Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR...Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6, IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology. 2005;2(2):130-135.展开更多
Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequ...Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.展开更多
基金Financial support for this work was provided by the National High Technology Research and Development Program of China(2004AA215242)by Science and Technology Commission of Shanghai Municipality(04XD14003).
文摘Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6, IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology. 2005;2(2):130-135.
基金This work supported by grants from the National Natural Science Foundation （No. 30771959）, Natural Science Research Foundation of Jiangsu Province （No. BK2006119） and Technology Research for Advanced Medicine of Jiangsu province （No. BG2007604）.
文摘Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.
