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

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

DENV-induced RLR triggering leads to antiviral type I IFN and type III IFN responses.

<p>IFNL1, IFNL2, IFNL3, IFNL4, IFN-α, IFN-β mRNA expression and DENV RNA expression in mock-infected or DENV-infected DCs over time in the presence or absence of blocking antibodies directed against IFNα/βR (A,C) or IFNLR (B,C) or in the presence or absence of DENV RNA replication inhibitor SDM25N (D) was measured by real-time PCR, normalized to GAPDH and set as 1 in DENV-infected DCs at 24 hours post infection. (E) Similar as in (A), but DCs were transfected with MAVS, RIG-I or MDA5 siRNA and IFNL1 and IFNL2 mRNA expression was analyzed 24h post infection. Data are collated (mean ± s.d.) of at least four (B,E), three (A,C) or two (D) different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test).</p

DENV RNA replication induces IL-27 expression in dermal CD14⁺ DCs.

<p>(A) CD14⁺ and CD1c⁺ dermal DCs were isolated from human skin and mock-treated or infected with DENV for indicated times before mRNA expression of IFN-β, MxA and IL-27p28 was analyzed using real-time PCR, normalized to GAPDH and set at 1 in 32h infected samples of CD14⁺ dermal DCs. (B) Similar to (A), but in the presence or absence of DENV replication inhibitor SDM25N. Data are collated (mean ± s.d.) of two independent experiments with different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test). N.S., not significant; N.D. not detected.</p

RLR activation by DENV or synthetic ligands drives T_FH formation via IL-27.

<p>Flow cytometry analysis of extracellular CXCR5, PD-1 (A,F) and intracellular Bcl-6 (B,E) expression of differentiated T cells resulting from cocultured naive CD4+ T cells with mock-treated DC or DCs infected with DENV (A,B) or stimulated with LPS, poly(I:C)LyoVec or 5’pppRNA-LyoVec (E,F) for 48h in the absence or presence of neutralizing antibodies against IL-27 (F) or after silencing MAVS (A,B). (C) IL-27p28 and IL-27EBI3 mRNA levels in DCs stimulated with poly(I:C)LyoVec or 5’pppRNA-LyoVec for 8h were analyzed using real-time PCR, normalized to GAPDH and set at 1 in 1.0 μg ml^-1 stimulated poly(I:C)LyoVec samples. (D) IL-27 in the supernatant of untreated, poly(I:C)LyoVec or 5’pppRNA-LyoVec stimulated DCs after 24h or 48h was analyzed by ELISA. (G) IgM and IgG in the supernatant of B cells cocultured for 7 days with differentiated T cells from cocultures of naive CD4+ T cells with untreated, LPS, poly(I:C)LyoVec or 5’pppRNA-LyoVec stimulated DCs was analyzed by ELISA. Data are representative of at least three (D,E) or two (B) independent experiments with different donors (mean ± s.d. of duplicates in, D) or are collated data (mean ± s.d.) of three (G), four (C,F) or two (A) different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test). -, unstimulated; p(I:C)LV, poly(I:C)LyoVec; pppRNA-LV, 5’pppRNA-LyoVec.</p