FIGURE 6.

Biofilm exacerbated inflammatory response and its control by WED. A, Human HaCaT keratinocytes were infected with a static biofilm infection as described in methods. A, DNA binding activity of NF-κB in human HaCaT keratinocytes measured using an ELISA-based (Trans-AM) method. Data are mean ± SEM (n = 4), *P < 0.001 compared with non-infected keratinocytes, ^†P < 0.001 compared with placebo-treated infected group (ANOVA, post-hoc Tukey HSD test). B, NF-κB transcription activity in human HaCaT keratinocytes transiently transfected with NF-κB dependent luciferase reporter gene (Ad5NF-κB-LUC) followed by static biofilm infection. Luciferase activity was determined. Data are mean ± SEM (n = 8), *P < 0.001 compared with non-infected keratinocytes, ^†P < 0.001 compared with placebo-treated group (ANOVA, post-hoc Tukey HSD test). C, D, mRNA expression of NF-κB directed pro-inflammatory genes: C, IL-1β and D, TNF-α in human HaCaT keratinocytes following 6 h of static biofilm infection. β-actin was used as housekeeping. Data are mean ± SEM (n = 6), *P < 0.001 compared with non-infected keratinocytes, ^†P < 0.001 compared with placebo-treated group (ANOVA, post-hoc Tukey HSD test). E, F, Protein expression: E, IL-1β and F, TNF-α in human HaCaT keratinocytes following 6 h of static biofilm infection. Data are mean ± SEM (n = 3), *P < 0.01 compared with non-infected keratinocytes, ^†P < 0.05 compared with placebo-treated group (ANOVA, post-hoc Tukey HSD test). G, H, Representative immunofluorescence images of active phospho-p65 of NF-κB (green) and DAPI (nuclear, blue) stained sections from porcine burn wounds subjected to induced infection (II) with P. aeruginosa PAO1 and A. baumannii 19606 followed by treatment with WED 2 h post-inoculation (prevention study). Bar graphs present quantitation of active phospho-p65 of NF-κB; scale bar 50 μm. Data are mean ± SEM (n = 4), *P < 0.005 compared with skin. ^†P < 0.05 compared with placebo (ANOVA, post-hoc Tukey HSD test). NF-κB indicates nuclear factor kappa B.