Discussion
Prevention of respiratory infections by vitamin D3 supplementation is controversial. Some reports show that vitamin D3 supplementation reduces the risk of developing respiratory infections34 while others do not show such protection.35 To our knowledge, this is the first in vivo study demonstrating that vitamin D3 supplementation increases baseline human ASL antimicrobial activity using a standardised sample and inoculum.
In our cohort, vitamin D3 supplementation for 90 days increased 25(OH)D3 serum concentration and increased ASL antimicrobial activity. To explain the later result, we presume that as calcium concentration increases several negative feedback loops tightly regulate vitamin D3 activation. For example, increased 1,25(OH)2D3 concentrations decreases 1-α hydroxylase and increases 24-hydroxylase, which is responsible for inactivation of both 25(OH)D3 and 1,25(OH)2D3.33
In addition, we found that ASL samples matched with a deficient vitamin D3 serum concentration had significantly lower antimicrobial activity compared with samples paired with sufficient vitamin D3. These results are consistent with epidemiological studies that report that low vitamin D3 levels increase the susceptibility to respiratory infections.11–14
We speculate that vitamin D3 increases the ASL antimicrobial activity by upregulating gene expression of antimicrobial peptides like cathelicidins.15 17–19 Since we did not find any difference in CAMP gene expression in macrophages, we hypothesised that the airway epithelium was responsible for the increase in ASL antimicrobial activity due to vitamin D3. Since we were limited by the volume and concentration of the samples, we took an indirect approach to confirm our hypothesis. As a proof of concept, we used an LL-37 antibody to neutralise the effect of LL-37 in the ASL.23 30 We found that the improvement in ASL antimicrobial activity after vitamin D3 supplementation was reversed when the ASL was treated with an LL-37 antibody. This effect can be in part due to both functional inhibition of this antimicrobial peptide and/or decreased synergism between LL-37 and other antimicrobial peptides such as hBDs.22 Future studies should consider sampling more volume of ASL to be able to measure directly AMPs. Nonetheless, He et al found that Vitamin D3 supplementation for 14 weeks increased the concentration of cathelicidin in both plasma and saliva in healthy subjects.36
We also did a subgroup analysis on the subjects who were smokers, and found compelling results that suggest that they did not increase their ASL antimicrobial activity after vitamin D3 supplementation. We acknowledge that our sample size was small and we might be underpowered to detect the difference. However, our in vitro study showing that human airway epithelial cells exposed to CSE in vitro had decreased cathelicidin gene expression in response to 25(OH)D3 strongly supports the feasibility of our in vivo finding, despite lack of power. In addition, it has been reported that cigarette smoke exposure is negatively correlated with LL-37 in human saliva.37 Furthermore, patients with COPD who have high risk of frequent exacerbations also have decreased plasma levels of LL-37 compared with control subjects.38 These results warrant further evaluation in larger sample sizes.
Our in vitro results also suggest that one of the mechanisms responsible for this finding is that cigarette smoke reduces gene expression coding for the enzyme responsible for converting vitamin D3 into its active form. This is consistent with a prior report that exposure to cigarette smoke was associated with impaired ability of human airway epithelia to hydroxylate 25(OH)D3 to 1,25(OH)2D3.7 When we used the active form of vitamin D3, we found a similar increase in cathelicidin gene expression regardless of CSE exposure. Heulens et al also reported that human macrophages exposed to CSE had a similar increase in cathelicidin gene expression in response to 1,25(OH)2D3compared with unexposed cells.39
Given that cigarette smoke exposure (1) decreased cathelicidin gene expression in response to 25(OH)D3, (2) decreased CYP27B1 gene expression and (3) did not affect the cathelicidin gene expression response to active vitamin D3, we propose that one mechanism by which CSE impairs airway innate immunity is by decreasing local vitamin D3 activation.
Another plausible explanation for decreased response to vitamin D3 in the smokers is different processing of hCAP18, the parent molecule of LL-37 before being processed or LL-37 itself. Smoking increases the concentration of proteolytic proteins such as cathepsin D and neutrophils elastase.40 41 Proteolysis by these enzymes can decrease LL-37 in the airways and its antimicrobial activity.23
Furthermore, although cathelicidin is a relevant antimicrobial peptide, other antimicrobials that have a vitamin D response element like hBDs might be also increased by vitamin D3 supplementation.42 Herr et al reported that airway epithelium exposed to smoke significantly reduced hBD-2 and antimicrobial activity in vitro. In addition, former or current smokers with pneumonia had decreased concentration of hBD-2 in pharyngeal washing fluid compared with non-smokers.43
Our study has several strengths, including analysing ASL samples from the same subjects before and after randomised intervention. We also recognise that we have several limitations such as the small number of samples in the subgroup analysis and the indirect approach to assess the role of LL-37 in the ASL antimicrobial activity using a neutralising antibody. Finally, it is conceivable that smokers require even higher doses of vitamin D3 and/or a longer duration of treatment.
Conclusions
We provide evidence that vitamin D3 supplementation for 90 days increases ASL antimicrobial activity in non-smoking humans. We presume that this effect occurs via an increase in ASL AMPs. Conversely, smoking participants did not improve their ASL antimicrobial activity, and we propose that the mechanism is, in part, due to impaired conversion of vitamin D3 to its active form leading to a decreased expression of airway AMPs. Future studies with a higher number of subjects are required to confirm the effect modification of smoking in the ASL antimicrobial activity in response to vitamin D3 in vivo, as well as the effect of active vitamin D3 in the airway antimicrobial activity of smokers.