Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease

Abstract

Background: Associations between vitamin D status and childhood asthma are increasingly reported, but direct causation and mechanisms underlying an effect remain unknown. We investigated the effect of early-life vitamin D deficiency on the development of murine neonatal allergic airways disease (AAD).

Methods: In utero and early-life vitamin D deficiency was achieved using a vitamin D-deficient diet for female mice during the third trimester of pregnancy and lactation. Offspring were weaned onto a vitamin D-deficient or vitamin D-replete diet, and exposure to intranasal house dust mite (HDM) or saline was commenced from day 3 of life for up to 6 weeks, when airway hyper-responsiveness (AHR), airway inflammation and remodelling were assessed.

Results: Neonatal mice that had in utero and early-life vitamin D deficiency had significantly increased pulmonary CD3(+) CD4(+) T1ST2(+) cells and reduced CD4(+) IL-10(+) cells. This effect was enhanced following HDM exposure. AHR in HDM-exposed mice was unaffected by vitamin D status. Introduction of vitamin D into the diet at weaning resulted in a significant reduction in serum IgE levels, reduced pulmonary eosinophilia and peri-bronchiolar collagen deposition.

Conclusion: Peri-natal vitamin D deficiency alone has immunomodulatory effects including Th2 skewing and reduced IL-10-secreting T regulatory cells, exaggerated with additional allergen exposure. Vitamin D deficiency in early life does not affect AHR, but contributes to disease severity with worse eosinophilic inflammation and airway remodelling. Importantly, supplementation with vitamin D improves both of these pathological abnormalities.

Keywords: animal models; asthma; eosinophils; lymphocytes; paediatric.

© 2014 The Authors. Allergy Published by John Wiley & Sons Ltd.

Figures

Figure 1

Early-life vitamin D insufficiency has no effect on airway resistance in pups at 6 weeks of age. Schematic representation of experimental protocol (A). Pregnant female Balb/c mice were fed a vitamin D-deficient diet from day-16 gestation or remained on a normal chow diet. Pups were challenged with house dust mite (HDM) or PBS for 6 weeks. Pups were weaned at 3 weeks of age and either fed a vitamin D-deficient diet or a normal chow diet. At 6 weeks of age, lung function was measured by the forced oscillation technique 4 h after last challenge of PBS (B) or HDM (C). + represents vitamin D intake and - represents a lack of vitamin D. Data are expressed as mean and SEM, n = 4–7.

Figure 2

Introduction of dietary vitamin D after in utero and early-life insufficiency can reduce eosinophilic lung inflammation. Pregnant female Balb/c mice were either fed a vitamin D-deficient diet from day-16 gestation or remained on a normal chow diet. Pups were exposed to house dust mite (HDM) or PBS and weaned on to either a vitamin D-deficient diet or a normal chow at 3 weeks of age. At 6 weeks of age, both total and eosinophil numbers in the BAL (A) and lung (B) were counted. + represents vitamin D intake; - represents a lack of vitamin D. Lung sections stained with haematoxylin and eosin from HDM-exposed pups with in utero vitamin D insufficiency (C). Postweaning vitamin D insufficiency (left) and postweaning vitamin D sufficiency (right) are shown. Data are expressed as box and whisker plots; horizontal bar represents median, n = 9–16 combined from 2 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney U-test. Significance refers to HDM and associated PBS control where no line is indicated.

Figure 3

In utero and early-life vitamin D insufficiency skews towards a Th2 environment and reduces T regulatory cells in the lungs of pups. Pregnant female Balb/c mice were fed a vitamin D-deficient diet from day-16 gestation or remained on a normal chow diet. Pups were challenged with house dust mite (HDM) or PBS for 6 weeks. Pups were weaned at 3 weeks of age and either fed a vitamin D-deficient diet or a normal chow. Lung cells were harvested 4 h after last challenge and analysed by flow cytometry. T-cell subsets were defined as CD3+CD8+ (A), CD3+CD4+ (B), CD3+CD4+T1ST2+ (C) and CD3+CD4+IL-10+ (D). + represents vitamin D intake; - represents a lack of vitamin D. Data are expressed as box and whisker plots; horizontal bar represents median, n = 4–6. *P < 0.05, **P < 0.01 by Mann–Whitney U-test. Significance refers to HDM and associated PBS control where no line is indicated.

Figure 4

Introduction of dietary vitamin D after in utero and early-life insufficiency can reduce IgE in pups at 6 weeks of age. Pregnant female Balb/c mice were fed a vitamin D-deficient diet from day-16 gestation or remained on a normal chow diet. Pups were challenged with house dust mite (HDM) or PBS for 6 weeks. Pups were weaned at 3 weeks of age and either fed a vitamin D-deficient diet or a normal chow. Total IgE and HDM-specific IgE (A) antibodies were measured in the serum of pups by ELISA. The cytokines IL-13, IL-5, IL-10 and IL-4 (B) were measured in the lung of pups by ELISA. + represents vitamin D intake; - represents a lack of vitamin D. Data are expressed as box and whisker plots; horizontal bar represents median, n = 9–16 combined from 2 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney U-test. Significance refers to HDM and associated PBS control where no line is indicated.

Figure 5

Introduction of dietary vitamin D after an in utero and early-life insufficiency reduces house dust mite (HDM)-induced lung collagen. Pregnant female Balb/c mice were fed a vitamin D-deficient diet from day-16 gestation or remained on a normal chow diet. Pups were challenged with HDM or PBS for 6 weeks. Pups were weaned at 3 weeks of age and either fed a vitamin D-deficient diet or a normal chow. At 6 weeks of age, lung sections were collected from pups 4 h after last challenge. Periodic acid–Schiff (PAS) staining was used to determine goblet cell hyperplasia in the lungs using a semiquantitative scoring system (A). Collagen deposition in the lungs of pups was measured using the Sircol assay for total lung collagen (B) and by image quantification to measure peri-bronchiolar-associated collagen (C). Lung sections stained with Sirius red (D); + represents vitamin D intake; - represents a lack of vitamin D. Data are expressed as box and whisker plots; horizontal bar represents median, n = 9–16 combined from 2 experiments. *P < 0.05, **P < 0.01 by Mann–Whitney U-test. Significance refers to HDM and associated PBS control where no line is indicated.