Figure 3

Analysis of bovine TLR5 activity in bovine epithelial cells. The bovine EBL cell line was stably transfected with a bTLR5 clone and CXCL8 secretion assayed following challenge with flagellin. (A) CXCL8 levels from EBLs with and without transfection of bTLR5. H7 flagellin at a range of concentrations was added to the cells and CXCL8 levels were measured by ELISA following overnight incubation. Transfection with bTLR5 resulted in significantly higher levels of CXCL8 being produced by the bTLR5+ cells with addition of 50-50 000 ng/mL of H7 flagellin (p < 0.001). The data shown are the means and 95% confidence intervals. (B) Analysis of CXCL8 production from EBL-TLR5 in response to addition of native H7 or an H7 flagellin preparation from which the majority of LPS has been removed. Medians and interquartile ranges are shown. (C) Secreted CXCL8 following addition of WT and mutated H7 flagellins to EBLs with transfected bTLR5. 50 ng/mL of WT-H7 and mutated flagellins were added to the EBLs transfected with bTLR5 and incubated overnight. CXCL8 was measured by ELISA. Addition of the R90T, R90A and L500A/I504A variants led to significantly lower levels of cytokine release (p < 0.001) relative to WT-H7 stimulation (asterisks). R90T showed a significantly lower induction than R90A (p < 0.001). Medians and interquartile ranges are shown. (D) Secreted CXCL8 following addition of WT H7 and the R90T flagellin mutant to EBL cells. A range of flagellin concentrations was incubated overnight with the cells and CXCL8 measured by ELISA. R90T flagellin demonstrated significantly reduced levels of CXCL8 activation at 50 ng/mL and 500 ng/mL (p < 0.001), marked by asterisks. The data shown are the means and 95% confidence intervals. All CXCL8 data shown is from a minimum of three biological replicates.