Differential Protein Pathways in 1,25-Dihydroxyvitamin D3 and

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Differential Protein Pathways in 1,25-Dihydroxyvitamin D3 and Dexamethasone Modulated Tolerogenic Human Dendritic Cells Gabriela Bomfim Ferreira,† Fleur S. Kleijwegt,‡ Etienne Waelkens,§,|| Kasper Lage,^,#,z Tatjana Nikolic,‡ Daniel Aaen Hansen,^ Christopher T. Workman,^ Bart O. Roep,‡ Lut Overbergh,*,†,4 and Chantal Mathieu†,4 †

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Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 902, B-3000 Leuven, Belgium ‡ Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands § ProMeta, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 901, B-3000 Leuven, Belgium Laboratory of Protein Phosphorylation and Proteomics, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 901, B-3000 Leuven, Belgium ^ Center for Biological Sequence Analysis, Kemitorvet building 208, Technical University of Denmark, DK-2800 Lyngby, Denmark # Pediatric Surgical Research Laboratories, MassGeneral Hospital for Children, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, United States z Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, United States

bS Supporting Information ABSTRACT: Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)2D3), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)2D3, dexamethasone (DEX), and a combination of both compounds (combi). Human CD14+ monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)2D3 and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3
7NL and 6
9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)2D3- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)2D3 is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)2D3 with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)2D3 effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)2D3, rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)2D3 and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation. KEYWORDS: 2D-DIGE, dendritic cell, dexamethasone, vitamin D, mass spectrometry, pathway analysis, protein interaction networks

’ INTRODUCTION Dendritic cells (DCs) play a dual role in the regulation of immune responses. As mature DCs (mDCs), they have an exceptional ability for antigen presentation and powerful capacity of T cell priming and activation. On the other hand, immature DCs (iDCs) are tissue resident cells disseminated throughout the peripheral tissues, have weak migratory and T cell priming r 2011 American Chemical Society

abilities, and more importantly, are able to induce T cell tolerance.1,2 In this regard, they exert an important role in maintaining peripheral tolerance by several mechanisms, such as silencing of differentiated antigen-specific T cells,3 activation and expansion Received: August 2, 2011 Published: November 21, 2011 941

dx.doi.org/10.1021/pr200724e | J. Proteome Res. 2012, 11, 941–971

Journal of Proteome Research of naturally occurring T regulatory cells (Tregs),4 transfer of regulatory properties to T cells5 and the differentiation of naïve cluster of differentiation (CD) 4+ cells into Tregs.6 However, under an inflammatory assault, these cells can differentiate toward mDCs, promoting strong pro-inflammatory immune responses. Moreover, the tolerogenic potential of nondifferentiated iDCs is limited and restricted to naïve/resting T cells, since they are unable to convert T effector cells into inducible Tregs.5 Thus, promoting the arrest of DC activation and maturation by using immunomodulatory drugs might represent a better strategy to obtain cells in a functionally locked and maturation-resistant DC phenotype, an important criterion for their ultimate clinical application in tolerance induction.1 A potentially interesting agent in this regard is the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), known to alter the behavior of different immune cells, for instance shifting T lymphocytes toward regulator cells7,8 and modulating monocyte cytokine profile. The main target for 1,25(OH)2D3 in a mixed immune cell population is however the DC.9,10 In this cell type, 1,25(OH)2D3 was extensively shown to impair in vitro and in vivo DC differentiation and maturation from either human peripheral blood monocytes9
13 or mouse bone marrow precursors,14 inducing the appearance of a different kind of DC, with a typical tolerogenic profile: less antigen presentation, down-regulation of costimulatory molecules (CD40, CD80, CD86) and almost annihilation of interleukin (IL)-12 secretion.9,13,15 Interestingly, the effects on surface marker expression and IL-12 secretion by DCs are only seen on the myeloid and not the plasmacytoid population.16 1,25(OH)2D3-modulated DCs are thus unable to fully activate T cells and initiate an immune response, as in the absence or low levels of costimulatory signals, stimulation of T cell receptors induces T cell anergy,17,18 decreased proliferation and cytokine secretion19 and increased apoptosis levels.9,10,17 Glucocorticoids (e.g., dexamethasone (DEX)) also alter the DC phenotype.20
28 Similar to 1,25(OH)2D3, DEX impairs the activation and maturation of murine29 and human DCs from monocytic precursors,23,24 as evidenced by the altered expression of antigen presenting and costimulatory molecules, reduced IL-12 production and T cell stimulatory capacity, and increased endocytic activity.30 Recently, in vitro DEX-treated human DCs were shown to induce the generation of Tregs, suppressing the proliferation of allospecific CD4+CD25
T cells, in a cell
cell contact manner.26 Furthermore, in vitro studies indicate that combining 1,25(OH)2D3 and DEX enhanced the effect of either compound alone on the modulation of DC surface marker expression, inhibition of DC pro-inflammatory cytokine production and on the attenuation of T cell stimulatory capacity.22,31 In addition, DCs treated with both steroids are more potent than IL-10-treated cells to suppress colitis in a SCID T cell transfer murine model.32 The aim of the present study was to characterize at the molecular level the separate and combined effects of 1,25(OH)2D3 and DEX in human DCs and to identify proteins and pathways affected by the combination of both compounds, in order to unravel the molecular basis for the cooperation of both steroids in the induction of tolerogenic human DCs. By using 2DDIGE and tandem mass spectrometry (MALDI-TOF/TOF), we identified several proteins with differential expression which reflect major changes in cytoskeleton organization, protein metabolism/biosynthesis/proteolysis and in metabolic pathways. In addition, we could show that 1,25(OH)2D3 is more potent than DEX in inducing changes on the protein profile of human DCs, skewing DCs from a more pro-inflammatory phenotype to a more

