Prenatal 25-hydroxyvitamin D deficiency affects development of atopic dermatitis via DNA methylation

Abstract

Despite epidemiologic evidences for the significance of cord blood (CB) 25-hydroxyvitamin D (25[OH]D) in atopic dermatitis (AD),1Baiz N. Dargent-Molina P. Wark J.D. Souberbielle J.C. Annesi-Maesano I. Group E.M.-C.C.S. Cord serum 25-hydroxyvitamin D and risk of early childhood transient wheezing and atopic dermatitis.J Allergy Clin Immunol. 2014; 133: 147-153Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar the underlying mechanism is not well understood. Recent studies have reported that epigenetic gene coding for a histone demethylase is induced by vitamin D (vitD).2Pereira F. Barbachano A. Silva J. Bonilla F. Campbell M.J. Munoz A. et al.KDM6B/JMJD3 histone demethylase is induced by vitamin D and modulates its effects in colon cancer cells.Hum Mol Genet. 2011; 20: 4655-4665Crossref PubMed Scopus (131) Google Scholar However, no previous studies have determined whether CB 25[OH]D deficiency contributes to AD development in offspring through epigenetic mechanisms, nor have any measured the vitD levels during the prenatal and postnatal periods in a large pediatric cohort. We here investigated the role of CB 25[OH]D deficiency in comparison with postnatal 25[OH]D levels in the development of AD within the first 3 years of life and evaluated whether CB 25[OH]D deficiency alters the DNA methylation profiles of CB leukocytes. The 25[OH]D levels were measured and categorized as severe deficiency (<10.0 ng/mL), deficiency (10.0-19.9 ng/mL), and sufficiency (≥20.0 ng/mL) in 955 infants at birth and 535 children at age 1 year from the COhort for Childhood Origin of Asthma and allergic diseases (COCOA) (see Fig E1 in this article's Online Repository at www.jacionline.org).3Yang H.J. Lee S.Y. Suh D.I. Shin Y.H. Kim B.J. Seo J.H. et al.The Cohort for Childhood Origin of Asthma and allergic diseases (COCOA) study: design, rationale and methods.BMC Pulm Med. 2014; 14: 109Crossref PubMed Scopus (49) Google Scholar We performed DNA methylation assessments in 3 population groups of 6-month infants (AD [n = 10], healthy controls [n = 10], and all subjects [n = 20]) in accordance with the CB 25[OH]D levels. To validate the methylation results, the mRNA expression levels for differentially methylated genes were assayed using real-time PCR (see this article's Methods section and Fig E2 in the Online Repository at www.jacionline.org). In terms of AD outcomes, severe CB 25[OH]D deficiency was associated with a higher risk of AD diagnosis and treatment at age 2 and 3 years, but serum 25[OH]D deficiency at 1 year was not (see Table E1 in this article's Online Repository at www.jacionline.org). Regarding AD prognosis, severe CB 25[OH]D deficiency significantly increased the risk of newly developed AD diagnosis and treatment within the first 3 years and reduced the chances of AD symptoms being missed, and a diagnosis and treatment not being received. This trend did not remain significant for serum 25[OH]D deficiency at 1 year (see Table E2 in this article's Online Repository at www.jacionline.org). The Cox proportional hazards model to examine the associations between 25[OH]D and AD prognosis revealed that severe CB 25[OH]D deficiency significantly increased the hazard ratio (HR) for AD symptoms (Fig 1, A; HR, 2.77; 95% CI, 1.14-6.72; P = .025), diagnosis (Fig 1, B; HR, 2.89; 95% CI, 1.09-7.96; P = .033) and treatment (Fig 1, C; HR, 1.46; 95% CI, 1.30-13.26; P = .016). However, serum 25[OH]D deficiency at 1 year was not associated with the HR for AD symptoms, diagnosis, or treatment (Fig 1, D; HR, 0.63; 95% CI, 0.08-4.73; P = .650; Fig 1, E; HR, 1.16; 95% CI, 0.27-5.08; P = .841; and Fig 1, F; HR, NA; P = .963, respectively). Of note, the CB 25[OH]D level significantly correlated with the CB eosinophil and total IgE levels at 1 year and eosinophil level at 3 years (see Fig E3 in this article's Online Repository at www.jacionline.org). In comparison between the differentially methylated CpG sites according to the sufficient and deficient CB 25[OH]D levels in each principal group (AD, healthy, and total), we identified their overlapping gene, which