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

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.

Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost

“…This type of prime-boost regime has previously been demonstrated to increase vaccination efficacy substantially [51,52]. In addition, Quil-A ® , used as adjuvant for the priming of the GP5-Mosaic DNA vaccine, was previously shown to further increase the immune responses [53][54][55]. Therefore, the prime-boost vaccination regime used here for testing GP5-Mosaic vaccine efficacy, in terms of broad protection, proved appropriate for testing the hypothesis.…”

Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement

“…Macrophages also play a role in antigen processing and presentation [42]. Most important functions depend on TLRs and inflammasomes [23,33,43]. Flagellin can activate the innate immune response via activation of inflammasomes [44,45].…”

“…Phase one flagella were prepared from S. typhimurium SL1344 phase 2 mutant by shearing according to the method described by Tahoun et al [33]. In brief, one colony of S. typhimurium SL1344 phase 2 mutants was stabbed in motility agar and grown overnight at 30 • C. Then, 10 µL of agar inoculums from the edge of the motility ring was cultured in LB broth overnight at 30 • C. The bacteria were harvested by centrifugation at 5000× g for 30 min at 4 • C. The pellet was suspended overnight at 4 • C in a volume of PBS that was 2% of the initial culture volume.…”

“…Endotoxin contamination of flagella was removed according to the method described by Tahoun [33]. Briefly, 1% triton X114 was added to the purified flagella at 1 in 500.…”

Giant flagellins form thick flagellar filaments in two species of marine γ-proteobacteria

“…Flagella were isolated by acid depolymerisation according to an existing method [23], then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% Novex Tris-Glycine gels (Fisher Scientific, Loughborough, U.K.).…”