title: Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.
abstract: Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.
authors.affiliation: Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: M
authors.identifier: http://orcid.org/0000-0002-6468-729X
authors.initials: M
authors.lastname: Bendix
authors.affiliation: Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Dige
authors.affiliation: Department of Immunology, Institute of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Deleuran
authors.affiliation: Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: J F
authors.initials: JF
authors.lastname: Dahlerup
authors.affiliation: Department of Medicine, Regional Hospital Horsens, Horsens, Denmark.
authors.forename: S P
authors.initials: SP
authors.lastname: Jørgensen
authors.affiliation: Department of Rheumatology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: L E
authors.initials: LE
authors.lastname: Bartels
authors.affiliation: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: L B
authors.initials: LB
authors.lastname: Husted
authors.affiliation: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: T
authors.initials: T
authors.lastname: Harsløf
authors.affiliation: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Langdahl
authors.affiliation: Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Agnholt
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics; D000328:Adult; D000368:Aged; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D045744:Cell Line, Tumor; D003424:Crohn Disease / Q000188:drug therapy*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010919:Placebos; D012333:RNA, Messenger / Q000096:biosynthesis; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D018167:Receptors, Calcitriol / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D047490:Retinoid X Receptor alpha / Q000096:biosynthesis / Q000235:genetics; D014807:Vitamin D / Q000627:therapeutic use*; D055815:Young Adult
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D010919:Placebos; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D018167:Receptors, Calcitriol; D047490:Retinoid X Receptor alpha; C579457:VDR protein, human; D014807:Vitamin D; D007371:Interferon-gamma; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase
keywords: Crohn's disease; T cells; VDR; flow cytometry; vitamin D