one CpG site in the microtubule associated monooxygenase, calponin and LIM domain containing 3 (MICAL3) gene was hypomethylated in all 3 groups with deficient CB 25[OH]D levels. Fig 2 shows the mRNA expression-level results. Children with AD and CB 25[OH]D deficiency showed a 3.15-fold greater MICAL3 mRNA expression in the placenta than did healthy children with CB 25[OH]D sufficiency (Fig 2, A; P = .001). The 4 groups, classified according to the CB 25[OH]D level and AD presence, differed significantly in terms of MICAL3 expression (Fig 2, A; P = .006). The CB 25[OH]D level was also found to be inversely associated with the MICAL3 (Fig 2, B; rho = −0.417; P = .015). Collectively, CB 25[OH]D deficiency, as a marker of the vitD status during pregnancy, may cause a loss of methylation in the MICAL3 gene in placenta (see Fig E4 in this article's Online Repository at www.jacionline.org). Because the MICAL3 is a member of the MICAL family of flavoprotein mono-oxygenases implicated in axon guidance and actin remodeling via the oxidation of actin molecules or the production of reactive oxygen species (ROS),4Terman J.R. Mao T. Pasterkamp R.J. Yu H.H. Kolodkin A.L. MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion.Cell. 2002; 109: 887-900Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar the MICAL3 overexpression-induced ROS could then overwhelm the fetal antioxidant defenses, leading to the subsequent development of AD within the first 3 years of life. This process likely also plays an important role in AD severity because there is a correlation between mRNA expression of the MICAL3 and the Scoring Atopic Dermatitis index (Fig 2, C; rho = 0.449; P = .024). In addition, MICAL3 expression levels were associated with the CB 25[OH]D levels regardless of AD presence (see this article's Results section and Fig E5 in the Online Repository at www.jacionline.org). Because this was a prospective birth cohort study, it means that there was already an interaction between MICAL3 expression and CB 25[OH]D levels at birth before the development of AD. To replicate the ROS-associated AD mechanism evidenced by our study of MICAL3, we selected another gene, 8-oxoguanine DNA glycosylase (OGG1), known to impact allergic disease in association with oxidative stress for the mRNA expression data.5Ba X. Bacsi A. Luo J. Aguilera-Aguirre L. Zeng X. Radak Z. et al.8-Oxoguanine DNA glycosylase-1 augments proinflammatory gene expression by facilitating the recruitment of site-specific transcription factors.J Immunol. 2014; 192: 2384-2394Crossref PubMed Scopus (86) Google Scholar Consistently, the children with AD and CB 25[OH]D deficiency had a 5.22-fold greater OGG1 mRNA expression compared with healthy subjects with CB 25[OH]D sufficiency (Fig 2, D; P = .051). The 4 groups differed significantly in terms of their OGG1 expression (Fig 2, D; P = .011). The OGG1 expression levels were found to be inversely associated with the CB [OH]D levels (Fig 2, E; rho = −0.432; P = .027) and Scoring Atopic Dermatitis index (Fig 2, F; rho = −0.641; P = .001). Furthermore, there was a significant correlation between the MICAL3 and OGG1 expression levels (Fig 2, G; rho = 0.563; P = .003). There have been no previous studies showing that MICAL3 and OGG1 are directly linked. However, previous reports have suggested that OGG1 is closely associated with vitD-induced oxidative stress in the development of asthma.6Lan N. Luo G. Yang X. Cheng Y. Zhang Y. Wang X. et al.25-Hydroxyvitamin D3-deficiency enhances oxidative stress and corticosteroid resistance in severe asthma exacerbation.PLoS One. 2014; 9: e111599Crossref PubMed Scopus (69) Google Scholar Future studies may thus be necessary to investigate the association between vitD and OGG1 in AD. Previous cohort studies of the association between CB 25[OH]D and AD have been limited due to factors such as the inclusion of a high-risk population,7Jones A.P. Palmer D. Zhang G. Prescott S.L. Cord blood 25-hydroxyvitamin D3 and allergic disease during infancy.Pediatrics. 