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

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)(2)D(3)), 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)(2)D(3), 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)(2)D(3) 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)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), 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)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.

High Mitochondrial Respiration and Glycolytic Capacity Represent a Metabolic Phenotype of Human Tolerogenic Dendritic Cells

“…Despite that both vitamin D 3 and dexamethasone alone have been reported to induce a tolerogenic effect on moDCs (23), we chose the combination of these compounds because this approach provides more potent tolerogenic effect than when individual drugs are used (14). This is currently an accepted model of human tolerogenic DC generation with a therapeutic utility (24).…”

“…In contrast to mature moDCs, proteomic analysis of tolerogenic moDCs, which are resistant to maturation (13), has shown a predominance of OXPHOS pathway proteins (14). Therefore, a comparative analysis across the spectrum of moDCs is required to explain the metabolic changes between immunogenic and tolerogenic moDC states.…”

1,25-Dihydroxyvitamin D3 Promotes Tolerogenic Dendritic Cells with Functional Migratory Properties in NOD Mice

“…Livers were processed further using a GentleMACS dissociator and C-tubes (Miltenyi Biotec), according to the manufacturer's specifications. All single-cell suspensions were passed through a mesh and washed extensively, and RBCs were lysed after incubation with NH 4 Cl. Cells were stained with the following conjugated Abs before identification of the PKH26 + CD11c + DC population: CD3, CD19, Ly6G, and CD11c (all from eBioscience).…”

“…Exposure of differentiating DCs in vitro to 1,25(OH) 2 D 3 initiates a complex and autonomous molecular process (4,5) that ultimately interferes with their differentiation and maturation (6)(7)(8)(9), locking the cells in a semimature state (10). Tolerogenic 1,25(OH) 2 D 3 -treated mature DCs (1,25D 3 -mDCs) lose their ability to activate autoreactive T cells and stimulate the generation of regulatory T cells (Tregs) (9,(11)(12)(13)(14).…”

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“…1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 )-conditioned tolerogenic DCs convert glucose to lactate for fast, although less efficient, ATP generation, accompanying high glucose consumption, even when oxygen is available for OXPHOS. Transcriptomics and proteomics data further support the early metabolic reprogramming of 1,25(OH) 2 D 3 -conditioned tolerogenic DCs which is controlled by the PI3K/Akt/mTOR pathway [4,5]. Also others documented a higher glycolytic capacity and reserve in tolerogenic DCs induced by 1,25(OH) 2 D 3 in combination with dexamethasone [6].…”