Introduction

Pattern-recognition receptors (PRRs) are important components of the innate immune system, enabling detection of infection. Defective PRR function has been implicated in several infectious and immune-mediated diseases in human beings, including Crohn's disease, which is associated with a genetic defect in the NOD2 gene that affects the function of this intracellular PRR. Anal furunculosis (AF), which occurs primarily in German shepherd dogs (GSD), shares several features with Crohn's disease and could occur through similar immunopathological mechanisms. The aim of the current study was to investigate canine PRR responses in vitro and to determine whether there was any defect in AF-affected GSD.

Materials and Methods

Pattern-recognition receptors that are involved in detection of bacteria or fungi (TLR1, TLR2, TLR4, TLR6, TLR9. NOD1 and NOD2) were evaluated in canine blood-derived monocyte/macrophages. PRR mRNA expression was quantified using real-time quantitative PCR. PRR function was evaluated by quantifying cytokine mRNA expression and TNFα synthesis in canine blood-derived monocyte/macrophages following stimulation of these cells with PRR-ligands using qualitative and quantitative PCR and a TNFα specific ELISA. PRR responses in blood-derived monocyte/macrophages from 12 healthy blood-donor dogs and 8 AF-affected GSD were compared.

Results

Canine blood-derived monocyte/macrophages were found to express mRNA for all the selected PRRs with TLR2 mRNA being the greatest and TLR5 mRNA being the least abundant. Stimulation of blood-derived monocyte/macrophages using specific PRR-ligands resulted in expression of pro-inflammatory cytokine mRNA. Quantification of TNFα mRNA and protein secretion from stimulated cells demonstrated variable responses with lipopolysaccharide (TLR4 ligand) and PAM3CSK4 (TLR1/2 ligand) proving to be the most potent and CpG DNA (TLR9 ligand) the least potent. Comparing PRR responses in blood-derived monocyte/macrophages from healthy blood-donor dogs with those from AF-affected GSD showed a deficiency in the latter in response to LD-MDP (NOD2 ligand) at the mRNA level but not at the protein level.

Conclusion

It is possible that dysfunctional NOD2 responses are involved in the pathogenesis of AF, similar to that seen in Crohn's disease.