A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2

Abstract

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by Orientia tsutsugamushi) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.

Keywords: BDCA-2; glycosaminoglycans; heparin; plasmacytoid dendritic cells; type I interferons.

Fig. 1.

Inhibitory effect of heparin or enoxaparin on TLR9-induced IFN-α production in hpDCs. hpDCs (n = 3) were treated with 100 U/mL heparin, 100 U/mL enoxaparin, or CpG-ODN 2216 at 0.1 µmol/L for 20 h, and IFN-α production was determined by ELISA (Left). Immune cells were treated with indicated concentrations of heparin (Middle) or enoxaparin (Right) for 1 h and then stimulated with CpG-ODN 2216 at 0.1 µmol/L for 20 h, and IFN-α production was determined by ELISA. Relative IFN-α production was defined as the percent ratio of IFN-α produced by cells treated with CpG-ODN 2216 and heparin or enoxaparin and compared to positive control (amount of CpG-ODN 2216-induced IFN-α production observed without heparin or enoxaparin). Each data point represents an individual donor. Statistical significance was analyzed using one-way ANOVA with Dunnett’s post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the positive control.

Fig. 2.

Inhibitory effect of heparin on type I IFN production depends on BDCA-2. (A) Representative flow cytometry histograms of BDCA-2 expression in CAL-1 cells, BDCA-2-deficient CAL-1 cells (CAL-1 KO BDCA-2), and BDCA-2-reconstituted CAL-1 cells (CAL-1-BDCA-2). Cells were stained with anti-BDCA-2-APC (AC144) labeled antibody and analyzed by flow cytometry. (B) Western blot analysis of BDCA-2 and β-ACTIN expression in CAL-1, CAL-1 KO BDCA-2, and CAL-1-BDCA-2 cells. Cell lysates were analyzed by SDS-PAGE and subsequent western blotting using anti-BDCA-2 (Cat# A58582, EpiGentek). (C) For the analysis of BDCA-2 surface expression, CAL-1-BDCA-2 and hpDCs were stained with anti-BDCA-2-APC (AC144) labeled antibody, and BDCA-2 mean fluorescence intensity (MFI) was determined by flow cytometry. (D) and (E) CAL-1-BDCA-2 cells (D) and CAL-1 KO BDCA-2 cells (E) were treated with 0.25 µg/mL anti-BDCA-2 or isotype control antibody, 4 U/mL heparin, and 0.1 µmol/L CpG-ODN 2216, and IFN-α production was determined after 20 h by ELISA (Left). Immune cells were treated with indicated concentrations of heparin and controls (0.25 µg/mL anti-BDCA-2 or isotype control antibody) for 1 h and then stimulated with 0.1 µmol/L CpG-ODN 2216 for 20 h (Right). IFN-α production was determined by ELISA. (F) CAL-1-BDCA-2 (Left) or CAL-1 KO BDCA-2 (Right) cells were treated with indicated concentrations of heparin and after 1 h, cells were washed and treated with 0.1 µmol/L CpG-ODN 2216 for 20 h, and IFN-α production was determined by ELISA. (G) CAL-1-BDCA-2 (Left) or CAL-1 KO BDCA-2 (Right) cells were treated with 0.1 µmol/L CpG-ODN 2216 and after 1 h, cells were washed and treated with indicated concentrations of heparin for 20 h, and IFN-α production was determined by ELISA. Relative IFN-α production was defined as the percent ratio of IFN-α produced by cells treated with CpG-ODN 2216 and heparin compared to positive control (amount of CpG-ODN 2216-induced IFN-α production observed without heparin). Each data point represents an experiment or donor. Statistical significance was analyzed using Student’s t test (C) or one-way ANOVA with Dunnett’s (D–G) post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the positive control (D–G).

Fig. 3.

