title: Decreased intracellular TLR9 confers hyporesponsiveness of RAW264.7 cells to subsequent CpG ODN challenge.
abstract: Low-dose CpG ODN pretreatment is known to induce effective protective immunity against acute infectious diseases. In the present study, using primary murine peritoneal macrophages and the macrophage-like cell line, RAW264.7, we investigated whether low-dose CpG ODN pretreatment would induce hyporesponsiveness in response to a subsequent high-dose CpG ODN challenge and further investigated the molecular mechanisms underlying this event. Our results showed that pretreatment with a low dose of CpG ODN inhibits TNF-alpha production stimulated by later high-dose CpG ODN stimulation in a dose- and time-dependent manner. Interestingly, anti-mouse TLR9 blocking antibody added prior to CpG ODN pretreatment did not affect TNF-alpha release, but antibody added after CpG ODN pretreatment augmented the pretreatment effect of CpG ODN. This difference suggests that cell-surface TLR9 is indeed functional on activated cells. Flow cytometry revealed that low-dose CpG ODN pretreatment decreased cell-surface binding and internalization of a subsequent high-dose stimulation, suggesting that decreased internalization of succeeding CpG ODN is associated with reduced TNF-alpha release. Although both intracellular and cell-surface TLR9 expression are observed, low dose of CpG ODN pretreatment increased only cell-surface TLR9 levels. Importantly, low-dose CpG ODN pretreatment also significantly inhibited the activation of NF-kappaB, an important downstream regulator of various proinflammatory cytokines. In summary, our results demonstrate that suppression of TNF-alpha production by low dose of CpG ODN pretreatment correlates with decreased binding and internalization of subsequent CpG ODN, decreased intracellular content of TLR9, and inhibition of NF-kappaB activation.
authors.affiliation: Department of Pharmacology, College of Medicine, The Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Liu
authors.initials: L
authors.lastname: Wei
authors.forename: Zhou
authors.initials: Z
authors.lastname: Hong
authors.forename: Zheng
authors.initials: Z
authors.lastname: Jiang
authors.forename: Ding
authors.initials: D
authors.lastname: Guofu
authors.forename: Cao
authors.initials: C
authors.lastname: Hongwei
authors.forename: Wang
authors.initials: W
authors.lastname: Liangxi
authors.forename: Lu
authors.initials: L
authors.lastname: Yongling
authors.forename: Li
authors.initials: L
authors.lastname: Bin
authors.forename: Luo
authors.initials: L
authors.lastname: Ping
mesh_terms: D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D051217:Toll-Like Receptor 9 / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; C423449:CpG ODN 1826; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D004247:DNA