title: The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.
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.
authors.affiliation: Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Georgina L
authors.initials: GL
authors.lastname: Hold
authors.affiliation: Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Susan
authors.initials: S
authors.lastname: Berry
authors.affiliation: Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Karin A
authors.initials: KA
authors.lastname: Saunders
authors.affiliation: Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Janice
authors.initials: J
authors.lastname: Drew
authors.affiliation: Biomathematics & Statistics Scotland, Aberdeen, United Kingdom.
authors.forename: Claus
authors.initials: C
authors.lastname: Mayer
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Heather
authors.initials: H
authors.lastname: Brookes
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Gay
authors.affiliation: Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Emad M
authors.initials: EM
authors.lastname: El-Omar
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Bryant
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D002460:Cell Line; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D006579:Heterozygote; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D008070:Lipopolysaccharides; D016328:NF-kappa B; C470409:TICAM1 protein, human; D051197:Toll-Like Receptor 4