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

Primers used for quantitative PCR analysis.

<p>Primers used for quantitative PCR analysis.</p

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

<p>Alveolar macrophages from non-smokers or smokers were stimulated for 48h with 10 nM 1,25(OH)₂D or vehicle. (A) Phagocytosis and (B) oxidative burst by alveolar macrophages from non-smokers (n = 7) and smokers (n = 11) 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) Cathelicidin levels were measured in supernatants of alveolar macrophage cultures from non-smokers (n = 5) and smokers (n = 5). mean±SEM. *p<0.05, **p<0.01.</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

Quantification and visualization of MIF and CD74-expressing cells in the pancreas of NOD mice.

<p>Quantification of the relative MIF and CD74 mRNA levels in the total pancreas of control and NOD mice by means of real-time reverse transcription PCR (RT-PCR) (Mean ± SEM; n = 4–5) (A &B) (*: p-values ≤ 0.05). (C) Pancreatic sections of NOD mice were stained for insulin (white), MIF (green), F4/80 (red) and DNA by DAPI (blue) and analyzed with confocal microscopy at ×40 original magnification. Cells featuring a co-localization of MIF and the macrophage marker F4/80 are indicated by the white arrows. (D) Confocal microscope image of pancreatic sections of NOD mice were stained for insulin (white), CD74 (green), F4/80 (red) and DNA by DAPI (blue) (×40 original magnification). The images depicted shows one representative microphotograph documenting the presence of multiple MIF⁺- or CD74⁺ cells within the pancreatic islets of 8-week-old NOD mice. Similar results were obtained with 12-week-old acutely diabetic NOD mice (not shown). (E) Quantification of the percentage of F4/80⁺CD11b⁺ macrophages within the live leukocyte gate (mean ± SEM; n = 4–5) (*: p-values ≤ 0.05). (F) Quantification of the percentage of CD74⁺ cells within the F4/80⁺CD11b⁺ macrophage population as determined on homogenized pancreas samples by flow cytometry (mean ± SEM; n = 4–5).</p

ISO-1-treated NOD- or C57BL/6-macrophages modulate T cell activation <i>in vitro</i>.

<p>(A-C) Ctr- or ISO-1-treated macrophages isolated from either C57BL/6 or NOD mice (5 × 10⁴ cells/well) were washed before addition of OVA_323-339 peptide or BDC2.5 mimotope (1 μg/mL) and culturing together with negatively isolated CD4⁺ T cells from OT-II or BDC2.5 Tg mice (1 × 10⁵ cells/well). The activation status of the co-cultured T cells was measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of CD44 and CD69 expression on viable CD4⁺ T cells after 3 days in co-culture (mean ± SEM) (n = 4) (*, p < 0.05; **, p < 0.01, ***p≤0.005). (C) To identify naïve T cells (defined as CD44^lowCD62L^high), memory T cells (defined as CD44^highCD62L^high) and effector T cells (CD44^highCD62L^low), multi-color flow cytometry was performed and the frequencies of these populations were quantified. (C) In CD4⁺ cells, the percentages of effector T cells significantly decreased in the presence of ISO-1-conditioned macrophages, while naïve T cells remained prominently visible compared with the control condition. A p-value < 0.05 indicate significant differences between groups (n = 4). The data shown are representative of four independent experiments.</p