The Toll-Like Receptor 4 Polymorphism Asp299Gly but Not Thr399Ile Influences TLR4 Signaling and Function

Abstract

The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.

TLR4 polymorphisms seem not to be associated with prediabetes and type 2 diabetes but predispose to diabetic retinopathy; TLR4 polymorphisms in glucose continuum

“…It is mainly Asp299Gly not Th r399Ile polymorphism that leads to modifi ed peptide structure, alters ligand-receptor interaction, post-receptor signaling and further cytokine production (Ferwerda et al 2008;Ohto et al 2012;Long et al 2014). TLR4 actively contributes to the infl ammatory response in development of microvascular complications, including diabetic retinopathy (Rajamani and Jialal 2014).…”

Genetic polymorphisms in Toll-like receptor 3 gene are associated with the risk of hepatitis B virus-related liver diseases in a Chinese population

“…The above data suggested that TLR3 genetic polymorphism could be a useful and promising target for the treatment of HCC. Genetic polymorphisms have been reported to play a role in regulating transcription activities and genetic expression levels (Long et al, 2014;Shen et al, 2015). Long et al found that cells expressing TLR4 Asp299Gly polymorphism produced less IL-8 (Long et al, 2014), while Shen et al reported that TLR4 polymorphism is associated with increased susceptibility to bladder cancer possibly by downregulating gene expression in various immune cells .…”

“…Genetic polymorphisms have been reported to play a role in regulating transcription activities and genetic expression levels (Long et al, 2014;Shen et al, 2015). Long et al found that cells expressing TLR4 Asp299Gly polymorphism produced less IL-8 (Long et al, 2014), while Shen et al reported that TLR4 polymorphism is associated with increased susceptibility to bladder cancer possibly by downregulating gene expression in various immune cells . Based on the above results, we speculate that the TLR3 3775290 polymorphism might contribute to HCC development by regulating the expression of TLR3 and activation in the TLR3 pathway.…”

TLR4 and TLR9 Polymorphisms Effect on Inflammatory Response in End-Stage Renal Disease Patients

“…The TLR4 polymorphism c.896A>G has been studied and it was related to an increased susceptibility to Gram-negative bacteremia and septic shock (16,17), by reducing LPS responsiveness. This hypo- responsiveness to LPS associated with the c.896A>G polymorphism is not due to a reduced surface TLR4 protein expression; instead, the polymorphism must alter the ability of TLR4 to interact with myeloid differentiation factor 2 (MD-2), with LPS and/or by inducing signal eliciting (18). Crystal structure of the tertiary TLR4/MD-2/LPS complex was recently elucidated for both wild-type and mutant TLR4 (19).…”