BDCA-2 interacts with heparin and heparin-derived GAGs. (A) Western blot analysis of the affinity protein precipitation assay of BDCA-2, DCIR, and dectin-2 ECDs using heparin-agarose. Sample input (Upper) and bound proteins (Lower) were analyzed by SDS-PAGE and subsequent western blotting using Strep-Tactin HRP conjugate (IBA). (B) Dose-dependent binding of BDCA-2, DCIR, and dectin-2 ECDs to uncoated or heparin-coated (1 mg/mL) ELISA plates. Bound lectins were detected by Strep-Tactin HRP conjugate (IBA). (C) Analysis of the interaction of BDCA-2 ECD (1 µg/mL) with different GAGs as indicated. ELISA plates were uncoated or precoated with individual GAGs at 1 mg/mL. Bound BDCA-2 ECD was detected by Strep-Tactin HRP conjugate (IBA). (D) Calcium-dependent binding of BDCA-2 ECD (WT) or calcium-binding BDCA-2 mutant ECD (APA) to heparin-coated (1 mg/mL) ELISA plates. Binding studies were performed in the absence or presence of 10 mmol/L Ca²⁺. Bound proteins were detected by Strep-Tactin HRP conjugate (IBA). (E) Main disaccharide of heparin, composed of glucosamine (GlcN) and iduronic acid (IdoA). Green squares represent the R groups (-H or -SO₃H) of the positions 2, 6 or N. (F) Binding of BDCA-2 ECD to ELISA plates coated with heparin or desulfated heparins (2-ODSH, 6-ODSH, and N-DSH) at 1 mg/mL. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA). (G) CAL-1-BDCA-2 cells were treated with different concentrations of 2-ODSH, 6-ODSH, and N-DSH or heparin followed by subsequent stimulation with 0.1 µmol/L CpG-ODN 2216. After 20 h, IFN-α production was determined by ELISA. Relative IFN-α production was defined as the percent ratio of IFN-α produced by cells treated with CpG-ODN 2216 and heparin derivatives compared to positive control (amount of CpG-ODN 2216-induced IFN-α production observed without heparin derivatives). The data represent the mean of four individual experiments. (H) Concentration-dependent binding of BDCA-2 ECD, 3 N-glycosylation mutants (N110Q, N137Q, and N164Q), and PNGase F fully deglycosylated BDCA-2 ECD to plate-bound heparin (1 mg/mL). Protein binding was detected with Strep-Tactin HRP conjugate (IBA). Each data point represents one experiment. Statistical significance was analyzed using one-way or two-way ANOVA with Dunnett’s (G) or Tukey’s (B–D, F, and H) post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the respectively control.

Fig. 4.

Heparin and plasma-mediated inhibition of CpG-ODN 2216–induced IFN-α production in hPBMCs, CAL-1-BDCA-2, or hpDCs and its modulation by BDCA-2 ECD. (A) CAL-1-BDCA-2 cells (n = 4) were treated with different concentrations of BDCA-2 ECD in the absence or presence of heparin (4 U/mL) (Left) or human plasma (0.5%) (Right) for 1 h and then treated with 0.1 µmol/L CpG-ODN 2216. After 20 h, the supernatant was harvested, and IFN-α production was determined by ELISA. (B) hPBMCs from four donors were treated with 4 µg/mL BDCA-2 ECD, 2.5% plasma, and 1 µmol/L CpG-ODN 2216 for 20 h, and IFN-α production was determined by ELISA (Left). hPBMCs (n = 4) were treated with indicated concentrations of BDCA-2 ECD with or without 2.5% plasma for 1 h and then stimulated with CpG-ODN 2216 1 µmol/L (Middle). hPBMCs (n = 3) were treated with indicated concentrations of dectin-2 with or without 2.5% plasma for 1 h and then stimulated with CpG-ODN 2216 1 µmol/L for 20 h (Right). After stimulation, IFN-α production was determined by ELISA. (C) hpDCs (n = 3) were treated with indicated concentrations of plasma for 1 h and then stimulated with CpG-ODN 2216 0.1 µmol/L (Left). After 20 h IFN-α production was determined by ELISA. hpDCs (n = 3) were treated with BDCA-2 ECD or dectin-2 (8 μg/mL) with or without 0.25% plasma for 1 h and then stimulated with CpG-ODN 2216 0.1 µmol/L for 20 h (Right). After stimulation, IFN-α production was determined by ELISA. (D) CAL-1-BDCA-2 cells were treated for 1 h with heparin (hep) or 1% plasma (P) untreated or overnight treated with a heparinase mixture consisting of heparinases I, II, and III (Hps) and chondroitinase ABC (Ch) and then stimulated with 0.1 µmol/L CpG-ODN 2216 for 20 h. After stimulation, IFN-α was determined by ELISA (Left). hpDCs (n = 6) were treated for 1 h with plasma 2% or 1%, either untreated or overnight treated with a heparinase mixture consisting of Hps I, II, and III, followed by stimulation with 0.1 µmol/L CpG-ODN 2216 for 20 h. After stimulation, IFN-α was determined by ELISA (Right). Relative IFN-α production was defined as the percent ratio of IFN-α produced by cells treated with CpG-ODN 2216 and heparin and compared to positive control (amount of CpG-ODN 2216-induced IFN-α production observed without heparin or plasma). Each data point represents one individual donor or experiment. Statistical significance was analyzed using one-way or two-way ANOVA with Dunnett’s (C Left panel) or Tukey’s (A–C Right panel and D) post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the respective controls of each experiment.

Fig. 5.

