Dataset Information

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

ABSTRACT: The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

SUBMITTER: Garcia-Gonzalez PA

PROVIDER: S-EPMC6547838 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

García-González Paulina A PA Maggi Jaxaira J Schinnerling Katina K Sepúlveda-Gutiérrez Alejandro A Soto Lilian L Neira Oscar O Mehdi Ahmed M AM Nel Hendrik J HJ Pesce Bárbara B Aravena Octavio O Molina María Carmen MC Catalán Diego D Thomas Ranjeny R Verdugo Ricardo A RA Aguillón Juan Carlos JC

Frontiers in immunology 20190528

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogen ...[more]

PMID: 31191540

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Project description:Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ?-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ?-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ?-cells arrested autoimmunity to ?-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ?-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.

Project description:BackgroundThe use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.MethodsTolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-?, IL-? and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.ResultsDifferences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-?) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-? production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.ConclusionsOur results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.

Project description:BackgroundDendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function.MethodsShort hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay.ResultsRIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-β1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-β1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-β1 antibody. TGF-β1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-β1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-β stimulation.ConclusionsOur results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-β1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-β1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.

Dataset Information

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

Publications

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

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