Recognition of LPS occurs via a heterodimeric complex formed between TLR4 and myeloid differentiation factor 2. LPS binds to the large hydrophobic pocket in MD2 to induce the formation of an m-shaped receptor multimer comprising at least two copies of the TLR4-MD2-LPS complex. The TLR4 Asp299Gly polymorphism lies within the extracellular domain of the receptor. Crystallography work shows that in the crystal structure of LPS bound TLR4 Asp299Gly/MD2 forms receptor dimers in the same way as wildtype TLR4, but there are local conformational changes. The effects of the Thr399Ile polymorphism on the LPS-TLR4/MD2 structure are minimal. SAR131675 Despite a body of excellent crystallographic and functional studies precisely how these SNPs alter TLR4 reactivity remains unclear. Upon ligand binding the TLR4/MD2 receptor complex ultimately recruits the adaptor proteins myeloid differentiation primary-response protein 88 and TIR-domain-containing adaptor protein-inducing IFN-B. MyD88 dependent signalling activates IKK and mitogen-activated protein kinase pathways. The IKK pathway, through regulation of early phase NF-kB, controls expression of proinflammatory cytokines and other immune related genes. MAPK pathway activation induces another transcription factor AP-1 which also plays a role in proinflammatory cytokine expression. NF-kB activation with delayed kinetics also occurs in the absence of MyD88 directed signalling through TRIF and TRIF-related adaptor molecule. This pathway also activates transcription factor interferon regulatory factor 3 to induce Type I interferons. Factors which alter the balance between these signalling pathways will impact on the immune response including appropriate adaptive immune cell recruitment. The effect of the TLR4 Asp299Gly polymorphism on adaptor protein recruitment is controversial. Analysis of human genetic data has led to the hypothesis that the TLR4 Asp299Gly polymorphism may primarily signal through TRAM/ TRIF rather than MyD88 which would alter the cytokine profile of patients with this SNP. Recent work using TLR42/2 mouse macrophages expressing human TLR4 Asp299Gly polymorphism suggest there is a deficit in both MyD88 and TRIF recruitment by the mutant receptor, although these experiments were performed in the absence of human MD2, meaning that complete receptor function was not permitted. The cosegregating polymorphism, Thr399Ile, in TLR4, affects the functional consequences of the Asp299Gly polymorphism. Early studies by Arbour and Schwartz showed that carriers of the Asp299Gly polymorphism had decreased NF-kB activity compared with wildtype TLR4. Later work showed that carrying the TLR4 Asp299Gly polymorphism alone was associated with increased disease risk and increased TNF-alpha production in comparison to having wildtype or the Thr399Ile polymorphism. The TLR4 Asp299Gly polymorphism therefore has important implications for disease outcome, yet the precise mechanisms by which this SNP has its effects remain undefined. Here we show, for the first time, that TLR4 carrying Asp299Gly and Thr399Ile polymorphisms have blunted responses to LPS compared to wild-type TLR4 as expected but, these constructs also have high levels of constitutive activity in reconstituted signalling assays. Unstimulated monocytes from patients carrying these mutations, compared to patients with wild-type TLR4, have downregulated expression of several genes in the TLR4 TRAM/ TRIF signalling pathway. LPS stimulation of monocytes from patients carrying the TLR4 mutations biases TLR4 signalling through the TRAM/TRIF pathway.