SolBDCA-2 is found in human plasma, and BDCA-2 shedding by ADAM proteases is increased upon cellular activation. (A and B) Correlation between solBDCA-2 concentration in plasma of different donors (n = 10) and IFN-α production after stimulation of CAL-1-BDCA-2 cells (A) or hpDCs (n = 8) (B) with 1% of plasma for 1 h and subsequent 0.1 µmol/L CpG-ODN 2216 stimulation for 20 h. Results were analyzed via ELISA. (C) Western blot analysis of solBDCA-2 in the supernatant of CAL-1, CAL-1-BDCA-2, CAL-1 KO BDCA-2, and CAL-1 KO BDCA-2-reconstituted with BDCA-2 cells (CAL-1 KO BDCA-2-BDCA-2) using heparin-agarose for precipitation. Samples were analyzed by SDS-PAGE and subsequent western blotting analysis using anti-BDCA-2 (Cat# A58582, EpiGentek). (D and E) CAL-1-BDCA-2 cells (D) or hpDCs (E) were treated with indicated concentrations of CpG-ODN 2216, 1 µmol/L ionomycin (IO) and 50 ng/mL phorbol myristate acetate (PMA). After 20 h of incubation, IFN-α production (Left) as well as solBDCA-2 (Right) concentrations were determined by ELISA. (F) CAL-1-BDCA-2 cells were treated with different concentrations of GW280264X (GW) for 1 h and then stimulated with CpG-ODN 2216 0.1 µmol/L or IO 1 µmol/L or PMA 50 ng/mL. After 20 h of incubation, solBDCA-2 production was determined by ELISA. (G) hpDCs (n = 7) were treated with GW (10 µmol/L) or GI (10 µmol/L) for 1 h and then stimulated or not with CpG-ODN 2216 0.1 µmol/L. After 20 h of incubation, solBDCA-2 production was determined by ELISA. (H) PCR analysis of isoforms of BDCA-2 mRNA. RNA from hpDCs (n = 3) was isolated and analyzed for the presence of BDCA-2 isoforms by RT-PCR amplification. The PCR products were analyzed by a 2% agarose gel. Relative solBDCA-2 production was defined as the percent ratio of solBDCA-2 in cells treated with GW or GI and CpG-ODN 2216/IO/PMA compared to positive control (amount of BDCA-2 production observed without the inhibitors). Each data point represents one individual donor or experiment. For correlation analysis, Pearson correlation coefficients were performed (A and B), and a P < 0.05 was considered significant. Statistical significance was analyzed using one-way ANOVA with Dunnett’s (D–F) or Tukey’s (G) post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the negative control (medium) (D and E) and compared to the positive control (F and G).

Fig. 6.

Dysregulation of solBDCA-2 in autoimmune diseases and scrub typhus (ST). (A) Determination of solBDCA-2 (Left) and heparin (Right) concentrations in plasma from 20 healthy controls (HC), 19 bullous pemphigoid (BP) patients, and 20 psoriasis (Pso) patients by ELISA and Anti IIa Assay Kit, respectively. (B) Determination of solBDCA-2 concentration in serum from 24 HC and 48 ST patients by ELISA (Left). Determination of heparin concentration in the serum from 15 HC and 15 ST patients by the Anti IIa Assay Kit (Right). (C) Correlation between solBDCA-2 and anti-dsDNA in the serum of HC (n = 19) together with ST patients (n = 24). (D) Correlation between solBDCA-2 and IFN-γ in the serum of healthy controls (n = 19) together with ST patients (n = 24). (E) SolBDCA-2 concentration in the serum of HC (n = 24), 16S-positive (n = 23), and 16S-negative (n = 24) ST patients. (F) Anti-dsDNA antibodies in the serum of HC (n = 47) and ST patients, both 16S-positive and 16S-negative (n = 169). The red dashed line represents the cutoff of 20 IU/mL. Each data point represents an individual donor/patient. *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the healthy control. For correlation analysis, Pearson correlation coefficients were performed (C and D), and a P < 0.05 was considered significant. Statistical significance was analyzed using Student’s t test (B) or one-way ANOVA with Dunnett’s (A, E, and F) post hoc test, *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the HC.

Fig. 7.

Graphical representation of the IFN regulatory network based on BDCA-2, solBDCA-2, and heparin. pDCs in circulation are in tight control on type I interferon production due to the presence of heparin, and this control is lifted once the cells leave the circulation and move to tissues where the concentration of heparin is lower and the activation of pDCs triggers the release of solBDCA-2. FcRγ: Fc receptor gamma-chain; an adapter protein containing immunoreceptor tyrosine-based activation motif (ITAM). SYK: tyrosine-protein kinase SYK. MYD88: myeloid differentiation primary response protein MyD88; an adapter protein involved in the signaling of TLRs.