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tolerogenic one. At the protein level, combining 1,25(OH)2D3 with DEX induced a unique protein expression pattern that was not merely the addition of alterations induced by the single treatments, however a major impact of 1,25(OH)2D3 was reflected in the combined treatment. Finally, the generation of protein interaction networks and pathway analysis suggest that 1,25(OH)2D3, rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose and oxidative phosphorylation, which may affect the production of or the response to ROS generation by modulated mDCs. Understanding the molecular processes behind the functional properties of DCs and indentifying molecular targets and new pathways of immunomodulation in these cells allows the identification of promising, yet underestimated, targets for therapeutic intervention to silence unwanted or deregulated immune responses.

’ METHODS Phenotypical and Functional Analysis of Human Dendritic Cells

In vitro Generation of Human Dendritic Cells. Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors (4 per experiment) by density gradient centrifugation (Ficoll, Axis-Shield, Oslo, Norway) and monocytes were purified subsequently by CD14-specific positive magnetic-activated cell sorting according to the supplier’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated monocytes (∼90% pure, 1  106 cells/mL) were cultured in RPMI 1640 medium (Gibco, Paisley, Scotland) supplemented with Glutamax-I (Gibco), 25 mM HEPES (Gibco), 5 μg/mL Geneticin (Life Technologies, Rockville MD) and 10% heatinactivated fetal calf serum (Gibco), in 6-well plates (TPP, Transadingen, Switzerland). For inducing DC differentiation, 500 U/mL recombinant human IL-4 (Gentaur, Brussels, Belgium) and 800 U/mL recombinant human granulocyte-macrophage colony-stimulating factor (Gentaur) were added. Medium and cytokines were refreshed on day 3. On day 6, iDCs were harvested for functional/morphological analysis or for protein extraction, and maturation was induced in the remaining cultures, via CD40 triggering by incubating 0.5  106 DCs with 0.2  106 CD154expressing L cells. After 48 h, activated DCs (mDCs) were harvested for functional/morphological analysis or for protein extraction. 1,25(OH)2D3 was added at the beginning of the culture at a final concentration of 10
8 M and refreshed at day 3. DEX was added on day 3 of culture at a final concentration of 10
6 M. Antibodies and Flow Cytometry. The following fluorochrome-conjugated antibodies for flow cytometry were used: antiHLA-DR (clone G46
6), anti-CD80 (clone L307.4), anti-CD86 (clone IT2.2), anti-CD1a (clone HI-149), anti-CD14 (clone M5E2), anti-CD274 (PD-L1; clone MIH1), anti-CD11c (clone S-HCL-3) and anti-CD127 (clone hIL-7R-M21). Anti-CD25 antibody (clone M-A251) was conjugated with PE-Cy7 and antiFoxP3 (clone PCH101) with APC. All conjugated antibodies were obtained from Pharmingen (San Diego, CA), except for anti-PD-L1 which was obtained from e-Bioscience. Flow cytometric staining was analyzed on a FACS Calibur (Becton Dickinson). Cytokine Analysis. Culture supernatants from control and treated mDCs were analyzed for the presence of cytokines using Luminex bead array (Biorad, Veenendaal, The Netherlands). 942

dx.doi.org/10.1021/pr200724e |J. Proteome Res. 2012, 11, 941–971

Journal of Proteome Research

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After isoelectric focusing, the IPG strips were equilibrated during two intervals of 15 min each in an equilibration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer, 0.02% bromophenol blue), and either 1% DTT (for IPG strips pH 3
7 NL) or 1.2% DeStreak (for IPG strips pH 6
9) and run on a 1 mm thick 11.5% polyacrylamide SDS gel (25  20  0.5 cm; Ettan Dalt Six, GE Healthcare). The gels were run overnight at 20 C with a constant current of 8 mA/gel, 9 W and 600 V for 1 h and then at 16 mA/gel, 15 W and 600 V until the bromophenol blue dye front reached the bottom of the gel. After the second dimension, the gels were scanned on a Typhoon 9400 gel imager (GE Healthcare). Initial scan parameters were press sample, depth plus 3 mm, 500 to 550 V photomultiplier tube setting and 1,000 μm pixel size, before repeating at 100 μm pixel size for a high-resolution scan. Spot detection and matching was performed automatically using the BVA module of DeCyder V7.0 software followed by careful manual rematching of unmatched spots and wrongly matched spots. Spot detection parameters were set as described by the manufacturer: spot detection algorithm 6.0, estimated number of spots set at 10000 and spot volumes below 30000 were excluded. The 20 spot maps corresponding to the 10 gels from the cytoplasmic and microsomal fractions and from each pH range were used to calculate average abundance. The protein spot expression levels which showed a statistically significant (p-value