Fig 2: DC-SIGN facilitates microbial DNA induced responses.(A,B,E,F) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA (A,E), ODN-2216 or control ODN (B,F) in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN (A,B) or after treatment with control or DC-SIGN siRNA (E,F) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with IgG1 isotype control or control siRNA. (C,D) DC-SIGN expression of monocyte-derived DCs after control or DC-SIGN siRNA treatment was measured by flow cytometry. (G) Expression of CD80, CD83 and CD86 expression by monocyte-derived DCs stimulated with control ODN or ODN-2216 in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of six (D), four (A,B,) or three (E,F) independent experiments with different donors or are representative of six (C) or two (G) independent experiments with different donors. *P<0.05, **P<0.01 (student’s t-test). EC-DNA: E. coli DNA.

Fig 3: CD11c+ DCs enhance IFN-α production of CD11c- cells via DC-SIGN.(A-D) mRNA expression of monocyte-derived DCs stimulated with EC-DNA (A), control ODN, ODN-2216 (B,D), or R837 (C) after treatment with control, TLR7, TLR9 (A-C), or IRF7 siRNA (D) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. (E) Immunoblot of monocyte-derived DCs or Raji cells whole cell lysate for TLR9. β-actin was used as loading control. (F) Analysis of TLR9 expression in monocyte-derived DC culture by flow cytometry. Number adjacent to gates indicates percentage of gated cells. (G) mRNA expression of sorted CD11c+ DCs and CD11c- cells stimulated with control ODN or ODN-2216 was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with ODN-2216 of CD11c- cells. (H) mRNA expression of sorted CD11c+ DCs and CD11c- cells stimulated with control ODN or ODN-2216 after treatment with control or DC-SIGN siRNA. Sorted cells were stimulated for 1h, washed and combined for 5h before mRNA expression was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of four (A,B,G), three (H) or two (C,D) independent experiments with different donors or are representative of four (F) or two (E) independent experiments with different donors. *P<0.05, **P<0.01 (student’s t-test). EC-DNA: E. coli DNA.

Fig 4: DC-SIGN binds class A ODN and microbial DNA.(A,B,D-H) E. coli DNA (A,B,D), human DNA (D) or indicated ODNs (E-H) were coated on high binding plates and recombinant DC-SIGN binding to coated ligands was measured by ELISA. (C) Recombinant DC-SIGN was coated on high binding plates and binding to DNAse-treated or untreated biotin-labeled E. coli DNA or Fucose was measured by ELISA. (I-K) Binding of parental Raji cells or Raji cells stably expressing DC-SIGN or Langerin to FITC-labeled E. coli DNA (I,J) or FITC-labeled ODN-2216 (K) was analyzed by flow cytometry. 10 μg/ml DNA or 5 μM ODN was used in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four independent experiments (G) or representative of at least four (I), three (E) or two (A-D,F,H,J,K) independent experiments (mean ± s.d. of duplicates in A-F,H). *P<0.05, **P<0.01 (student’s t-test). EC-DNA: E. coli DNA.