title: The immunostimulatory activity of CpG oligonucleotides on microglial N9 cells is affected by a polyguanosine motif.
abstract: Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.
authors.affiliation: Institute of Brain Research, University of Tuebingen, Calwer Str. 3, D-72076 Tuebingen, Germany. zhangzhiren@yahoo.com
authors.forename: Zhiren
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ketai
authors.initials: K
authors.lastname: Guo
authors.forename: Hermann J
authors.initials: HJ
authors.lastname: Schluesener
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D007211:Indoles; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D053762:Interleukin-12 Subunit p40; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000276:immunology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009838:Oligodeoxyribonucleotides / Q000276:immunology / Q000494:pharmacology*; D011068:Poly G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D011134:Polysaccharides / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: C408982:CPG-oligonucleotide; D004268:DNA-Binding Proteins; D007211:Indoles; D053762:Interleukin-12 Subunit p40; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides; D021122:Protein Subunits; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D011068:Poly G; C007293:DAPI; C007789:fucoidan; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse