DCs facilitate B cell responses against microbial DNA via DC-SIGN

<div><p>Microbial DNA is highly immunostimulatory and is sensed by endosomal pattern recognition receptors after release from internalized microbes. It is unclear how extracellular DNA released from dead microbes is delivered to endosomal PRRs to induce immune responses. Here we have investigated the ability of DCs to bind and internalize extracellular <i>E</i>.<i>coli</i> DNA as well as synthetic DNA. DCs internalized <i>E</i>.<i>coli</i> and synthetic DNA, which was dependent on the C-type lectin receptor DC-SIGN. Notably, endosomal uptake of DNA by DCs enhanced TLR9-dependent responses of B cells against DNA. Hence, we have identified DC-SIGN as a cell surface receptor for DNA that facilitates immune responses directed against DNA.</p></div

Dendritic cells interact with both class A ODN and microbial DNA via DC-SIGN.

<p>(<b>A</b>-<b>D</b>). Flow cytometry analysis of monocyte-derived DCs incubated with EC-DNA-FITC (<b>A,B</b>) or ODN-2216-FITC (<b>C,D</b>) for 10 min in the presences or absence of EGTA, IgG1 isotype control or blocking antibodies directed against DC-SIGN. (<b>E</b>,<b>F</b>) Confocal imaging of EC-DNA-FITC (green, <b>E</b>) or ODN-2216-FITC (green, <b>F</b>), early endosome antigen 1 (EEA1, red), DC-SIGN (turquoise) and DNA (Hoechst, blue) in monocyte-derived DCs stimulated with EC-DNA-FITC (<b>E</b>) or ODN-2216-FITC (<b>F</b>). 10 μg/ml DNA or 5 μM ODN was used in all experiments. Data are collated (mean ± s.d.) of three (<b>B</b>,<b>D</b>) independent experiments with different donors or are representative of at least three (<b>A,C</b>) or two (<b>E,F</b>) independent experiments with different donors. *P<0.05, **P<0.01 (student’s t-test).EC-DNA: <i>E</i>.<i>coli</i> DNA, ROI: region of interest.</p

Dendritic cells produce type I IFN or cytokines in response to synthetic and microbial DNA.

<p>(<b>A</b>,<b>B</b>,<b>D</b>,<b>E</b>) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA (<b>A</b>,<b>B</b>,<b>D</b>), human DNA (<b>D</b>), ODN-2216 or control ODN (<b>E</b>) for indicated time points was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with the highest expression. (<b>C</b>) Similar as in (<b>A</b>), but EC-DNA was treated with DNAse before stimulation. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four (<b>C</b>), three (<b>A</b>,<b>B</b>) or two (<b>D</b>,<b>E</b>) independent experiments with different donors *P<0.05, **P<0.01 (student’s t-test). EC-DNA: <i>E</i>. <i>coli</i> DNA.</p

DC-SIGN binds class A ODN and microbial DNA.

<p>(<b>A</b>,<b>B</b>,<b>D</b>-<b>H</b>) <i>E</i>. <i>coli</i> DNA (<b>A</b>,<b>B</b>,<b>D</b>), human DNA (<b>D</b>) or indicated ODNs (<b>E-H</b>) were coated on high binding plates and recombinant DC-SIGN binding to coated ligands was measured by ELISA. (<b>C</b>) Recombinant DC-SIGN was coated on high binding plates and binding to DNAse-treated or untreated biotin-labeled <i>E</i>. <i>coli</i> DNA or Fucose was measured by ELISA. (<b>I-K</b>) Binding of parental Raji cells or Raji cells stably expressing DC-SIGN or Langerin to FITC-labeled <i>E</i>. <i>coli</i> DNA (<b>I,J</b>) or FITC-labeled ODN-2216 (<b>K</b>) 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 (<b>G</b>) or representative of at least four (<b>I</b>), three (<b>E</b>) or two (<b>A-D,F,H</b>,<b>J</b>,<b>K</b>) independent experiments (mean ± s.d. of duplicates in <b>A</b>-<b>F,H</b>). *P<0.05, **P<0.01 (student’s t-test). EC-DNA: <i>E</i>. <i>coli</i> DNA.</p