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

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

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

DENV-mediated RLR activation induces IL-27 via IFNα/βR signaling.

<p>DCs were mock-treated or infected with DENV for 30 (E,F,G,H,I), 32 (B,C,D,K) or 48 hours (A) in the presence or absence of blocking antibodies directed against IFNα/βR (C),IFNLR (D) or after MAVS, RIG-I, MDA5, IKKε, or IRF9 silencing (A,B,K) and secretion of IL-27 into the supernatant was measured by ELISA (A) or mRNA expression was analyzed using real-time PCR (B,C,D,K), normalized to GAPDH and set at 1 in DENV-infected samples treated with control siRNA (B,K) or isotype antibody (C,D). (E-G) STAT1 phosphorylation at Ser708 or Ser727 in mock-treated or DENV-infected DCs was analyzed by flow cytometry (E,F) or immunoblot (G) using phospho-specific antibodies. Total STAT1 served as loading control in (G). Relative mean fluorescent intensity (MFI) shown in (F). (H) Confocal imaging of DNA (Hoechst, blue), DENV Envelope protein (E protein, green) or IRF9 (red) in mock-treated or DENV-infected DCs 30 hours post infection. White arrowheads indicate cells with nuclear IRF9. (I) Immunoblot for IRF9 of nuclear (NE) or cytoplasmic (CE) extracts of DCs infected with DENV at indicated times. β-actin served as loading control. (J) Similar as in (I) but IRF9 levels in nuclear extracts were measured by ELISA. Data are representative of at least five (E), four (H), or two (A,G,I) independent experiments with different donors (mean ± s.d. of duplicates in A) or are collated data (mean ± s.d.) of four (D,F,K),three (B,C) or two (J) 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

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

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

DENV RNA replication triggers RIG-I and MDA5.

<p>IFN-β mRNA expression 24 hours post infection (h.p.i.) of DCs that were infected with DENV (A,B,F,K) after MAVS, TRIF, MYD88 (A), RIG-I, MDA5 (B), TBK1, IKKε (F) or IRF3 (K) silencing by RNA interference was measured by real-time PCR, normalized to GAPDH and set as 1 in DENV-infected DCs treated with control siRNA. (C,D) Flow cytometry analyses of TBK1 or IKKε phosphorylation at Ser172 in mock-treated or DENV-infected DCs 14 h.p.i. using phospho-specific antibodies. Data in (D) show relative mean fluorescent intensity (MFI). (E) Immunoblot of whole cell lysates of DCs mock-treated or infected with DENV in the presence or absence of DENV replication inhibitor SDM25N for TBK1 p-S172 and IKKε p-S172. Total TBK1 or IKKε served as loading control. (G) IFN-β mRNA of DENV-infected DCs 24 h.p.i. in the presence or absence of TBK1/IKKε inhibitor BX795 was measured by real-time PCR, normalized to GAPDH and set as 1 in DENV-infected DCs. (H) Confocal imaging of DNA (Hoechst, blue), DENV Envelope protein (E protein, green) or IRF3 (red) in mock-treated or DENV-infected DCs 18 h.p.i. White arrowheads indicate cells with nuclear IRF3. (I) Immunoblot for IRF3 of nuclear (NE) and cytoplasmic extracts (CE) of DCs infected with DENV at indicated times. β-actin served as loading control. (J) Similar as in (I) but IRF3 levels in nuclear extracts were measured by ELISA. Data are representative of at least five (H), four (C) or two (E,I) independent experiments with different donors or are collated (mean ± s.d.) of at least six (K), five (A) or four (B,D,F), three (G) or two (J) different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test).</p

DENV infection of DCs induces Bcl-6⁺CXCR5⁺PD-1⁺ T_FH formation.

<p>Flow cytometry analysis of extracellular CXCR5, PD-1 (A,C) and intracellular Bcl-6 (B,D) expression of differentiated T cells after coculture of naive CD4+ T cells with mock-treated DC or DCs infected with DENV for 48h in the absence or presence of DENV replication inhibitor SDM25N. Numbers in zebra plots of (A) indicate percentage of gated cells. Histograms in (B) represent all T cells from mock coculture (grey), DENV coculture (black) or CXCR5⁺PD-1⁺ T cells from DENV coculture (red) as gated in (A). Results in (D) are relative to fluorescent intensity of DENV samples set as 1. (E) IL-21 in supernatant of differentiated T cells as described in (A) was measured by ELISA. (F) IgM and IgG in the supernatant of B cells cocultured for 7 days with differentiated T cells from mock-treated or DENV-infected DC-T cell cocultures was analyzed by ELISA. Data are representative of at least five (A) or four (B) independent experiments with different donors or are collated data (mean ± s.d.) of five (C), four (D), three (F) or two (E) different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test). MFI: mean fluorescent intensity.</p