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Published on: May 2, 2018
TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous
Abhijit Mukhopadhyay1, Shawna R Cook2, Phillip SanMiguel3
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
Abstract:
Gram-negative bacterial septicemia is mediated through binding of lipopolysaccharide (LPS) to mammalian toll-like receptor protein 4 (TLR4). TLR4 and its cognate protein, myeloid differentiation factor 2 (MD2) form a heterodimeric complex after binding LPS. This complex induces a cascade of reactions that results in increased proinflammatory cytokine gene expression, including TNFα, which leads to activation of innate immunity. In horses, the immune response to LPS varies widely. To determine if this variation is due to differences in TLR4 or MD2, DNA from 15 healthy adult horses with different TNFα dynamics after experimental intravenous LPS infusion was sequenced across exons of TLR4 and MD2. Haplotypes were constructed for both genes using all identified variants. Four haplotypes were observed for each gene. No significant associations were found between either TNFα baseline concentrations or response to LPS and haplotype; however, there was a significant association (P value = 0.0460) between the baseline TNFα concentration and one MD2 missense variant. Three-dimensional structures of the equine TLR4-MD2-LPS complex were built according to haplotype combinations observed in the study horses, and the implications of missense variants on LPS binding were modeled. Although the sample size was small, there was no evidence that variation in TLR4 or MD2 explains the variability in TNFα response observed after LPS exposure in horses.
Insights
Lipopolysaccharide (LPS) triggers immune responses via toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD2). In horses, genetic variations in TLR4 or MD2 do not fully explain differing TNFα responses to LPS, though one MD2 variant associates with baseline TNFα.
Area of Science:
- Veterinary Immunology
- Equine Genetics
- Infectious Disease Pathogenesis
Background:
- Gram-negative bacterial septicemia involves lipopolysaccharide (LPS) binding to toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD2).
- This interaction activates innate immunity, leading to proinflammatory cytokine release, such as TNFα.
- Horses exhibit significant variability in their immune response to LPS.
Purpose of the Study:
- To investigate if genetic variations in equine TLR4 and MD2 genes contribute to the observed variability in TNFα response to LPS.
- To identify specific TLR4 and MD2 haplotypes and variants associated with differential immune responses.
Main Methods:
- DNA sequencing of TLR4 and MD2 exons in 15 healthy adult horses with known TNFα dynamics post-LPS infusion.
- Haplotype construction for TLR4 and MD2 genes based on identified variants.
- Computational modeling of equine TLR4-MD2-LPS complex structures to assess missense variant impact on LPS binding.
Main Results:
- Four haplotypes were identified for both TLR4 and MD2 genes.
- No significant association was found between TLR4 or MD2 haplotypes and baseline TNFα concentrations or LPS response.
- A significant association was observed between a specific MD2 missense variant and baseline TNFα concentration (P=0.0460).
Conclusions:
- Genetic variations in TLR4 and MD2, including identified haplotypes and most missense variants, do not appear to be the primary drivers of TNFα response variability in horses after LPS exposure.
- A single MD2 missense variant may influence baseline TNFα levels, warranting further investigation.
- The study suggests other factors likely contribute to the diverse equine immune response to LPS.
