Abstract
Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.
Highlights
Flagella have been shown to play a significant role in bacterial pathogenesis, primarily through their function as motility organelles, and as adhesins and as proinflammatory agonists
Toll like receptors (TLRs) have an intracellular domain that is relatively conserved between the different TLRs including the presence of a toll/interleukin-1 (TIR) region that contains specific amino acids that are phosphorylated upon microbial associated molecular patterns (MAMPs) binding and can interact with different adaptor proteins leading to signalling cascades resulting in pro-inflammatory cytokine release [16,11]
Signalling from HEK293 cells transfected with human and bovine toll-like receptor 5 (TLR5) clones The bovine TLR5 and human TLR5 clones were obtained from InvivoGen
Summary
Flagella have been shown to play a significant role in bacterial pathogenesis, primarily through their function as motility organelles, and as adhesins and as proinflammatory agonists. TLRs have an intracellular domain (endodomain) that is relatively conserved between the different TLRs including the presence of a toll/interleukin-1 (TIR) region that contains specific amino acids that are phosphorylated upon MAMP binding and can interact with different adaptor proteins leading to signalling cascades resulting in pro-inflammatory cytokine release [16,11]. It is evident that specific residues within the more conserved D1 domains are required for binding to the TLR5 ectodomain, with the more variable D2 and D3 regions responsible for the antigenic variability of flagellins [17]. While the D0 and D1 domains of flagellin are relatively conserved, variation in these regions has been shown to limit innate responses to flagellin expressed by α and ε Proteobacteria, including Helicobacter pylori [18,19]
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