We have shown for the first time that children with STRA have significantly lower serum 25(OH)D3 levels than do children with MA. Lower serum 25(OH)D3 levels were associated with worse lung function, poor asthma control, and more steroid use in MA and STRA. Within STRA, low 25(OH)D3 levels were associated with increased ASM mass, but not with other parameters of airway remodeling, nor with airway inflammation despite an association with aeroallergen sensitization.
Importantly, the children in this study with STRA had been carefully assessed such that their basic management had been optimized, and any “difficult asthmatics” (whose asthma was uncontrolled because of modifiable factors such as poor adherence to treatment) had been excluded. The detailed multidisciplinary assessment to ensure as far as possible that basic management is correct is one of the novel features of this study.
In this group with STRA, a significant negative association was present between volume fraction of ASM and 25(OH)D3
levels. Of note, however, there was no association between RBM thickness or epithelial shedding and vitamin D. Although a negative relationship between ASM mass and lung function has been reported in pediatric cases with difficult asthma (10
), this is the first demonstration of an association between low serum 25(OH)D3
, poor lung function and asthma control, increased BDR and ASM mass. It is therefore plausible that the link between ASM mass and lung function in severe asthma may partly be explained by low 25(OH)D3
levels. The association between increased ASM mass and low 25(OH)D3
is supported by in vitro
studies, which have shown that vitamin D inhibits smooth muscle proliferation (11
). Vitamin D blocked smooth muscle proliferation in a concentration-dependent manner in human smooth muscle cells sensitized with asthmatic serum (12
), and it inhibited ASM cell proliferation by preventing progression of the cell cycle, not by inducing apoptosis (13
). Furthermore, vitamin D inhibits cell growth in muscle cell cultures (13
). Moreover, vitamin D increases glucocorticoid bioavailability in bronchial smooth muscle cells (25
). In contrast to the published in vitro
studies, there was no relationship between serum 25 (OH)D3
levels and ASM proliferation assessed by myocyte PCNA staining in our subjects. However, all in vitro
work has been done in adult ASM, and mechanisms may be different in children. For example, ASM apoptosis may be reduced. Importantly, ASM mass is still increasing as part of normal growth and development in children (26
), and therefore the influence of superimposed pathological abnormalities is likely to be different from those in adults. Further work is needed to determine the mechanistic effects of 25(OH)D3
on pediatric ASM.
The cross-sectional nature of the biopsy data prevent us from being certain whether the relationship between increased ASM mass and vitamin D is a result of severe asthma, or whether the increased ASM mass may have been present before the development of disease and caused the asthma. It is possible that a developmental structural defect of the airway wall, such as ASM hypertrophy in children with STRA, results from vitamin D deficiency in utero
, and that may have led to asthma in the first place. It may be that exaggerated ASM hypertrophy is a cause of their asthma, rather than a consequence, as a result of in utero
) and postnatal vitamin D deficiency. The effects of vitamin D deficiency in utero
) could be in addition to, or independent of, airway remodeling. This is especially important as a randomized controlled trial of vitamin D therapy in children with STRA could potentially reverse the ASM hypertrophy and change the course or natural history of these patients’ asthma. However, if vitamin D induces a smooth muscle developmental defect in utero
, then it may prove more challenging to reverse. Importantly, it should be noted that in a previous study of infants with severe wheeze at a median age of 1 year (29
), there was no increase in ASM mass on biopsy (30
). Vitamin D levels were not measured in that study, but the findings mitigate against, although do not exclude, the developmental hypothesis.
Interestingly, we did not find an association between any of the inflammatory cells quantified (eosinophils, neutrophils, or mast cells) and serum 25(OH)D3 levels. This remained true for both luminal inflammation (BAL and sputum) and tissue inflammation (endobronchial biopsy). It is possible that the substantial antiinflammatory treatment prescribed for these children may have masked a relationship between vitamin D and airway inflammation in STRA. Having established a link between serum 25(OH)D3 levels and lung function and asthma control, and importantly, having now seen a novel link between serum vitamin D levels and airway smooth muscle alone, we suggest that vitamin D supplementation in children with STRA and low 25(OH)D3 levels may be a novel therapeutic target directed against some aspects of remodeling. Of note, there are currently no treatments that inhibit or prevent airway remodeling.
