Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood.
The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revealed that all three are susceptible to polymorphic alterations, influencing the response to LPS. Previous experiments in the horse showed large inter-individual variations in the response to LPS. With the aim to assess this inter-individual variation, we performed a whole blood assay in 10 healthy horses as a functional assay to study the responsiveness to LPS. In 3 out of the 10 horses, LPS-induced TNF-alpha production was significantly lower compared to the overall mean. Subsequently the entire cDNA sequence encoding for the TLR-4, MD-2 and CD-14 protein was documented for each horse. Although mutations were observed in the sequence of TLR-4, these could not be related to an altered response to LPS in the concentration used in this study, as determined in the whole blood assay. Despite the various mutations found in the TLR-4 receptor protein, no alterations could be found in either the MD-2 or CD-14 gene, which are obviously more conserved structures.
Department of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 16, 3584 CM, Utrecht, The Netherlands. a.h.werners@vet.uu.nl
A H
AH
Werners
S
S
Bull
J C
JC
Vendrig
T
T
Smyth
R R
RR
Bosch
J
J
Fink-Gremmels
C E
CE
Bryant
D000818:Animals; D003601:Cytotoxicity Tests, Immunologic / Q000662:veterinary; D005260:Female; D006736:Horses / Q000097:blood / Q000235:genetics* / Q000276:immunology*; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics* / Q000276:immunology; D008297:Male; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D012313:RNA