TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous lipopolysaccharide infusion

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.

Figure 1

Percent change in plasma TNFα concentration. The percent change in plasma TNFα concentration one hour after IV LPS infusion compared to baseline values in 15 horses (H1–H15).

Figure 2

Construction of Equine TLR4-MD2-LP structure. (a) Structure of the human TLR4-MD2-LPS complex (PDB ID 3FXI), with TLR4 proteins (cyan blue), MD2 proteins (green), and LPS endotoxin (orange). (b) Merged equine (magenta) and human (cyan blue) TLR4 subunit, and merged equine (cornflower blue) and human (green) MD2 protein; LPS endotoxin (orange). (c) The complete structure of the equine TLR4-MD2-LPS complex, with TLR4 (sky blue), MD2 (magenta), and LPS endotoxin (orange).

Figure 3

Complex 3: equine TLR4-MD2-LPS with TLRF-2:TLR4-2:MD2-1:MD2-2. a. The relevant amino acids are shown in a ball-and-stick model. b. The distance between MD2’s Thr106 and TLR4’s Phe263 (4.42 Å) is shown.