeruginosa-induced activation of NF-kappa B and secretion of proinflammatory cytokines by airway epithelial cells require that the bacteria express flagellin. We tested whether P. aeruginosa and human airway epithelial cells secrete factors that modulated this response. Experiments were performed with both the Calu-3 cell line and primary cultures of tracheal epithelial cells. P. aeruginosa strain PAK Delta fliC (flagellin knockout) did not activate NF-kappa B or interleukin-8 (IL-8) but inhibited flagellin-activated NF-kappa B by 40 to 50% and IL-8 secretion by 20 to 25%. PAK Delta fliC also inhibited NF-kappa B induced by IL-1 beta and Toll-like receptor 2 agonist Pam3CSK4. Similar inhibitions were observed with strains PAK, PAO1, and PA14. The inhibitory factor was present in conditioned medium isolated from PAK Delta fliC or Calu-3 plus PAK Delta fliC, but it was not present in conditioned medium isolated from Calu-3 cells alone or from PAK Delta fliC that had been heat treated. Inhibition by PAK Delta fliC-conditioned medium was exerted from either the apical or the basolateral side of the epithelium, was enhanced in simple Ringer's solution over that in tissue culture medium, and did not result from altered pH or depletion of glucose. The inhibitory effect of conditioned medium was abolished by boiling and appeared from filtration studies to result from effects of a factor with a molecular mass of <3 kDa. These and further studies with isogenic mutants led to the conclusion that the NF-kappa B and IL-8 response of airway epithelial cells to P. aeruginosa results from a balance of proinflammatory effects of flagellin and antiinflammatory effects of a small (<3-kDa), heat-sensitive factor(s) that is not lipopolysaccharide, C12 homoserine lactone, alginate, CIF, or exotoxin A, S, T, U, or Y.