Effects of CpG-ODN on TLR-9 expression and Th1/Th2 cytokines secretion from mouse macrophages

Objective To investigate the effects on Toll like receptor-9(TLR-9) expression and Th1/Th2 cytokines secretion from mouse macrophages(RAW264.7) induced by CpG motifs.Methods RAW264.7 cells grown in 24-well plates in vitro were randomly divided into 4 groups,namely CpG-ODN group,Non-CpG-ODN group,CpG-ODN+chloroquine group and control group.CpG-ODN group was treated with CpG-ODN (0.3 μg/ml).Non-CpG-ODN group was treated with Non-CpG-ODN (0.3 μg/ml).CpG-ODN+chloroquine group was treated with CpG-ODN (0.3 μg/ml) and chloroquine (0.3 μg/ml).Control group was treated with DMEM.After 24 hours,the expressions of TLR-9 mRNA and protein were analyzed by RT-PCR and Western blot respectively.The concentration of TNF-αand IL-6 in the supernatants of each group were examined by Radioimmunoasssy(RIA) kits and the level of IL-12 was analyzed by ELISA.Results After treatment for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group was statistically different as compared with the Non-CpG-ODN group(t=7.12,P0.01).The CpG-ODN group was statistically different as compared with the control group(t=8.04,P0.01);The CpG-ODN+chloroquine group was statistically different as compared with the Non-CpG-ODN group(t=4.25,P0.05);The CpG-ODN+chloroquine group was statistically different as compared with the control group (t=5.58,P0.01).The expression of TLR-9 protein in CpG-ODN group (1.35±0.18) was statistically different as compared with the Non-CpG group (1.01±0.04) and control group(0.91±0.10),(t=3.21、3.67,all P0.05).CpG-ODN+chloroquine group (1.34±0.15),was statistically different as compared with the Non-CpG group and control group(t=3.71、4.10,all P0.05).But CpG-ODN group was not statistically different as compared with the CpG-ODN+chloroquine group(t=0.10,P0.05).The level of TNF-α in the supernatant in the CpG-ODN group [(0.48±0.12)ng/ml] was higher than Non-CpG-ODN group [(0.29±0.23)ng/ml ]、CpG-ODN- chloroquine group [(0.32±0.24)ng/ml] and control group [(0.29±0.19)ng/ml,P0.05].(t=2.84、2.38、3.25 respectively at Non-CpG-ODN group,CpG-ODN-chloroquine group,control group,all P0.05)The levels of IL-6 and IL-12 in the supernatants in the CpG-ODN group [(27.69±15.61)pg/ml and(48.59±25.17)pg/ml] had no significant difference compared with that in Non-CpG-ODN group [(17.02±10.66)pg/ml and (59.58±32.89)pg/ml]、CpG-ODN+chloroquine group [(28.58±24.08)pg/ml and(41.30±13.17)pg/ml] and control group [(16.12±17.70)pg/ml and (61.54±39.69)pg/ml](t=1.94、0.75 all P0.05).Conclusions CpG-ODN can enhance the expressions of TLR-9 and secretion of Th1 cytokines from macrophages.