1. Vitamin D Controls Murine and Human Plasmacytoid Dendritic Cell Function 
Nina Karthaus, Annemiek B. van Spriel, Maaike W.G. Looman, Shuo Chen, Lisanne M. Spilgies, Liesbet Lieben, Geert Carmeliet, Marleen Ansems, Gosse J. Adema 
Journal of Investigative Dermatology 
Volume 134, Issue 5, Pages (May 2014)
DOI: /jid
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2. Figure 1
Murine plasmacyoid dendritic cells (pDCs) express vitamin D receptor (VDR) in vitro and ex vivo. (a) DCs were generated in vitro from murine bone marrow (BM) or freshly isolated from BM and spleen. Cells were sorted into pDC and conventional DC (cDC) fractions by magnetic activated cell sorting. Murine VDR (mVDR). VDR mRNA was detected by quantitative PCR, and mRNA levels are shown relative to the expression of mouse PBGD. VDR protein expression was detected by (b) western blot and (c) intracellular antibody staining and subsequent FACS on in vitro−cultured pDCs and cDCs or primary cells from BM and spleen. Geometric mean fluorescence intensities (gMFI) shown are corrected for isotype control staining. Data shown are mean values (a) or representative (b, c) of at least three independent experiments. *P<0.05. Data are shown as mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Murine plasmacyoid dendritic cells (pDCs) and conventional DCs (cDCs) react to stimulation with calcitriol and its precursor calcifediol. pDCs and cDCs were sorted from Flt3L bone marrow cultures. Expression of (a) the co-regulators Drip205 and Hairless, (b) the vitamin D–metabolizing enzymes Cyp27A1, Cyp2R1, and Cyp27B1, and the vitamin D receptor (VDR) target genes VDR (c) and Cyp24A1 (d) in pDCs and cDCs were detected with quantitative PCR. mRNA levels are shown relative to expression of mouse PBGD. Data shown are mean values of at least three independent experiments. *P<0.05; **P<0.01. Data are shown as mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Vitamin D–stimulated murine plasmacyoid dendritic cells (pDCs) and conventional DCs (cDCs) induce markedly impaired T-cell proliferation. (a) pDCs and (b) cDCs were sorted from Flt3L BM cultures of either wild-type (WT) or vitamin D receptor deficient (VDR-/-) mice. Purified pDCs and cDCs were prestimulated for 2hours with 100nM calcitriol and 100nM calcifediol. Subsequently, cells were stimulated again with 100nM calcitriol, 100nM calcifediol, and vehicle (no stim) or 100ngml−1 CpG for an additional 16hours. Cells were washed and co-incubated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled splenocytes (CD11c, B220 depleted) for 5 and 6 days. T-cell proliferation was measured as indicated by CFSE dilution. Numbers indicate the percentage of proliferation. Data shown are representative of at least three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Vitamin D impairs the capacity of murine plasmacyoid dendritic cells (pDCs) and conventional DCs (cDCs) to activate T cells. (a) pDCs and (b) cDCs were sorted from Flt3L bone marrow (BM) cultures of either wild-type (WT) or vitamin D receptor deficient (VDR-/-) mice. Purified pDCs and cDCs were prestimulated for 2hours with 100nM calcitriol and 100nM calcifediol. Subsequently, cells were stimulated again with 100nM calcitriol, 100nM calcifediol, and vehicle (no stim) or 100ngml−1 CpG for an additional 16hours. Cells were washed and co-incubated with splenocytes (CD11c, B220 depleted) for 5 and 6 days. IFNγ levels in the supernatant were measured with ELISA. Data shown are mean values of at least three independent experiments. ns, nonsignificant; *P<0.05; **P<0.01; ***P< Data are shown as mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Human plasmacyoid dendritic cells (hPDCs) and monocyte-derived dendritic cells (moDCs) react to stimulation with calcitriol. hPDCs were sorted from peripheral blood leukocytes (PBLs). moDCs were cultured from monocytes for 6 days in the presence of IL-4 (300Uml−1) and GM-CSF (450Uml−1). Expression of (a) the human vitamin D receptor (hVDR) protein was detected by intracellular antibody staining. (b) The vitamin D (VitD)-metabolizing enzymes Cyp27A1, Cyp2R1, and Cyp27B1 and (c) the VDR target gene Cyp24A1 in hPDCs and moDCs were detected with quantitative PCR. (c) moDCs and hPDCs were stimulated with 100nM calcitriol and 100nM calcifediol for the indicated time points. mRNA levels are shown relative to the expression of human PBGD. Data shown are (a) representative or (b, c) mean values of at least three independent experiments. *P<0.05; ***P< Data are shown as mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Vitamin D stimulation of human plasmacyoid dendritic cells (hPDCs) reduces their ability to activate T cells. hPDCs were sorted from peripheral blood leukocytes (PBLs) and prestimulated for 2hours with 100nM calcitriol and 100nM calcifediol. Subsequently, cells were stimulated again with 100nM calcitriol, 100nM calcifediol, and vehicle (no stim) or 100ngml−1 CpG for an additional 5hours. Cells were washed and co-incubated with allogeneic PBLs for 7 days. (a) T-cell proliferation was measured as indicated by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution and (b) IFNγ production by PBLs was measured by ELISA. Data shown are mean values of at least three independent experiments. *P<0.05; **P<0.01; ***P< Data are shown as mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions