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Figure 7 | Respiratory Research

Figure 7

From: Airway epithelial cell tolerance to Pseudomonas aeruginosa

Figure 7

Tolerance and loss of tolerance in IL-8 secretion and NF-κB driven transcription is similar in hTBE cells and the AALEB cell line, and dnIRAK1 inhibits responses to Ps. a. and IL-1β but not TNFα. A-D) The apical surface of well-differentiated, day-21 hTBE cell cultures (A, C) or AALEB cells in conventional culture on plastic (B, D) were challenged with boiled Ps. a. filtrate (P) or TSB (T) in the absence of human serum as indicated. In A and B, cultures were incubated for 24 hours, and following washing and media change, the apical surfaces were re-challenged when indicated. IL-8 was measured in the media 24 hours following the second challenge. C and D) Cells were transfected with adenoviral vectors expressing constitutive or NF-κB driven reporter genes as described in Methods and were challenged with TSB or Ps. a. and lysed for reporter gene assay as indicated. Note the steep slope of the Ps. a. response in naive or TSB-pretreated cells versus the attenuated slope in Ps.a.-pretreated cells. E, F) hTBE and AALEB cells were transfected with adenoviral vectors expressing constitutive and NF-κB driven reporter genes and dnIRAK1 or a control vector as described in the Methods and were lysed for reporter gene assay 8 hours after treatment as indicated. dnIRAK1 inhibited approximately 50% of the Ps. a. response in both cell types, and, in AALEB cells, >90% of the IL-1β response, but not the TNFα response. All points represent the mean of triplicate wells ± SD. The results in panel E ands F are representative of 2 and 3 separate experiments, respectively

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