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Keywords:

  • Plasmacytoid dendritic cells;
  • Cell activation;
  • HMGB1;
  • RAGE;
  • Immunity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Dendritic cells (DC) are key components of innate and adaptive immune responses. Plasmacytoid DC (PDC) are a specialized DC subset that produce high amounts of type I interferons in response to microbes. High mobility group box 1 protein (HMGB1) is an abundant nuclear protein, which acts as a potent pro-inflammatory factor when released extracellularly. We show that HMGB1 leaves the nucleus of maturing PDC following TLR9 activation, and that PDC express on the plasma membrane the best-characterized receptor for HMGB1, RAGE. Maturation and type I IFN secretion of PDC is hindered when the HMGB1/RAGE pathway is disrupted. These results reveal HMGB1 and RAGE as the first known autocrine loop modulating the maturation of PDC, and suggest that antagonists of HMGB1/RAGE might have therapeutic potential for the treatment of systemic human diseases.

Abbreviations:
HMGB1:

High mobility group box 1 protein

PDC:

Plasmacytoid DC

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Dendritic cells (DC) are potent antigen-presenting cells bridging the innate and adaptive immune responses. To date, two subsets of DC have been identified in humans: myeloid DC and plasmacytoid DC (PDC), the latter also referred to as type I interferon (IFN-α)-producing cells 16. PDC recognize viral or bacterial DNA patterns, consisting of unmethylated CpG motifs in the context of species-dependent surrounding sequences (CpG) 7. As a consequence they produce, within a few hours, large amounts of IFN-α 810. Based on their ability to induce IFN-α secretion by PDC, two types of CpG have been described: CpG 2216 (CpG-A), which induces high amounts of IFN-α, and CpG 2006 (CpG-B), which promotes survival, maturation and migration of PDC to the lymph nodes, but induces lower amounts of IFN-α 1114. PDC sustain the priming of naive T cells and their differentiation into Th1/Th2 effectors 1517 or into regulatory T cells 16, 1822.

Recent studies shed light on the events involved in the recognition of microbial DNA by human PDC, the only subset of human DC that expresses the Toll-like receptor 9 (TLR9) 12. Both CpG-A and CpG-B have been shown to activate PDC via TLR9 23. Upon phagocytosis PDC lyse microorganisms in phagolysosomes, where microbial DNA interacts with and activates TLR9 derived from the endoplasmic reticulum 24. TLR9 activation leads to recruitment of the MyD88 adaptor protein and phosphorylation of the IL-1R–associated kinase (IRAK). Phosphorylated IRAK associates with the adaptor molecule TNF–associated factor 6 (TRAF6) 25. Two separate signaling pathways, JNK and NF-κB, are then activated, promoting PDC maturation and survival. IFN-α induction, an event exclusively occurring in PDC, depends on the formation of a complex consisting of MyD88, TRAF6 and the IRF7 transcription factor, as well as on TRAF6-dependent ubiquitination 26.

The events that determine the outcome of the interaction of PDC with T cells, and in particular the induction of effector or regulatory immune responses, are poorly characterized. Environmental signals that control the maturation of PDC in response to microbial DNA play a crucial role 27, 28. Recent data suggest that the high mobility group box 1 protein (HMGB1) is involved in the function of antigen-presenting cells (monocyte-derived DC) in vitro 2931. In vivo, HMGB1 has been shown to increase the immunogenicity of soluble or corpusculate antigens 29. HMGB1 is abundantly expressed in the nuclei of mammalian cells (>106 molecules/nucleus), facilitating interaction of chromatin with various nuclear proteins 32, 33. HMGB1 reaches the extracellular environment when monocytes/macrophages actively secrete it following acetylation and transfer to secretory endolysosomal compartments 3436 or by passive release from necrotic cells 37. Once in the extracellular milieu, HMGB1 has been shown to activate the receptor for advanced glycation end-products (RAGE) 3840 and possibly TLR2 in neutrophils and macrophages 41. Extracellular HMGB1 mediates endotoxin lethality and inflammation 32, 4246. These findings suggest that HMGB1 may be involved in regulating the function of PDC, which have been implicated in the pathogenesis of cancer 47, 48 and chronic inflammatory conditions like systemic lupus erythematosus and allergy 4951.

