TLR5 Risk-Associated Haplotype for Canine Inflammatory Bowel Disease Confers Hyper-Responsiveness to Flagellin

Aarti Kathrani,Lorena Alvarez,Angela Holder,Brian Catchpole,Dirk Werling,Karin Allenspach,Kenneth Simpson,Rachel Louise Allen

doi:10.1371/journal.pone.0030117

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.

Highlights

The healthy gut is able to regulate inflammatory responses to commensal bacteria and food antigens whilst maintaining the capacity to respond to pathogens [1][2][3]
In order to compare the functional response of the indentified polymorphisms, the TLR5 sequence of the inflammatory bowel disease (IBD) risk-protective haplotype (G22A haplotype GSD carrying the risk-protective TLR5 haplotype (GTT)) and the IBD risk-associated haplotype (G22A haplotype ACC) [16] were cloned in to pcDNA3.1 plasmids containing YFP to monitor successful expression
As initial association studies suggested a significant effect of canine TLR5 Single nucleotide polymorphisms (SNP) with the occurrence of IBD in German shepherd dogs (GSDs) [16], we investigated whether these SNPs resulted in functional differences of the expressed protein constructs

Summary

Introduction

The healthy gut is able to regulate inflammatory responses to commensal bacteria and food antigens whilst maintaining the capacity to respond to pathogens [1][2][3]. This distinction relies in part on appropriately functioning pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs); which are ideally situated in intestinal epithelial cells and recognise microbe associated molecular patterns (MAMPs) [4]. The breakdown in this immunological tolerance to commensal and dietary antigens is thought to play an important role in the pathogenesis of inflammatory bowel disease (IBD) [2] and the same may be true in dogs. We have recently shown an upregulation of TLR4 and a down-regulation of TLR5 at the mRNA level in German shepherd dogs (GSDs) with IBD [15]

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