Even after adjusting for confounding factors including age, sex, body mass index, FEV1
, and ethnicity, a significant relationship between serum vitamin D levels and asthma control, exacerbations, inhaled and oral steroid use, and positive BDR remained (). Some of our findings concur with reports in children and adults with much less severe asthma. These include the associations found between low 25(OH)D3
levels and asthma control and exacerbations (5
), lower lung function (6
), increased reversibility to bronchodilator (5
), and greater antiinflammatory medication (inhaled corticosteroid, oral steroid, and leukotriene receptor antagonist) use (5
SERUM VITAMIN D LEVELS AND DISEASE SEVERITY IN SUBJECTS WITH MODERATE ASTHMA AND SUBJECTS WITH SEVERE THERAPY-RESISTANT ASTHMA
Although total serum IgE levels and specific IgE to cat, dog, pollen, Dermatophagoides pteronyssinus
, and Aspergillus fumigatus
were inversely related to 25(OH)D3
levels, there was no relationship between 25(OH)D3
and serum, BAL, or biopsy eosinophils. Some (5
), but not all (6
), investigators have found correlations between lower 25(OH)D3
levels and markers of allergy in childhood asthma. A U-shaped association between low and high 25(OH)D3
levels and serum total IgE levels has been demonstrated (34
). An association between 25(OH)D3
deficiency and increased sensitization to 11 of 17 environmental and food allergens in children (n = 3,136), but not in adults (n = 3,454), in the National Health and Nutrition Examination Survey has also been shown (35
). Our results are in agreement with this report (35
) that children with low serum 25(OH)D3
levels are more likely to have allergic sensitization. Interestingly, we have only found an association between sensitization to aeroallergens and 25(OH)D3
levels, but not food allergens.
In terms of ethnicity, because numbers were small we allocated the children into “white” and “nonwhite” groups, but did not attempt to classify ethnicity in more detail. There were more nonwhite children in the STRA group (36%) compared with the MA group (16%) and control subjects (12%) (P
= 0.056). Children with nonwhite skin had lower serum vitamin D levels (Figure E3A
), as reported by others (4
). Moreover, nonwhite children had more severe asthma compared with white children (Figure E4
). However, the ANOVA results have shown that there is no interaction between ethnicity and disease severity for levels of vitamin D (P
= 0.20). This could be due to the small number of subjects in the study. Importantly, race is a proxy for skin color, and although skin color is a proxy for low vitamin D levels, our conclusions relate to the relationship between low serum vitamin D levels, whether driven by diet, sunlight, ethnicity or another factor, and asthma severity and pathology.
The cross-sectional design of this study made it impossible to determine whether low 25(OH)D3 levels result in severe asthma in children, or whether children with severe asthma have low 25(OH)D3 levels because, for example, they are unable to go outside and exercise normally. Also, because of the ethics of performing bronchoscopy for research in children, we could not perform invasive endobronchial biopsies in control subjects and subjects with MA, and this is a potential source of information bias. One of the other limitations of this study was that BDR and ACT were not performed in normal control subjects. Although the potential to correlate ASM mass with the results of airway challenge would be of interest, given the subjects’ disease severity, this was thought to be both unsafe and unethical.
It is challenging to propound a unifying hypothesis to account for ASM changes being the sole manifestation of vitamin D deficiency. In terms of remodeling, we speculate that this may be due to a heightened sensitivity of ASM to vitamin D deficiency as compared with other airway wall components. The lack of any effect on inflammation may in part be due to high-dose inhaled and oral steroid therapy. Although vitamin D deficiency causes a degree of steroid insensitivity, the high doses used in the children with STRA may have overcome this. However, we acknowledge that these ideas remain speculative at the present time and the determination of the exact mechanism between low 25(OH)D3 and airway remodeling in STRA will require intervention studies.
In summary, in this cross-sectional study, children with genuine severe asthma had significantly lower serum vitamin D levels than children with MA and control subjects. Lower serum vitamin D levels were associated with worse parameters of asthma severity, and we propose that a contributory mechanism may be via an effect on ASM. As numbers are small, and represent a selected population, the conclusions drawn must be tentative. However, these findings suggest that detecting and treating low serum vitamin D levels in children with STRA may aid in the treatment of specific structural airway changes.