Here we report that HMGB1 is exported from the nucleus of PDC upon selective engagement of TLR9 and that its availability in the environment is required for PDC maturation and IFN-α secretion.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The selective activation of TLR9 by CpG induces PDC maturation, IFN-α secretion and extra-nuclear relocation of HMGB1

Human PDC can be traced and isolated with relative ease as they are CD4+HLA-DR+CD11cCD123+BDCA2+BDCA4+. We isolated PDC with a purity that was routinely >95% (Fig. 1A). PDC were incubated with different TLR ligands and the expression of co-stimulatory molecules and the secretion of IFN-α analyzed. As expected, PDC secreted IFN-α in response to CpG 2006 (Fig. 1B) and not when incubated with other TLR ligands. As shown in Fig. 1C, PDC efficiently matured upon triggering TLR9 by CpG 2006, as demonstrated by up-regulation of CD40, CD83 and CD86. In contrast, treatment with lipopolysaccharide (LPS) did not elicit detectable changes in the phenotype of PDC (Fig. 1C).

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Figure 1. PDC maturation and IFN-α secretion is specifically triggered by CpG. (A) Freshly isolated PDC were identified as BDCA-2+, CD123+, CD11c cells. Purity was above 95% routinely. (B) Purified PDC were incubated alone (w/o) or stimulated with CpG 2006 (CpG), LPS, heat shock protein (Hsp) 70, Poly I:C or peptidoglycan (PGN) for 48 hours. All cultures were supplemented with IL-3. IFN-α was measured in the supernatant by ELISA. CpG 2006 induces production of IFN-α from PDC. Samples were analyzed in duplicates. Data are shown as mean ± SD. (C) Freshly isolated PDC were incubated with CpG 2006 (CpG), lipopolysaccharide (LPS) or alone (w/o). Mean fluorescence intensity (MFI) of CD40, CD83 and CD86 was analyzed by flow cytometry 40 h later. All cultures were supplemented with IL-3. The histograms on the left display the isotype controls (dashed line), untreated cells (dotted line), LPS (thin line) and CpG (gray). Triggering of TLR9 with CpG leads to up-regulation of co-stimulatory molecules (CD40, CD86) and of maturation markers (CD83). Data are representative for three independent experiments with cells from different donors.

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HMGB1 was mainly present in the nucleus of freshly isolated PDC, as visualized by confocal microscopy (Fig. 2A, upper row). Upon TLR9 activation with CpG 2006, a dramatic relocalization in the cytosol took place (Fig. 2A, middle row). This effect was not observed following treatment with LPS (Fig. 2A, lower row). In activated monocytes this phenomenon has been shown to precede secretion of HMGB1 in the extracellular environment 35, 52. We then checked if HMGB1 is released in the extracellular milieu by PDC upon stimulation with CpG 2006. The relocalization of nuclear HMGB1 into the cytosol was accompanied by its release in the medium (Fig. 2B). Furthermore, we tested the effect of recombinant HMGB1 on the production of IFN-α by PDC. On stimulation with recombinant HMGB1 PDC secreted low amounts of IFN-α (Fig. 2C, p<0.01).

