1,25(OH)₂D₃ and TX527 reduce IFN-γ, IL-4, and IL-17 but increase IL-10 in expanded human CD4⁺CD25^highCD127^low T cells.

<p>Peripheral blood CD3⁺ T cells from control donors (n = 5) or type 1 diabetes patients (n = ) were cultured for 8 days in the presence of 10^-8 M 1,25(OH)₂D₃ (1,25D₃) or TX527 or corresponding concentration of vehicle (CTR). CD4⁺CD25^highCD127^low T cells were sort-purified and mRNA expression of IFN-γ, IL-4, IL-17, and IL-10 was quantified by real-time RT-PCR using B2M and RPL27 as normalization genes. Bar graphs represent the mean ± SEM. Significance was tested using a two-tailed Mann-Whitney test. *<i>P</i><0.05; **<i>P</i><0.01. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃- or TX527-exposed human T cells from control donors and type 1 diabetes patients can suppress autologous CD4 and CD8 T cell responses.

<p><b>A, B</b>: CFSE-labeled responder cells from control (Control, n = 5–7) and type 1 diabetes (T1D, n = 7–10) donors were stimulated for 4 days with anti-CD3/CD28 mAbs and co-cultured with autologous unsorted CD4⁺ (<b>A</b>) or sorted CD4⁺CD25^highCD127^low (<b>B</b>) T cell populations (day 8) from control-, 1,25(OH)₂D₃- or TX527-treated cultures, as indicated. Shown are bar graphs summarizing the percentage suppression of proliferation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109194#s2" target="_blank">Methods</a> section) of CD4⁺ (top) and CD8⁺ (bottom) T cells without or with Tregs at a 2∶1 (in case of unsorted CD4⁺ T cells, <b>A</b>) or 1∶1 (in case of sorted CD4⁺CD25^highCD127^low T cells, <b>B</b>) Treg:Tresponder ratio. Significance was tested using a two-tailed Mann-Whitney test, all not significantly different. * <i>P</i><0.05. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 reduce T helper cytokines in human T cell cultures.

<p>Human peripheral blood CD3⁺ T cells, isolated from control subjects (n = 19) and type 1 diabetes patients (n = 20), were activated using anti-CD3/CD28 and treated with vehicle (CTR), 10⁻⁸ M 1,25(OH)₂D₃ (1,25D₃) or 10⁻⁸ M TX527. Concentrations of indicated cytokines were determined in the supernatant of day 8 cultures. Results are shown as bar graphs of mean ± SEM, data are grouped per treatment and donor type. Significance was calculated using a two-tailed Mann-Whitney test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 trigger a stable Treg phenotype in T cells from human type 1 diabetic patients.

<p>T cells from control subjects (Control) or type 1 diabetes patients (T1D) were cultured in the presence of vehicle (CTR; white boxes), 10⁻⁸ M 1,25(OH)₂D₃ (1,25D₃, grey boxes) or 10⁻⁸ M TX527 (black boxes). On day 6, the T cell cultures were exposed to normal T cell medium (left panel: <b>A-D</b>) or a cytokine cocktail (right panel: <b>E-H</b>) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109194#s2" target="_blank">methods</a> section. T cells were harvested 48 h later and stained for flow cytometry. Box and Tukey wisker plot summarizes the frequencies of positive cells in the CD4+ T cell gate. <b>A</b>: Surface expression of OX-40 (CD134) by activated CD4⁺ T cells of control donors (Control, n = 43) or type 1 diabetes patients (T1D, n = 58). <b>B</b>: Frequency of CD25^highCD127^low cells in the CD4⁺ T cell gate from control subjects (Control, n = 4) and type 1 diabetes patients (T1D, n = 7). CTLA-4 (<b>C</b>) and FOXP3 (<b>D</b>) expression in CD4⁺CD25^highCD127^low T cells of control donors (Control, n = 28) and type 1 diabetes patients (T1D, n = 45). <b>E</b>: Frequency of OX-40 expression on CD4⁺ T cells from control subjects (Control, n = ) or type 1 diabetes patients (T1D, n = 7) after additional stimulation with a cytokine cocktail. <b>F</b>: Frequency of CD25^highCD127^low cells in CD4⁺ T cells. CTLA-4 (<b>G</b>) and FOXP3 (<b>H</b>) expression in the CD4⁺ CD25^highCD127^low T cell gate. Data are grouped per donor type and treatment, cross-bars indicate median ± SEM. Significance was calculated using a two-tailed Mann-Whitney test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. All other comparisons were not significantly different.</p

Additional file 1: of Vitamin D deficiency exacerbates COPD-like characteristics in the lungs of cigarette smoke-exposed mice

The effect of vitamin D deficiency on additional lung function parameters in air- and CS-exposed mice after 6 and 12 weeks. (PDF 96 kb

Effect of 1,25(OH)₂D on the <i>ex vivo</i> inflammatory response of alveolar macrophages from non-smoking and smoking subjects.

<p>Alveolar macrophages from non-smokers (n = 5) or smokers (n = 5) were stimulated for 48h with 10 nM 1,25(OH)₂D or vehicle, followed by an additional stimulation for 24h with LPS/IFN-γ. Levels of (A) IL-8, (B) TNF-α, (C) MCP-1 and (D) IL-6 were measured in supernatants of alveolar macrophage cultures. mean±SEM. *p<0.05, ***p<0.001.</p

Effect of CSE on vitamin D metabolism in THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. mRNA expression levels of (A) VDR, (B) CYP27B1 and (C) CYP24A1 were determined in cell lysates. mean±SEM. *p<0.05, ***p<0.001</p

Effect of 1,25(OH)₂D on antibacterial response in CSE-treated THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% or 25% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. (A) Phagocytosis and (B) oxidative burst by THP-1 macrophages following bacterial challenge. Graphs show the percentage of alveolar macrophages that have internalized <i>E</i>. <i>coli</i> bacteria (A) or have produced reactive oxygen species (B). (C) mRNA and protein levels of cathelicidin were determined in cell lysates and culture supernatants, respectively. Independent experiments were performed in triplicate. mean±SEM. **p<0.01, ***p<0.001, ****p<0.0001.</p

Effect of 1,25(OH)₂D on the inflammatory response in CSE-treated THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. mRNA and protein levels of (A) IL-8, (B) TNF-α and (C) MCP-1 were determined in cell lysates and culture supernatants, respectively. Independent experiments were performed in triplicate. mean±SEM. *p<0.05, **p<0.01, ****p<0.0001.</p

1,25-Dihydroxyvitamin D Modulates Antibacterial and Inflammatory Response in Human Cigarette Smoke-Exposed Macrophages

<div><p>Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)₂D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)₂D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)₂D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)₂D did not improve defects in phagocytosis of <i>E</i>. <i>coli</i> bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)₂D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.</p></div