Differential Proteomic Analysis of Hepatocellular Carcinomas from Ppp2r5d Knockout Mice and Normal (Knockout) Livers.

BACKGROUND: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Mice lacking the tumor-suppressive protein phosphatase 2A subunit B56δ (Ppp2r5d) spontaneously develop HCC, correlating with increased c-MYC oncogenicity. MATERIALS AND METHODS: We used two-dimensional difference gel electrophoresis-coupled matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify differential proteomes of livers from wild-type, non-cancerous and HCC-affected B56δ knockout mice. RESULTS: A total of 23 proteins were differentially expressed/regulated in liver between wild-type and non-cancerous knockout mice, and 119 between non-cancerous and HCC knockout mice ('cancer proteins'). Overlap with our reported differential transcriptome data was poor. Overall, 56% of cancer proteins were reported before in HCC proteomics studies; 44% were novel. Gene Ontology analysis revealed cancer proteins mainly associated with liver metabolism (18%) and mitochondria (15%). Ingenuity Pathway Analysis identified 'cancer' and 'gastrointestinal disease' as top hits. CONCLUSION: We identified several proteins for further exploration as novel potential HCC biomarkers, and independently underscored the relevance of Ppp2r5d knockout mice as a valuable hepatocarcinogenesis model.

Vitamin D-modulated dendritic cells delay lethal graft-versus-host disease through induction of regulatory T cells.

Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.

Vitamin D controls the capacity of human dendritic cells to induce functional regulatory T cells by regulation of glucose metabolism.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3.

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs.

Vitamin D3 Induces Tolerance in Human Dendritic Cells by Activation of Intracellular Metabolic Pathways.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH)2D3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH)2D3, hypoxia did not change the tolerogenic function of 1,25(OH)2D3-treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH)2D3-conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH)2D3, because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress.

Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes.

Posttranslational modifications of self-proteins play a substantial role in the initiation or propagation of the autoimmune attack in several autoimmune diseases, but their contribution to type 1 diabetes is only recently emerging. In the current study, we demonstrate that inflammatory stress, induced by the cytokines interleukin-1ß and interferon-γ, leads to citrullination of GRP78 in ß-cells. This is coupled with translocation of this endoplasmic reticulum chaperone to the ß-cell plasma membrane and subsequent secretion. Importantly, expression and activity of peptidylarginine deiminase 2, one of the five enzymes responsible for citrullination and a candidate gene for type 1 diabetes in mice, is increased in islets from diabetes-prone nonobese diabetic (NOD) mice. Finally, (pre)diabetic NOD mice have autoantibodies and effector T cells that react against citrullinated GRP78, indicating that inflammation-induced citrullination of GRP78 in ß-cells generates a novel autoantigen in type 1 diabetes, opening new avenues for biomarker development and therapeutic intervention.

1α,25-Dihydroxyvitamin D3 and its analogs as modulators of human dendritic cells: a comparison dose-titration study.

The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), presents pronounced immunomodulatory effects, mainly mediated through its actions on different immune cells such as dendritic cells (DC) and T lymphocytes. Because of the high concentrations needed to obtain immune effects, a major limitation in using 1α,25(OH)2D3 in clinical immune therapy is its calcemic side effects. TX527 (19-nor-14,20-bis-epi-23-yne-1α,25(OH)2D3) is a structural 1α,25(OH)2D3 analog showing reduced calcemic activity with maintained immunomodulatory effects in vitro and in vivo. The aim of the present study was to establish the relative potency of TX527 versus the parent molecule as an immunomodulator in vitro. In this regard, we evaluated the morphology, surface marker expression and reactive oxygen species (ROS) production in in vitro-generated human DCs treated with TX527 or 1α,25(OH)2D3 at different concentrations. Human CD14(+) monocytes were differentiated toward immature DCs, in the presence or absence of 1α,25(OH)2D3 or TX527 in a dose range from 10(-7)M to 10(-10)M. Mature DCs (mDC) were obtained after exposure of cells to LPS/interferon (IFN) γ or cluster of differentiation (CD) 40 ligand (L). Both compounds potently inhibited down-regulation of the monocytic marker CD14 in mDCs. Interestingly, CD80 and HLA-DR were down-regulated after TX527 treatment, whereas this effect was lost when using 1α,25(OH)2D3 at the lowest concentration (10(-10)M). ROS production was especially induced in TX527-treated DCs, without any adverse effects on cell survival. Finally, this altered DC surface phenotype was accompanied by typical morphological features, with control cells forming large clusters of non-adherent cells, whereas TX527 and, to a lesser extent, 1α,25(OH)2D3-modulated cells yielding small clusters of mostly adherent spindle-shaped cells. This more pronounced immune potential in vitro combined with the previously shown decreased side effects on calcium and bone metabolism, makes TX527 a promising 1α,25(OH)2D3 analog for in vivo applications in autoimmune diseases and transplantation. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Differential protein pathways in 1,25-dihydroxyvitamin d(3) 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)(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.

