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Interferon-Gamma DNA Methylation Is Affected by Mycophenolic Acid but Not by Tacrolimus after T-Cell Activation.

ABSTRACT: Immunosuppressive drug therapy is required to treat patients with autoimmune disease and patients who have undergone organ transplantation. The main targets of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA; the active metabolite of mycophenolate mofetil) are T cells. It is currently unknown whether these immunosuppressive drugs have an effect on DNA methylation-an epigenetic regulator of cellular function. Here, we determined the effect of tacrolimus and MPA on DNA methylation of the gene promoter region of interferon gamma (IFN?), a pro-inflammatory cytokine. Total T cells, naive T cells (CCR7⁺CD45RO^-), and memory T cells (CD45RO⁺ and CCR7^-CD45RO^-) were isolated from CMV seropositive healthy controls and stimulated with ?-CD3/CD28 in the presence or absence of tacrolimus or MPA. DNA methylation of the IFN? promoter region was quantified by pyrosequencing at 4?h, days 1, 3, and 4 after stimulation. In parallel, T-cell differentiation, and IFN? protein production were analyzed by flow cytometry at days 1 and 3 after stimulation. Our results show that MPA induced changes in IFN? DNA methylation of naive T cells; MPA counteracted the decrease in methylation after stimulation. Tacrolimus did not affect IFN? DNA methylation of naive T cells. In the memory T cells, both immunosuppressive drugs did not affect IFN? DNA methylation. Differentiation of naive T cells into a central-memory-like phenotype (CD45RO⁺) was inhibited by both immunosuppressive drugs, while differentiation of memory T cells remained unaffected by both MPA and tacrolimus. IFN? protein production was suppressed by tacrolimus. Our results demonstrate that MPA influenced IFN? DNA methylation of naive T cells after stimulation of T cells, while tacrolimus had no effect. Both tacrolimus and MPA did not affect IFN? DNA methylation of memory T cells.

SUBMITTER: Peters FS

PROVIDER: S-EPMC5506181 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Interferon-Gamma DNA Methylation Is Affected by Mycophenolic Acid but Not by Tacrolimus after T-Cell Activation.

Peters Fleur S FS Peeters Annemiek M A AMA Hofland Leo J LJ Betjes Michiel G H MGH Boer Karin K Baan Carla C CC

Frontiers in immunology 20170712

Immunosuppressive drug therapy is required to treat patients with autoimmune disease and patients who have undergone organ transplantation. The main targets of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA; the active metabolite of mycophenolate mofetil) are T cells. It is currently unknown whether these immunosuppressive drugs have an effect on DNA methylation-an epigenetic regulator of cellular function. Here, we determined the effect of tacrolimus and MPA on DNA methylatio ...[more]

PMID: 28747916

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Project description:Granulomatosis with polyangiitis (GPA) is an autoimmune disease affecting mainly small blood vessels. B-cells are important in the GPA pathogenesis as precursors of autoantibody-producing cells but likely also contribute (auto)antibody-independently. This has been underlined by the effectiveness of B-cell-depletion (with Rituximab) in inducing and maintaining disease remission. Mycophenolate-mofetil (MMF) and azathioprine (AZA) are immunosuppressive therapies frequently used in GPA-patients. Interestingly, MMF-treated GPA-patients are more prone to relapses than AZA-treated patients, while little is known about the influence of these drugs on B-cells. We investigated whether MMF or AZA treatment (or their active compounds) alters the circulating B-cell subset distribution and has differential effects on in vitro B-cell proliferation and cytokine production in GPA-patients that might underlie the different relapse rate. Circulating B-cell subset frequencies were determined in samples from AZA-treated (n = 13), MMF-treated (n = 12), untreated GPA-patients (n = 19) and matched HCs (n = 41). To determine the ex vivo effects of the active compounds of MMF and AZA, MPA and 6-MP respectively, on B-cell proliferation and cytokine production, PBMCs of untreated GPA-patients (n = 29) and matched HCs (n = 30) were cultured for 3-days in the presence of CpG-oligodeoxynucleotides (CpG) with MPA or 6-MP. After restimulation (with phorbol myristate acetate, calcium-ionophore), cytokine-positive B-cell frequencies were measured. Finally, to assess the effect of MMF or AZA treatment on in vitro B-cell proliferation and cytokine production, PBMCs of MMF-treated (n = 18), and AZA-treated patients (n = 28) and HCs (n = 41) were cultured with CpG. The memory B-cell frequency was increased in AZA- compared to MMF-treated patients, while no other subset was different. The active compounds of MMF and AZA showed in vitro that MPA decreased B-cell proliferation in GPA-patients and HCs. B-cell proliferation in MMF- and AZA-treated patients was not different. Finally, the IL-6+ B-cell frequency was decreased by MPA compared to 6-MP. No differences in IL-10+, IL-6+ or TNFα+ B-cell proportions or proliferation were found in MMF- and AZA-treated patients. Our results indicate that MMF could be superior to AZA in inhibiting B-cell cytokine production in GPA-patients. Future studies should assess the effects of these immunosuppressive drugs on other immune cells to elucidate mechanisms underlying the potential differences in relapse rates.

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Interferon-Gamma DNA Methylation Is Affected by Mycophenolic Acid but Not by Tacrolimus after T-Cell Activation.

Publications

Interferon-Gamma DNA Methylation Is Affected by Mycophenolic Acid but Not by Tacrolimus after T-Cell Activation.

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