title: Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9.
abstract: Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4-6 nt from the 5'-end. Activation is augmented by a 5'TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5'-end and the ODN's length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia;
authors.forename: Jelka
authors.initials: J
authors.lastname: Pohar
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia;
authors.forename: Duško
authors.initials: D
authors.lastname: Lainšček
authors.affiliation: Division of Innate Immunity, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan;
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Fukui
authors.affiliation: Division of Innate Immunity, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan;
authors.forename: Chikako
authors.initials: C
authors.lastname: Yamamoto
authors.affiliation: Division of Innate Immunity, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Laboratory of Innate Immunity, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; and.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Miyake
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia; EN-FIST Centre of Excellence, SI-1000 Ljubljana, Slovenia.
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia; EN-FIST Centre of Excellence, SI-1000 Ljubljana, Slovenia roman.jerala@ki.si mojca.bencina@ki.si.
authors.forename: Mojca
authors.initials: M
authors.lastname: Benčina
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology; D051940:B7-2 Antigen / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D016898:Interferon-alpha / Q000276:immunology / Q000378:metabolism; D018664:Interleukin-12 / Q000276:immunology / Q000378:metabolism; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D059372:Nucleotide Motifs / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D013045:Species Specificity; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000819:agonists / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D051940:B7-2 Antigen; D016898:Interferon-alpha; D018664:Interleukin-12; D015850:Interleukin-6; D037181:Lectins, C-Type; D009838:Oligodeoxyribonucleotides; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D000099192:CD69 Antigens