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Figure 2. PDC relocate HMGB1 into the cytosol and secrete it upon triggering of TLR9 by CpG. (A) Expression of HMGB1 was assessed by staining with polyclonal antibodies anti-HMGB1 followed by a second antibody conjugated with Alexa Fluor 594 (red). Nuclei were visualized by staining with Hoechst 33342 (blue). In freshly isolated PDC (w/o) HMGB1 is present mainly in the nucleus (upper row). In cells stimulated with CpG 2006 (CpG) for 40 hours HMGB1 staining is observed predominantly in the cytoplasm (middle row). As a control, in cells stimulated with LPS, no relocation of HMGB1 was observed. Results are from representative routine experiments. (B) Cells were incubated alone (w/o) or stimulated with CpG 2006, and supernatants tested for HMGB1 at the indicated time points. HMGB1 was determined by sandwich ELISA. Cells stimulated with CpG 2006 secrete HMGB1. (C) Cells were incubated alone (w/o) or stimulated with 8 μg/ml recombinant HMGB1 (HMG) or 1 μg/ml CpG 2006 (CpG) for 48 h. All cultures were supplemented with IL-3. IFN-α was measured in the supernatant by ELISA. Recombinant HMGB1 induces secretion of IFN-α. Samples were analyzed in duplicates. Data are shown as mean ± SD. The asterisk denotes statistically significant values (p<0.01).

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Endogenous HMGB1 tunes the maturation of PDC via its receptor RAGE

To analyze if the relocation of HMGB1 into the extracellular milieu upon triggering of TLR9 by CpG has a role in the maturation of PDC, we committed PDC to maturation with CpG 2006 in the absence or presence of reagents that antagonize HMGB1. We used anti-HMGB1 polyclonal antibodies, and a recombinant N-terminal fragment of HMGB1 (box A) that attenuates HMGB1-induced release of pro-inflammatory cytokines by macrophages and behaves as an HMGB1 antagonist 53, 54. The antibodies we used recognize the sequence 166–181 of HMGB1, which is part of the domain that interacts with the RAGE receptor 5355. We then evaluated by flow cytometry the membrane expression of CD40, CD83 and CD86. Anti-HMGB1 antibodies and box A hindered the up-regulation of CD40, CD83 and CD86 induced by CpG 2006 (Fig. 3A, B). No differences were detected in the presence of irrelevant antibodies (Fig. 3A, B) or a GST control protein (not shown). Blockade of HMGB1 with antibodies inhibited the up-regulation of CD40 and CD86 on stimulation of PDC with CpG 2216 as well (Fig. 3C). In parallel, we assessed the IFN-α secretion from PDC stimulated with CpG. HMGB1 blockade either by anti-HMGB1 antibodies or by box A decreased the production of IFN-α induced by CpG 2006 (Fig. 4B, p<0.05). Irrelevant antibodies or a GST control protein did not influence the cytokine release (Fig. 4B). The secretion of IFN-α by PDC stimulated with CpG 2216, a stronger inducer of IFN-α, was also found to be reduced by blocking HMGB1 with antibodies (Fig. 3D, p<0.05).

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Figure 3. Maturation of PDC induced by CpG depends on HMGB1. PDC were either stimulated with CpG 2006 alone (-) or in presence of anti-HMGB1 antibodies (anti HMG), box A (boxA) or a control immunoglobulin (Ig). (A) Mean fluorescence intensity of unstimulated cells (w/o) or of cells stimulated with CpG 2006 in presence or absence of HMGB1 blockade was analyzed. Blockade of HMGB1 hinders the up-regulation of CD40, CD83 and CD86. (B) The histograms display the CD40 fluorescence of cells stimulated with CpG 2006 alone (gray) or in presence of anti-HMGB1 antibodies, box A or a control immunoglobulin, respectively (thin line). The staining with isotype controls is represented by the dotted line. The mean fluorescence values for CpG 2006 alone and in presence of HMGB1 blockade or a control immunoglobulin are given adjacent to the histogram. (C) The histograms display the CD40 and CD86 fluorescence of cells stimulated with CpG 2216 alone (gray) or in presence of anti-HMGB1 antibodies (thin line). The staining with isotype controls is represented by the dotted line. The mean fluorescence values for CpG 2216 alone and in presence of HMGB1 blockade are given adjacent to the histogram. Blockade of HMGB1 hinders the up-regulation of CD40 and CD86. D. PDC were left untreated (w/o) or stimulated with CpG 2216 alone (-) or in presence of anti-HMGB1 antibodies (anti HMG). Blockade of HMGB1 decreases significantly the production of IFN-α. Data are representative of three independent experiments performed in different donors. Data are shown as mean ± SD. The asterisks denote statistically significant values (p<0.05).

