Increased vitamin D levels at birth and in early infancy increase offspring allergy risk—evidence for involvement of epigenetic mechanisms
2015; Elsevier BV; Volume: 137; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2015.06.040
ISSN1097-6825
AutoresKristin M. Junge, Tobias Bauer, Stefanie Geißler, Frank Hirche, Loreen Thürmann, Mario Bauer, Saskia Trump, Matthias Bieg, Dieter Weichenhan, Lei Gu, Jan‐Philipp Mallm, Naveed Ishaque, Oliver Mücke, Stefan Röder, Gunda Herberth, Ulrike Diez, Michael Borte, Karsten Rippe, Christoph Plass, Carl Hermann, Gabriele I. Stangl, Roland Eils, Irina Lehmann,
Tópico(s)Digestive system and related health
ResumoAlthough a beneficial effect of vitamin D on health is widely accepted, its role in allergy development has been controversial. Both allergy-preventing and allergy-promoting effects have been reported. Thus, a deeper mechanistic understanding of how vitamin D is related to the regulation of immune reactivity and allergic inflammation is required. Vitamin D was shown to modify gene expression1Ramagopalan S.V. Heger A. Berlanga A.J. Maugeri N.J. Lincoln M.R. Burrell A. et al.A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution.Genome Res. 2010; 20: 1352-1360Crossref PubMed Scopus (697) Google Scholar through binding of the vitamin D receptor to vitamin D response elements. However, only 26% of the genes identified as regulated by vitamin D have a vitamin D response element in proximity to their transcription start site (TSS),1Ramagopalan S.V. Heger A. Berlanga A.J. Maugeri N.J. Lincoln M.R. Burrell A. et al.A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution.Genome Res. 2010; 20: 1352-1360Crossref PubMed Scopus (697) Google Scholar indicating that additional mechanisms are involved in the transcriptional control by vitamin D. As an additional mechanism, epigenetically mediated transcriptional deregulation through vitamin D–induced changes in DNA methylation was suggested.2Fetahu I.S. Hobaus J. Kallay E. Vitamin D and the epigenome.Front Physiol. 2014; 5: 164Crossref PubMed Scopus (194) Google Scholar Here, we studied DNA-methylation pattern on a genomewide scale at base-pair resolution in healthy newborn children with high and low vitamin D levels to elucidate the role of vitamin D in epigenetic programming of an allergy-protective or allergy-promoting immune reactivity. Within the LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) mother-child cohort,3Herberth G. Hinz D. Roder S. Schlink U. Diez U. Borte M. et al.Innate versus adaptive immune response in newborns and their mothers: results from the LINA birth cohort study.Allergy. 2010; 65: 645-646Crossref PubMed Scopus (4) Google Scholar differential DNA methylation was assessed by using whole genome bisulfite sequencing in 6 cord blood samples comparing 3 children with high to 3 children with low 25-hydroxyvitamin D3 (25[OH]D3) levels. We assessed blood cell type composition on the basis of promoter methylation level from 5 lineage markers in each sample (see Methods in this article's Online Repository at www.jacionline.org). The analysis showed that the variation in high versus low vitamin D samples was in a nonsignificant range of 1% to 2% for all cell types analyzed in cord blood (see Table E2 in this article's Online Repository at www.jacionline.org). To omit differentially methylated regions (DMRs) solely caused by differences in cellular blood composition between low and high vitamin D samples, we used an additional threshold of 10% methylation difference for DMR calling. Differential methylation was validated in the entire cohort by target-specific methylation analysis. To decipher the regulatory role of 25(OH)D3-induced DNA-methylation changes, we performed histone modification Chromatin Immuno Precipitation sequencing to segment the genome into distinct regulatory elements and linked differential DNA methylation to transcription (see this article's Online Repository). Furthermore, vitamin D levels and their link to allergic outcomes at different time points in early childhood were studied. Serum 25(OH)D3 levels were measured in 378 newborns, 466 1-year-old children and 304 2-year-old children (see Fig E1 in this article's Online Repository at www.jacionline.org) by using high-performance liquid chromatography tandem mass spectrometry as described earlier.4Weisse K. Winkler S. Hirche F. Herberth G. Hinz D. Bauer M. et al.Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study.Allergy. 