title: TLR5 risk-associated haplotype for canine inflammatory bowel disease confers hyper-responsiveness to flagellin.
abstract: Single nucleotide polymorphisms (SNP) in the TLR5 gene have been associated with human inflammatory bowel disease (IBD) and animal models of this disease. We recently demonstrated a significant association between three non-synonymous SNPs in the canine TLR5 gene and IBD in German shepherd dogs (GSDs). However, so far, no direct link between these SNPs and a disturbance in TLR5 function was shown. In the present study, we determined the functional significance of the canine TLR5 SNPs by transfecting the identified risk-protective and risk-associated haplotype into human embryonic kidney cells (HEK) and assessed nuclear factor-kappa B (NF-κB) activation and CXCL8 production after stimulation. In addition, a whole blood assay for TLR5 activation was developed using blood derived from carrier dogs of either haplotype. There was a significant increase in NF-kB activity when cells transfected with the risk-associated TLR5 haplotype were stimulated with flagellin compared to the cells expressing the risk-protective TLR5 haplotype. This difference in NFkB activation correlated with CXCL8 expression in the supernatant measured by ELISA. Furthermore, whole blood taken from carrier dogs of the risk-associated TLR5 haplotype produced significantly more TNF after stimulation with flagellin compared to that taken from carriers of the risk-protective haplotype. Thus, we show for the first time a direct functional impact of the canine IBD risk-associated TLR5 haplotype, which results in hyper-responsiveness to flagellin compared to the IBD risk-protective TLR5 haplotype. Our data potentially suggest that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease.
authors.affiliation: Department of Veterinary Clinical Sciences, Royal Veterinary College, University of London, Hatfield, Hertfordshire, United Kingdom.
authors.forename: Aarti
authors.initials: A
authors.lastname: Kathrani
authors.forename: Angela
authors.initials: A
authors.lastname: Holder
authors.forename: Brian
authors.initials: B
authors.lastname: Catchpole
authors.forename: Lorena
authors.initials: L
authors.lastname: Alvarez
authors.forename: Kenneth
authors.initials: K
authors.lastname: Simpson
authors.forename: Dirk
authors.initials: D
authors.lastname: Werling
authors.forename: Karin
authors.initials: K
authors.lastname: Allenspach
mesh_terms: D000818:Animals; D001769:Blood / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D005408:Flagellin / Q000494:pharmacology; D020022:Genetic Predisposition to Disease / Q000235:genetics; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000097:blood / Q000235:genetics* / Q000473:pathology; D016209:Interleukin-8 / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012307:Risk Factors; D051198:Toll-Like Receptor 5 / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D016209:Interleukin-8; D008164:Luminescent Proteins; D016328:NF-kappa B; D051198:Toll-Like Receptor 5; D014409:Tumor Necrosis Factor-alpha; D005408:Flagellin