title: A naturally occurring variant in human TLR9, P99L, is associated with loss of CpG oligonucleotide responsiveness.
abstract: The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.
authors.affiliation: Department of Toll-like Receptors and Cancer, German Cancer Research Centre (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Andriy V
authors.initials: AV
authors.lastname: Kubarenko
authors.forename: Satish
authors.initials: S
authors.lastname: Ranjan
authors.forename: Anna
authors.initials: A
authors.lastname: Rautanen
authors.forename: Tara C
authors.initials: TC
authors.lastname: Mills
authors.forename: Sunny
authors.initials: S
authors.lastname: Wong
authors.forename: Fredrik
authors.initials: F
authors.lastname: Vannberg
authors.forename: Michael
authors.initials: M
authors.lastname: Neumaier
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bekeredjian-Ding
authors.forename: Adrian V S
authors.initials: AV
authors.lastname: Hill
authors.forename: Parviz
authors.initials: P
authors.lastname: Ahmad-Nejad
authors.forename: Alexander N R
authors.initials: AN
authors.lastname: Weber
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005838:Genotype; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051217:Toll-Like Receptor 9 / Q000235:genetics* / Q000378:metabolism
chemical_list: C408982:CPG-oligonucleotide; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D008156:Luciferases