Differential Protein Pathways in 1,25-Dihydroxyvitamin D₃ and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

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 <i>in vitro</i> 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 (<i>p</i> < 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

Differential Protein Pathways in 1,25-Dihydroxyvitamin D₃ and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

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 <i>in vitro</i> 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 (<i>p</i> < 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

Differential Protein Pathways in 1,25-Dihydroxyvitamin D₃ and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

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 <i>in vitro</i> 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 (<i>p</i> < 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

Differential Protein Pathways in 1,25-Dihydroxyvitamin D₃ and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

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 <i>in vitro</i> 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 (<i>p</i> < 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

Summary of the benchmarks conducted in this study and the corresponding findings.

<p>The scheme, output, benchmark, validation and results are shown.</p

Validation of the performance of the spindle prediction platform (SPIP) in the human proteome.

<p>Validation of the predictions using the text mined, manually curated dataset, EXPERT, as true positives. (A) ROC curves: Sensitivity (also called Recall; y-axis) versus 1-Specificity (x-axis). And (B) PR curves: Precision (y-axis) versus Recall (x-axis) retrieved by each method.</p

Summary of the methods used in this study.

<p>The class, method, type and laboratory where the methods were developed is shown.</p

Number of predictions retrieved by each method.

<p>Number of predictions retrieved by each method.</p

Network model of the hidden spindle hubs.

<p>Hidden spindle hubs (rectangular nodes) and associated known spindle proteins (pink circle nodes). Enriched functional classes related to spindle clusters are indicated – see Methods (black labels). For the spindle interacting proteins IDs see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031813#pone.0031813.s020" target="_blank">Table S10</a></b>.</p

The Spindle Prediction Integrated Platform (SPIP) workflow.

<p>Left panel, “Prediction”: describes three different approaches (dashed boxes, NNI, DGC, LM) which include seven independent methods for predicting spindle associated proteins from all proteins in the human proteome. Each method has its own associated confidence score (red: the less confident). NNI group of methods includes the MLNN method that integrates different spindle protein features to predict new spindle proteins using Neural Network technology; The DGC approach includes the following methods: DORA that searches for domains characteristic of known spindle proteins in target proteins; hiPPI that scores potential interactions between putative and spindle proteins based on their homology to known interacting protein pairs; CODA that scores putative spindle proteins if there is a homologous domain fused to a homologue of a domain typically associated with spindle proteins; the GOSS method that measures semantic similarity of the GO terms for known and putative spindle proteins, and finally the GECO method that measures the correlation of gene expression profiles between known and putative spindle proteins. The LM approach includes the COCITE method that detects pairs of spindle and target proteins co-cited in the literature. The left panel of the figure represents the following: For a given set of proteins (labelled with numbers) each method scores the same protein at a different rank, for example protein 1 is top-ranked in NNI but ranked in second place by Hippi i.e. depending on the method we could have different rankings for the same protein. Central box, “Integration”: The scores within each prediction dataset were translated into p-values and combined in a target prediction matrix. The prediction p-values from the 3 approaches, LM, NNI and DGC were then integrated into the Spindle Prediction Integrated Platform score (SPIP) for every protein target, again using Fisher's method (for more details see the Material and Methods section). Upper box, Validation”: SPIP was validated using two different schemes, a computational one using the whole human proteome, and an experimental one using a subset of selected “unknown proteins” to conduct experimental validation (see the text). Lower box, “Context analyses”: to identify relevant targets potentially involved in “hidden hubs”.</p