1,25-Dihydroxyvitamin D3 alters murine dendritic cell behaviour in vitro and in vivo.

BACKGROUND: Differentiation and maturation of dendritic cells yield a cell type with the ability to prime immune responses towards defence and destruction. 1,25(OH)2D3, the active form of vitamin D3, fosters the development of tolerogenic dendritic cells. This study aimed to evaluate the effects of 1,25(OH)2D3 on murine dendritic cell behaviour in vitro and in vivo. METHODS: Dendritic cells were differentiated from bone marrow cells of female C57Bl/6 mice in the presence or absence of 10(-8) M 1,25(OH)2D3 for 8 days (IL4 and GM-CSF). Maturation was induced for 48 h (IFNγ, LPS and BALB/C islet homogenate antigen). RESULTS: Bone marrow-derived dendritic cells displayed a different surface marker profile in the presence of 1,25(OH)2D3 with decreased MHC II, CD86 and CD80 and increased CCR5, DEC205, F4/80 and CD40, as well as lower IL6 and IL12 expression upon LPS/IFNγ stimulation. T-cell proliferation was significantly reduced when exposed to islet antigen-loaded 1,25D3-DCs as compared to control dendritic cells and IL4, IL10, TNFα and TGFß levels were increased. In vivo, transfer of islet antigen-loaded control dendritic cells resulted in priming of the immune system and hyperacute islet allograft rejection (4/4), whereas this was prevented in 5/7 mice treated with islet antigen-loaded 1,25D3-DCs. CONCLUSION: We conclude that in vitro 1,25(OH)2D3 exposure alters dendritic cell behaviour, converting them into a cell type that drives T cells away from destruction towards a regulatory phenotype.

Understanding dendritic cell biology and its role in immunological disorders through proteomic profiling.

Dendritic cells (DC) have always been present on the bright spot of immune research. They have been extensively studied for the last 35 years, and much is known about their different phenotypes, stimulatory capacity, and role in the immune system. During the last 15 years, great attention has been given to studies on global gene and protein expression profiles during the differentiation and maturation processes of these cells. It is well understood that studying the proteome, together with information on the role of protein post-translational modifications (PTM), will reveal the real dynamics of a living cell. The rapid increase of proteomic studies during the last decade describing the differentiation and maturation process in DCs, as well as modifications brought by the use of different compounds that either increase or decrease their immunogenicity, reflects the importance of understanding the molecular processes behind the functional properties of these cells. In the present review, we will give an overview of proteomic studies focusing on DCs. Thereby we will concentrate on the importance of these studies in understanding DC behavior from a molecular point of view and how these findings have aided in understanding the differences in functional properties of these cells.

Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D.

Structural analogues of vitamin D have been put forward as therapeutic agents able to exploit the immunomodulatory effects of vitamin D, without its undesired calcemic side effects. We have demonstrated that TX527 affects dendritic cell (DC) maturation in vitro, resulting in the generation of a tolerogenic cell. In the present study, we aimed to explore the global protein changes induced by the analogue in immature DC (iDC) and mature human DC and to correlate them with alterations in DC morphology and function. Human CD14(+) monocytes were differentiated toward iDC or mature DCs, in the presence or absence of TX527 (10(-8) M) (n=4). Protein samples were separated into two different pH ranges (pH4-7 and 6-9), analyzed by 2-D DIGE and differentially expressed spots (p<0.01) were identified by MALDI-TOF/TOF (76.3 and 70.7% in iDC and mature DCs, respectively). Differential protein expression revealed three protein groups predominantly affected by TX527 treatment, namely proteins involved in cytoskeleton structure, in protein biosynthesis/proteolysis and in metabolism. Moreover, protein interactome-network analysis demonstrated close interaction between these different groups (p<0.001) and morphological and functional analyses confirmed the integrated effect of TX527 on human DCs, resulting in a cell with altered morphology, cell surface marker expression, endocytic and migratory capacity.

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach.

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14(+) peripheral blood monocytes was investigated using two pH ranges (pH 4-7 and 6-9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein-interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune-associated disorders.