Abstract
Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test) and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.
Highlights
Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases [1,2]
These results suggested that unstimulated monocyte/macrophages from patients carrying the TLR4 SNPs may have basally altered levels of pro-inflammatory gene expression in comparison to cells from patients with wild-type TLR4 if the results of our transient transfection analysis were physiologically relevant
For the first time, that the TLR4 Asp299Gly and Thr399Ile polymorphisms confer altered constitutive activity upon the TLR4 receptor and this is associated with basal downregulation of many pro-inflammatory genes in unstimulated monocytes from patients carrying these SNPs
Summary
Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases [1,2]. A functional polymorphism that alters the amino acid at position 299 in TLR4, the lipopolysaccharide (LPS) receptor, (TLR4 Asp299Gly - rs4986790) [3,4] reduces carrier responsiveness to LPS challenge [3]. Recognition of LPS occurs via a heterodimeric complex formed between TLR4 and myeloid differentiation factor 2 (MD2). 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 [10], but there are local conformational changes. The effects of the Thr399Ile polymorphism on the LPS-TLR4/MD2 structure are minimal. Despite a body of excellent crystallographic and functional studies precisely how these SNPs alter TLR4 reactivity remains unclear
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