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thumbnail image

Figure 4. Secretion of IFN-α from PDC upon CpG stimulation depends on HMGB1 and RAGE. (A) Freshly isolated PDC express RAGE (gray). Expression of RAGE by PDC was studied by flow cytometry. The dot plots represent cells stained with control isotype-matched antibodies or with anti-RAGE antibodies. (B) PDC were stimulated with CpG 2006 alone (-) or in presence of anti-HMGB1 (anti HMG), anti-RAGE antibodies, box A (boxA), a control immunoglobulin (Ig) or a GST control protein. Blockade of HMGB1 or RAGE decreases significantly the production of IFN-α. The asterisks denote statistically significant values (p<0.05).

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HMGB1 is a specific ligand for RAGE, the best characterized receptor for HMGB1 on hematopoietic cells 38. We checked if this receptor is present on the plasma membrane of PDC. As shown in Fig. 4A, freshly isolated PDC expressed RAGE, as evaluated by flow cytometry. We used anti-RAGE antibodies to directly test whether RAGE was involved in the effect of HMGB1 on PDC maturation. These antibodies are directed against the sequence 42–59 of the human RAGE, and hinder the interaction of RAGE with its ligand HMGB1 56. Blockade of RAGE significantly decreased the production of IFN-α (Fig. 4B; p<0.05). No effects were observed in the presence of control antibodies (Fig. 4B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The ability of DC to orchestrate adaptive immune responses stems from their capacity to integrate the information provided by maturation stimuli and the microenvironment in which they are located 57, 58. In humans, the myeloid DC and PDC complement each other in terms of location, pathogen-recognition receptors and sets of cytokines produced in response to challenge 59, 60. TLR trigger the maturation of DC and the secretion of numerous cytokines and chemokines upon recognition of pathogens, representing the most important class of pathogen-recognition receptors 25, 61. Expression of TLR9 strictly by PDC endows them with specific recognition of viral or bacterial DNA sequences 9. In contrast to other TLR, TLR9 is not expressed on the membrane but in intracellular compartments, where recognition of microbial DNA occurs after internalization and degradation of microbes 25.

We report here that HMGB1 is expressed in the nucleus of freshly purified PDC. Upon triggering of TLR9, HMGB1 leaves the nucleus and is secreted in the extracellular environment. Its blockade hinders the maturation of PDC and the secretion of IFN-α (Fig. 3, 4). In addition, recombinant HMGB1 induced a low production of IFN-α by PDC. The best characterized receptor for HMGB1 on inflammatory cells is RAGE 38, 40. We observed that freshly isolated PDC express RAGE, which makes them potentially responsive to HMGB1 (Fig. 4). This is indeed the case, since the selective hindrance of RAGE abolished the secretion of IFN-α (Fig. 4). A similar role for RAGE in mediating HMGB1 functional activities has been demonstrated in different models, including regeneration of peripheral nerves 38, 62 and tumor growth/metastasis 63.

Our data suggest that triggering of TLR9 with CpG leads to maturation of PDC by activation of an endogenous loop, represented by HMGB1 and its receptor, RAGE. HMGB1 and RAGE are used to decode signals derived from dying cells: necrotic cells trigger an inflammatory response via HMGB1, while apoptotic cells are swept off silently because they do not release HMGB1 32, 43, 64. PDC might have evolved a similar decoding mechanism. Microbial signals trigger mobilization of the abundant nuclear HMGB1 in PDC, which in turn is required for PDC maturation and for induction of a protective inflammatory response governed by IFN-α. Self-sustaining, clinically evident autoimmunity has been proposed to depend on the failure of the balance between cytokines that drive the differentiation of the major subsets of DC, like TNF-α and IFN-α 6. The latter factor in particular plays a leading role in the pathogenesis of the prototypic systemic autoimmune disease, systemic lupus erythematosus, a disease in which the deregulated clearance of dying cells is crucial 65. Further investigation is warranted to determine the potential of the regulated release of HMGB1 in human disease.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Media and reagents

