Table 1.
Properties of INH-ODNs.
Fig 1.
Dose response relationship of TLR7- and TLR9-ligands.
(A) Human PBMCs (2x105 cells/well) were stimulated with the TLR9-ligands CpG-ODN 2006 (1, 5, 10 μM) or CpG-ODN 2216 (0.5, 1, 5, 10 μM) or with the TLR7-ligands imiquimod (0.5, 1, 5, 10 μM) or R848 (0.5, 1, 5, 10 μM). After a culture period of 4 days IL-6 was quantified in the culture supernatant by ELISA. (B) The intracellular ATP content of the cells stimulated in (A) was analyzed. Error bars represent SD of three individual cultures from one donor.
Fig 2.
INH-ODNs are not toxic for human PBMCs.
(A) PBMCs (4x105 cells/well) were cultured with INH-ODNs alone (10 μM) or with a combination of CpG-DNA 2006 (100 nM) and titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM). Medium in the absence of cells (medium) and cells cultured in the absence of CpG-DNA 2006 or INH-ODNs (mock) served as negative controls. Bovine LDH was used as positive control (pos. contr.). The extracellular LDH-content was determined after 48h of culture. Error bars represent SD of three individual cultures. The experiment was repeated with imiquimod (5 μg/ml) using cells from a different donor (data not shown). INH-ODNs were again not toxic. (B) PBMCs (4x105 cells/well) were not stimulated (mock) or stimulated with CpG-ODN 2006 (100 nM) or a combination of CpG-ODN 2006 (100 nM) and titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM) as indicated. The intracellular ATP-content was determined after 48h of culture. Error bars represent SD of three individual cultures. The experiment was repeated twice with cells from another donor with similar results.
Fig 3.
INH-ODNs impair efficiently TLR9-mediated release of IFN-α by PBMCs.
(A) Human PBMCs (5–6x105 cells/well) were stimulated with CpG-ODN 2216 (3 μM) in the absence (CpG) or presence of titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM) for 24h. IFN-α was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IFN-α release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 6–9 cultures). #p<0.05, ANOVA compared to CpG-ODN 2216; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888 and compared to INH-ODN 21158 for G-modified INH-ODN 24987 and 24991. (B) For comparison bone marrow-derived pDCs (2x105 cells/well) from lupus-prone MRL/Mp-lpr/lpr mice were treated as described in (A) with the exception that instead of INH-ODN 20959 G-modified INH-ODN 105870 was used. INH-ODN were used at a concentration of 0.1, 1, 10 μM. Data represent mean and SD of two separate experiments (each bar represents n = 2–6 cultures). #p<0.05, ANOVA compared to CpG-ODN 2216; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888.
Fig 4.
IFN-α release triggered via TLR7 is also efficiently impaired by INH-ODNs.
The experiment was performed as described in Fig. 3A with the exception that PBMCs were stimulated with the TLR7-ligand RNA-ORN 22075 (5 μM). Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 9 cultures). #p<0.05, ANOVA compared to RNA-ORN; *p<0.05, ANOVA compared to INH-ODN 20959 for G-modified INH-ODN 105871.
Fig 5.
G-modified INH-ODNs were significantly more effective in preventing IL-6 release by CpG-ODN-stimulated human PBMCs.
PBMCs (3–4x105 cells/well) were stimulated with CpG-ODN 2006 (100 nM) in the absence (CpG) or presence of titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM) for 48h. IL-6 was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IL-6 release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 8–9 cultures). #p<0.05, ANOVA compared to CpG-ODN 2006; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888; §p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.
Fig 6.
G-modification significantly improves the ability of INH-ODNs to impair imiquimod-induced IL-6 release by human PBMCs.
PBMCs (4x105 cells/well) were stimulated with imiquimod (5 μg/ml) in the absence (imiquimod) or presence of titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM) for 48h. IL-6 was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IL-6 release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD of two independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 6 cultures). #p<0.05, ANOVA compared to imiquimod; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888 or compared to INH-ODN 21158 for G-modified INH-ODN 24987 and 24991 or compared to INH-ODN 20959 in case of G-modified INH-ODN 105871.
Fig 7.
IL-6 release by CpG-ODN-activated human B-cells is significantly more prevented by G-modified INH-ODNs.
(A) B-cells (5–10x104 cells/well) were stimulated with CpG-ODN 2006 (100 nM) in the absence (CpG) or presence of titrated amounts of INH-ODNs (0.1, 1, 10 μM) for 24h. IL-6 was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IL-6 release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 4–9 cultures). (B) IL-6 content of the culture supernatants of the experiment described in (A) was additionally determined after 5 days of culture. (C) depicts the intracellular ATP-content of the cells described in (B). Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 4–9 cultures). #p<0.05, ANOVA compared to CpG-ODN 2006; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888 or compared to INH-ODN 21158 for G-modified INH-ODN 24987 and 24991; §p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.
Fig 8.
G-modified INH-ODNs are significantly more effective to inhibit IL-6 release by imiquimod-stimulated human B-cells.
(A) B-cells (1x105 cells/well) were stimulated with imiquimod (5 μg/ml) in the absence (imiquimod) or presence of titrated amounts of INH-ODNs (0.1, 1, 10 μM) for 24h. IL-6 was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IL-6 release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD from two independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 3–6 cultures). (B) IL-6 content of the culture supernatants of the experiment described in (A) was additionally determined after 6 days of culture. #p<0.05, ANOVA compared to imiquimod; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888 or compared to INH-ODN 21158 for G-modified INH-ODN 24987 and 24991 or compared to INH-ODN 20959 in case of G-modified INH-ODN 105871; §p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.
Fig 9.
G-modified INH-ODNs prevent CpG-ODN or imiquimod induced CD86 expression by CD20+ B-cells.
PBMCs (4x105 cells/well) were stimulated with CpG-ODN 2006 (100 nM) or imiquimod (5 μg/ml) in the absence or presence of INH-ODNs (1, 10 μM). After 48h cells were harvested, stained with CD20 and CD86 antibodies and CD20+ cells were analyzed by flow cytometry for CD86 expression. (A) shows CD86 expression profiles upon treatment with individual stimulator/inhibitor combinations as indicated. (B) and (C) depict CD86 expression post stimulation with CpG-ODN 2006 (B) or imiquimod (C) from two independent experiments. Data represent mean and SD of these two experiments, each experiment was performed with cells from a different donor (each bar represents n = 4 cultures). #p<0.05, ANOVA compared to CpG-ODN 2006; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595 and 24888 §p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.
Fig 10.
G-modified INH-ODNs partially prevent CpG-ODN or imiquimod induced HLA-DR expression by CD20+ B-cells.
The cells used in Fig. 9 were also stained with an HLA-DR antibody and analyzed by flow cytometry for HLA-DR expression. (A) shows HLA-DR expression profiles upon treatment with individual stimulator/inhibitor combinations as indicated. (B) and (C) depict HLA-DR expression post stimulation with CpG-ODN 2006 (B) or imiquimod (C) from two independent experiments. Data represent mean and SD of these two experiments, each experiment was performed with cells from a different donor (each bar represents n = 4 cultures). #p<0.05, ANOVA compared to CpG-ODN 2006; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595 and 24888 §p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.