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Figure 4 | Veterinary Research

Figure 4

From: Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella

Figure 4

Responses of bovine monocyte-derived macrophages (bMDMs) to flagellin. (A) mRNA levels of CXCL8 and TLR5 following addition of flagellin to bMDMs. Transcript levels are plotted relative to time 0 and increase significantly at 4 h and 24 h (p < 0.001), while TLR5 transcript levels are lower (p < 0.001) 4 h after addition of flagellin but not significantly different (p = 0.073) at 24 h, significance marked by asterisks. (B) Assessment of TLR5 transcript levels in bMDMs. Transfected cells were pre-incubated with siRNAs for 48 h then WT H7 flagellin (26.5 ng/mL) added (time 0). TLR5 transcripts were reduced by treatment with both TLR5#2 and TLR5#3 relative to controls at both time 0 and 24 h (p < 0.001), marked by asterisks. (C) Assessment of CXCL8 transcript levels in bMDMs. CXCL8 mRNA levels were measured in cells challenged with WT H7 flagellin as above. CXCL8 transcript levels were significantly reduced in the cells treated with the TLR5#2 and TLR5#3 siRNAs relative to the controls at 24 h (p < 0.001), indicated by asterisks. Data is from three biological repeats with a minimum of three technical replicates. (D) Determination of released CXCL8 from bMDMs with reduced TLR5 expression. bMDMs were treated as above and supernatant CXCL8 determined by ELISA. TLR5 #3 siRNA significantly reduced secreted levels of CXCL8 relative to un-transfected controls at 24 h (p < 0.001), while a significant reduction was evident for TLR5 #2 at 24 h (p = 0.012), marked with asterisks. (E) CXCL8 release from bMDMs challenged with WT and altered H7 flagellin. CXCL8 was measured in the supernatants of the bMDMs 24 h after stimulation with WT or R90T H7 flagellin. The CXCL8 levels released were significantly lower for R90T compared with WT flagellin (p = 0.022, asterisk). All plots show medians with upper and lower quartiles; outliers as dark circles.

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