The medium used throughout was RPMI 1640 (Gibco) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1.5 mM L-glutamine and 10% heat-inactivated fetal calf serum and 10 ng/ml recombinant human IL-3 (R&D Systems) 66. Rabbit polyclonal anti-HMGB1 antibody raised against peptide 166–181 was purchased from BD Biosciences PharMingen. Expression and purification of recombinant HMGB1, box A and GST vector protein (used as control) was performed as previously described 67. Goat anti-RAGE polyclonal antibody was from Chemicon.

Culture of PDC

Human peripheral blood mononuclear cells were isolated from healthy donor blood by Ficoll density gradient centrifugation. Blood PDC were purified by immunomagnetic selection using BDCA-4 cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. Purity of freshly isolated PDC was typically more than 95%. Purity and maturation of PDC was routinely assessed by staining with antibodies to BDCA-2 (Miltenyi Biotec), CD11c, CD40, CD83, CD86, CXCR4 and CD123 (BD Biosciences PharMingen) 4. The samples were analyzed on a FACScan (Becton Dickinson). CpG oligodeoxynucleotides (ODN 2006, ODN 2216; 5 μg/ml, InvivoGen) 11, endotoxin (LPS, 1 μg/ml, Sigma Aldrich), rabbit anti-HMGB1 (1:200 dilution), goat anti-RAGE antibody (1:200 dilution), box A (10 μg/ml), Hsp70 (10 μg/ml), recombinant HMGB1 (8 μg/ml), poly I:C (Amersham Biosciences 10 μg/ml) and PGN (Sigma Aldrich 20 μg/ml) were added as indicated. For detection of cytokines, supernatants were collected after 40 h and either used immediately or stored frozen until analysis.

Immunoassays for HMGB1

Cellular localization of HMGB1 was assessed by immunostaining of DC using affinity-purified rabbit anti-HMGB1 polyclonal antibodies as described 37. Briefly, DC were transferred onto adhesion slides, and incubated at 37°C for 1 h to allow adherence. The adherent cells were fixed with phosphate-buffered formaldehyde (4%, pH 7.4 for 15 min), and permeabilized with saponin. After blocking the slides with 10% BSA, cells were sequentially incubated with anti-HMGB1 antibodies and goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular Probes). Nuclei were counterstained with Hoechst 33342 stain. The levels of HMGB1 in the culture medium were assessed by Western blotting analysis using rabbit polyclonal antibodies as previously described 37.

Detection of IFN-α and HMGB1 by ELISA

The levels of IFN-α in the culture medium were determined using a two-site sandwich ELISA (Bender MedSystems). HMGB1 levels in the supernatants of cultured cells were assessed using a two-site sandwich ELISA using rabbit polyclonal (BD Biosciences PharMingen) and mouse monoclonal (R&D Systems) antibodies anti-HMGB1. The concentration of cytokines in the samples was calculated with reference to standard curves of purified recombinant cytokines at various dilutions. Samples were analyzed in serial twofold dilutions in duplicate.

Statistical analysis

Statistical analysis was performed using the two-tailed Student's t-test for unpaired samples with unequal variance. p values of less than 0.05 were considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank Julia Hering and Francesco Demarchis for recombinant HMGB1 and box A production and quality control, respectively. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (to A.A.M., P.R.-Q. and M.E.B.), by the European Community (to .R.-Q.), by the Fondazione Berlucchi and by the Ministero della Sanità (to A.A.M. and M.E.B.).

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