Figure 1.
Low levels of vitamin D3 in postmenopausal women.
Serum vitamin D3 levels in postmenopausal women are low compared to premenopausal women, P<0.05 (Mann-Whitney test). Individual data are presented with median levels (56 nmol/L and 74 nmol/L). The recommended level of 75 nmol/L is marked with a dotted line.
Figure 2.
25D3 and cathelicidin in serum and bladder tissue of women during a three-month period of 25D3 supplementation.
Postmenopausal women were treated with 2000 units of oral 25D3 daily for 12 weeks. Serum samples were collected before, at 6 and 12 weeks. Serum 25D3 levels increased from a baseline median value of 68.5 nmol/L to 104.5 nmol/L at 6 weeks, P<0.001 (Wilcoxon rank test), and 117 nmol/L at 12 weeks, P<0.01 (Wilcoxon rank test). The dotted line denotes the recommended serum 25D3 level of 75 nmol/L and median values are indicated (A). Despite treatment with 25D3, serum cathelicidin levels did not increase (B). E. coli-infected bladder biopsies from women receiving 25D3 supplementation responded with higher expression of CAMP mRNA, P<0.05 (Mann-Whitney test) (C) and cathelicidin protein, P <0.05 (Mann-Whitney test) (D) compared to infected pieces prior to supplementation. Fluorescence immunohistochemistry showed increased cathelicidin protein expression in infected bladder biopsies. Tissue samples shown are from the same patient before (E) and after (F) 12 weeks of 25D3 treatment. Cathelicidin is localized in the uroepithelium including the larger umbrella cells closest to the bladder lumen. Staining without primary antibody serves as negative control (G). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm. Cathelicidin is stained with AlexaFluor 594 (red), the cell nucleus with DAPI (blue).
Figure 3.
Bladder epithelial cells are equipped with the machinery for local 25D3 conversion and activity.
Expression of CYP27B1, VDR and CYP24A1 was detected on mRNA level in normal bladder cells (HBEP) and three bladder cells lines (TERT-NHUC, 5637 and T24) by RT-PCR (A, C, E) and on protein level in tissue from bladder biopsies by fluorescence immunohistochemistry (B, D, F). mRNA expression results were normalized to expression in the kidney epithelial cell line A498. The proteins are stained with AlexaFluor 594 (red), the cell nucleus with DAPI (blue). CYP27B1 protein was detected in the bladder mucosa where it was evenly distributed in the bladder epithelial layers with an abundance of staining seen in the peri-nuclear area, consistent with its association to mitochondria (B). VDR protein was localized in the cytoplasm and nucleus (D). Low levels of the CYP24A1 protein were detected in bladder mucosa (F). All images are captured at ×63 magnification and the right panels of images B, D and F show controls stained without primary antibodies against CYP27B1, VDR and CYP24A1, respectively.
Figure 4.
Human bladder cells respond with CYP24A1 and CAMP induction to 1,25D3 and 25D3.
Normal bladder cells (HBEP, upper panel) and TERT-NHUC bladder cells (lower panel) were stimulated with 10−7 M 25D3 or 10−8 M 1,25D3 for 24 hours. Levels of gene-specific mRNA for CYP24A1 (A, B), CAMP (C, D), VDR (E, F) and CYP27B1 (G, H) in stimulated cells were determined and are presented as fold change compared to vector-treated control cells. Data presented are pooled from three independent experiments and shown as mean and standard deviation; ***P<0.001, **P<0.01, *P<0.05 (ANOVA with Dunnett's post-test).
Figure 5.
1,25D3 or 25D3 in combination with E. coli increased cathelicidin protein in bladder epithelial cells.
Production of cathelicidin increased in TERT-NHUC bladder cells after treatment with 1,25D3 or 25D3, and was further increased by infection with E. coli CFT073 (UPEC). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm (A). The control image (Neg control) is shown in the right upper panel. Fluorescence intensity was quantified and is presented as mean values with standard deviation; ***P<0.001, *P<0.05 (ANOVA with Dunnett's post-test) (B). An increase in cathelicidin protein was also recorded by flow cytometry in E. coli-infected TERT-NHUC bladder cells treated with 1,25D3 or 25D3. Grey is vector +E. coli, red is 1,25D3 10−8 M+E. coli and black is 25D3 10−7 M+E. coli (C).
Figure 6.
Vitamin D increases the cathelicidin-mediated antibacterial effect of bladder epithelial cells.
Cell-associated UPEC are killed as determined with Live/Dead staining where live bacteria are stained green and dead bacteria are stained red (A). UPEC attach and invade bladder cells and are colocalized with cathelicidin. GFP-labelled E. coli CFT073 is green and cathelicidin is stained with AlexaFluor 594 (red); regions of colocalization are yellow (B). The antibacterial effect induced by 1,25D3 was measured in medium from TERT-NHUC, 5637 and T24 bladder cells after stimulation for 48 hours. E. coli CFT073 was incubated in conditioned medium from treated cells for 90 minutes (Supernatant). Cells from the same experiment were harvested and lysed (Cell lysate). The resulting bacterial count is shown as percentage of vector treated control cells. Mean values with standard deviation are shown from three independent experiments; ***P<0.001, **P<0.01 and *P<0.05 (ANOVA with Dunnett's post-test) (C). Adding a neutralizing anti-cathelicidin antibody (α-cathelicidin ab) to the conditioned medium from TERT-NHUC bladder cells blocked the antibacterial effect, whereas conditioned medium in the presence of an irrelevant antibody maintained its antibacterial activity. Mean values with standard deviation are shown from four independent experiments; *P<0.05 (ANOVA with Dunnett's post-test) (D).