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Vitamin D3 treatment of vitamin D–insufficient asthmatic patients does not alter immune cell function  Brandy Reid, MS, Pierre-Olivier Girodet, MD, PhD,

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Presentation on theme: "Vitamin D3 treatment of vitamin D–insufficient asthmatic patients does not alter immune cell function  Brandy Reid, MS, Pierre-Olivier Girodet, MD, PhD,"— Presentation transcript:

1 Vitamin D3 treatment of vitamin D–insufficient asthmatic patients does not alter immune cell function  Brandy Reid, MS, Pierre-Olivier Girodet, MD, PhD, Jonathan S. Boomer, PhD, Azza Abdel-Gadir, PhD, Kathy Zheng, MPH, Michael E. Wechsler, MD, Leonard B. Bacharier, MD, Susan J. Kunselman, MA, Tonya S. King, PhD, Elliot Israel, MD, Mario Castro, MD, Manuela Cernadas, MD, Jonathan M. Green, MD  Journal of Allergy and Clinical Immunology  Volume 138, Issue 1, Pages e9 (July 2016) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Intracellular cytokine analysis of CD4+ T cells. Peripheral blood samples were obtained before (V3) and after (V6) 12 weeks of treatment with placebo or vitamin D. Intracellular cytokine staining of CD4+ T cells was performed, and the percentage of CD4+ T cells expressing IL-4, IFN-γ, IL-10, or IL-17A was determined. Shown is the fold change from V3 to V6, which was calculated as the ratio of the V6 value divided by the V3 value for each sample pair. There was no difference in this ratio between vitamin D– and placebo-treated participants. The TH1/TH2 ratio, which was calculated by dividing the percentage of CD4/IFN-γ–positive by the percentage of CD4/IL-4–positive cells, was also not different. Shown are individual data points and lines representing means ± SDs. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Cytokine secretion by stimulated PBMCs and monocytes. A, PBMCs were isolated from samples collected either before (V3) or after (V6) vitamin D supplementation or placebo and stimulated with α-CD3 and α-CD28 for 48 hours. Secreted cytokines were analyzed in the culture supernatant by using a cytokine bead array or ELISA (TGF-β only). The fold change from V3 to V6 was calculated by dividing the value at V6 by the value at V3 for each sample pair. B, CD14+ monocytes were isolated from PBMCs from vitamin D–deficient asthmatic patients before (V3) and after (V6) vitamin D supplementation or placebo and stimulated with LPS for 48 hours. IL-6, IL-10, and IL-12p40 production was determined by using an ELISA. The fold change from V6 to V3 (V6/V3) for each sample pair was calculated, and individual data points and lines representing means ± SDs are shown. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Participant flow. Participants in the VIDA trial were approached for optional enrollment in this substudy. Samples from the first 100 eligible participants consenting to the study were collected at each participating site and shipped to the research laboratories at Washington University (T-cell studies) or Brigham and Women's Hospital (myeloid cell studies). Because the sample requirements were different between laboratories, a sample might have been usable at one site but not the other, accounting for the different numbers available for final analysis. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Intracellular cytokine analysis of CD4+ T cells. Shown are individual data points for intracellular cytokine staining of CD4+ T cells at V3 and V6, which were used to generate the fold change data presented in Fig 1. Samples were obtained before (V3) and after (V6) 12 weeks of treatment with placebo or vitamin D as described for Fig 1 in the body of the article. There was no effect of vitamin D repletion on the percentage of CD4+ T cells expressing IL-4, IFN-γ, IL-10, or IL-17A. There was a small but statistically significant decrease in the percentage of IL-4–secreting cells in the placebo-treated group (P = .03) but no change in the ratio of TH1 to TH2 cells and no difference in the fold change of IL-4–secreting cells from V6 to V3 (see Fig 1). The TH1/TH2 ratio was calculated by dividing the percentage of CD4/IFN-γ–positive by the percentage of CD4/IL-4–positive cells. Data shown are individual sample values, as well as means ± SDs for the group. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 Cytokine secretion of stimulated PBMCs. Shown are individual values used to determine the fold change in cytokine secretion from V3 to V6 in Fig 2, A. PBMCs were isolated from samples collected either before (V3) or after (V6) vitamin D supplementation or placebo and stimulated with α-CD3 and α-CD28 for 48 hours. Secreted cytokines were analyzed in the culture supernatant by using a cytokine bead array or ELISA (TGF-β only). Data shown are individual sample values, as well as means ± SDs for the group. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Monocyte cytokine production is not altered by vitamin D supplementation. Shown are individual values used to determine the fold change in cytokine secretion from monocytes shown in Fig 2, B. CD14+ monocytes were isolated from PBMCs from vitamin D–deficient asthmatic patients before (V3) and after (V6) vitamin D supplementation or placebo and stimulated with LPS for 48 hours. IL-6, IL-10, and IL-12p40 production was determined by means of ELISA. Data shown are individual sample values, as well as means ± SDs for the group. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Gating strategy for DC subsets. Live PBMCs were forward versus side scatter gated. We gated on the lineage Lin1 (CD3, CD14, CD16, CD19, CD20, and CD56)–FITC negative population (Lin−). Next, We then gated on the HLA-DR positive population. Myeloid DCs were identified by plotting HLA-DR versus CD11c. The myeloid DC population was identified as the Lin−HLA-DR+CD11c+ population. Plasmacytoid DCs were identified by plotting HLA-DR versus CD123. Plasmacytoid DCs were identified as the Lin−HLA-DR+CD123+ population. Isotype controls were used to determine background levels. Rectangular gates indicate selected population. Please see representative plots below. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 Gating strategy for monocyte subsets. Live PBMCs were gated on forward versus side scatter. We then gated on the CD14+ population (monocytes). The 3 human peripheral blood monocyte subsets (CD14+CD16−, CD14+CD16+, and CD14lowCD16+) were identified by quadrants on the CD14 versus CD16 plot. Isotype controls were used to determine background levels. See representative plots below. Rectangular gates indicate the selected population. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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