2012; 130: e1128-e1135Crossref PubMed Scopus (120) Google Scholar, 8Palmer D.J. Sullivan T.R. Skeaff C.M. Smithers L.G. Makrides M. Higher cord blood 25-hydroxyvitamin D concentrations reduce the risk of early childhood eczema: in children with a family history of allergic disease.World Allergy Organ J. 2015; 8: 28Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar small sample sizes,1Baiz N. Dargent-Molina P. Wark J.D. Souberbielle J.C. Annesi-Maesano I. Group E.M.-C.C.S. Cord serum 25-hydroxyvitamin D and risk of early childhood transient wheezing and atopic dermatitis.J Allergy Clin Immunol. 2014; 133: 147-153Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Jones A.P. Palmer D. Zhang G. Prescott S.L. Cord blood 25-hydroxyvitamin D3 and allergic disease during infancy.Pediatrics. 2012; 130: e1128-e1135Crossref PubMed Scopus (120) Google Scholar, 8Palmer D.J. Sullivan T.R. Skeaff C.M. Smithers L.G. Makrides M. Higher cord blood 25-hydroxyvitamin D concentrations reduce the risk of early childhood eczema: in children with a family history of allergic disease.World Allergy Organ J. 2015; 8: 28Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar use of parentally reported AD,1Baiz N. Dargent-Molina P. Wark J.D. Souberbielle J.C. Annesi-Maesano I. Group E.M.-C.C.S. Cord serum 25-hydroxyvitamin D and risk of early childhood transient wheezing and atopic dermatitis.J Allergy Clin Immunol. 2014; 133: 147-153Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Jones A.P. Palmer D. Zhang G. Prescott S.L. Cord blood 25-hydroxyvitamin D3 and allergic disease during infancy.Pediatrics. 2012; 130: e1128-e1135Crossref PubMed Scopus (120) Google Scholar and a lack of both prenatal and postnatal vitD measurements.1Baiz N. Dargent-Molina P. Wark J.D. Souberbielle J.C. Annesi-Maesano I. Group E.M.-C.C.S. Cord serum 25-hydroxyvitamin D and risk of early childhood transient wheezing and atopic dermatitis.J Allergy Clin Immunol. 2014; 133: 147-153Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Jones A.P. Palmer D. Zhang G. Prescott S.L. Cord blood 25-hydroxyvitamin D3 and allergic disease during infancy.Pediatrics. 2012; 130: e1128-e1135Crossref PubMed Scopus (120) Google Scholar, 8Palmer D.J. Sullivan T.R. Skeaff C.M. Smithers L.G. Makrides M. Higher cord blood 25-hydroxyvitamin D concentrations reduce the risk of early childhood eczema: in children with a family history of allergic disease.World Allergy Organ J. 2015; 8: 28Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Our present study has addressed these concerns by using a large, general population–based birth cohort in which the AD diagnosis was made by pediatric allergy specialists and prenatal and postnatal vitD measurements were also made. We thereby obtained consistent results in our risk factor analysis and the epigenetic assessments of CB 25[OH]D. Our study was limited by the fact that approximately 50% of the COCOA participants were excluded from the final analysis, and the placental analyses were performed in only a small number of subjects. However, the included and excluded participants did not differ with regard to demographic characteristics (see Table E3 in this article's Online Repository at www.jacionline.org), and the effect of vitD in the fetus was confirmed in the human placental samples. Furthermore, we did not investigate CB methylation and expression of genes. But, we alternatively measured gene expression levels in the placental tissue of each child. A recent study has shown however that placental tissue and umbilical CB are both rich in hematopoietic stem/progenitor cells and that a significantly positive correlation exists between the gene expression levels in the placental tissue and umbilical CB.9Chen S. Liu S. Xu L. Yang L. Jin Z. Ma Y. et al.The characteristic expression pattern of BMI-1 and SALL4 genes in placenta tissue and cord blood.Stem Cell Res Ther. 2013; 4: 49Google Scholar In conclusion, our present study is the first to demonstrate that AD development in offspring induced by CB 25[OH]D deficiency is associated with ROS-associated gene methylation. Further studies are warranted to determine whether 25[OH]D supplementation during pregnancy may have a modifying effect on subsequent AD development and severity through these epigenetic mechanisms. 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