2013; 68: 220-228Crossref PubMed Scopus (186) Google Scholar Median 25(OH)D3 levels from birth until age 2 years, seasonal variation, correlations between different ages, and the impact of vitamin D supplementation are presented in Table E1 and Fig E2 (see this article's Online Repository at www.jacionline.org). As described earlier,4Weisse K. Winkler S. Hirche F. Herberth G. Hinz D. Bauer M. et al.Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study.Allergy. 2013; 68: 220-228Crossref PubMed Scopus (186) Google Scholar, 5Miyake Y. Tanaka K. Okubo H. Sasaki S. Arakawa M. Maternal consumption of dairy products, calcium, and vitamin D during pregnancy and infantile allergic disorders.Ann Allergy Asthma Immunol. 2014; 113: 82-87Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar we found that cord blood 25(OH)D3 level was positively associated with physician-diagnosed food allergy and atopic dermatitis until the children were 3 years old (Table I). In addition, an association was found between 25(OH)D3 levels at age of 2 years and an enhanced risk of wheezing within the third year of life (Table I).Table IAssociation between cord blood, year 1 and year 2 25(OH)D3 levels, and atopic outcomes of the child in the months following the vitamin D measurementN25(OH)D3, quartilesn (%)OR (95% CI)FirstSecondThirdFourthRawP valueAdjustedP valueVitamin D birth → outcome month 0-36 Atopic eczema (symptoms)32027 (8.4)21 (6.6)15 (4.7)27 (8.4)0.98 (0.79-1.22).8851.05 (0.82-1.35).673 Atopic eczema (diagnosed)30515 (4.9)13 (4.3)13 (4.3)20 (6.6)1.17 (0.91-1.50).2161.34 (1.00-1.80).047 Food allergy (diagnosed)2914 (1.4)3 (1.0)5 (1.7)9 (3.1)1.48 (0.97-2.25).0661.86 (1.08-3.20).023 Wheezing ever32438 (11.7)34 (10.5)25 (7.7)34 (10.5)0.94 (0.77-1.15).5420.97 (0.78-1.22).817 Wheezing recurrent36719 (5.2)23 (6.3)16 (4.4)20 (5.4)0.96 (0.77-1.20).7011.07 (0.83-1.38).606Vitamin D year 1 → outcome month 12-36 Atopic eczema (symptoms)40925 (6.1)17 (4.2)30 (7.3)19 (4.6)0.97 (0.79-1.20).7971.01 (0.98-1.04).690 Atopic eczema (diagnosed)37410 (2.7)7 (1.9)14 (3.7)13 (3.5)1.21 (0.91-1.62).1911.04 (0.99-1.09).064 Food allergy (diagnosed)3703 (0.8)5 (1.4)5 (1.4)7 (1.9)1.33 (0.87-2.03).1861.03 (0.97-1.09).291 Wheezing ever40925 (6.1)37 (9.0)33 (8.1)30 (7.3)1.06 (0.87-1.28).5611.01 (0.99-1.04).312 Wheezing recurrent40813 (3.2)17 (4.2)21 (5.1)22 (4.9)1.25 (0.99-1.58).0641.03 (0.99-1.06).097Vitamin D year 2 → outcome month 24-36 Atopic eczema (symptoms)28912 (4.2)15 (5.2)9 (3.1)7 (2.4)0.78 (0.58-1.05).1010.92 (0.65-1.29).623 Atopic eczema (diagnosed)2894 (1.4)5 (1.7)7 (2.4)6 (2.1)1.16 (0.78-1.72).4691.37 (0.87-2.14).168 Food allergy (diagnosed)2894 (1.4)2 (0.7)2 (0.7)1 (0.4)0.64 (0.34-1.22).1700.95 (0.47-1.93).881 Wheezing ever2885 (1.7)15 (5.2)17 (5.9)15 (5.2)1.35 (1.02-1.78).0351.38 (1.01-1.90).044 Wheezing recurrent2883 (1.0)5 (1.7)9 (3.1)7 (2.4)1.34 (0.91-1.97).1421.50 (0.95-2.38).080N = cases with questionnaire data and vitamin D measurement in the given combination. Odds ratios (ORs) with 95% CI and P value are shown either for raw data or adjusted for sex of the child, number of siblings, family history for atopy, maternal urine cotinine level (34th week of pregnancy), keeping of a cat, month of birth, and breast-feeding.Significant values are in boldface. Open table in a new tab N = cases with questionnaire data and vitamin D measurement in the given combination. Odds ratios (ORs) with 95% CI and P value are shown either for raw data or adjusted for sex of the child, number of siblings, family history for atopy, maternal urine cotinine level (34th week of pregnancy), keeping of a cat, month of birth, and breast-feeding. Significant values are in boldface. Next, we identified 508 significantly differentially methylated regions associated with 483 genes in children with high compared with low 25(OH)D3 levels. Thereby, 25(OH)D3-dependent methylation changes were predominantly associated with loss of DNA methylation (see Fig E3, A, and Table E3 in this article's Online Repository at www.jacionline.org), which is in line with earlier reported data.2Fetahu I.S. Hobaus J. Kallay E. Vitamin D and the epigenome.Front Physiol. 2014; 5: 164Crossref PubMed Scopus (194) Google Scholar Among those 508 DMRs, an intergenic DMR spanning 6 CpGs was identified, located 52,400 base pairs upstream of the TSS of group-specific component (vitamin D binding protein; Δmethylation, 17.3%; see Fig E3, B, and Table E3). However, validation in low versus high vitamin D cord blood samples in the entire LINA cohort using MassARRAY-based target-specific methylation analysis barely missed the significance level (P = .063; see Fig E5 in this article's Online Repository at www.jacionline.org). No DMRs were called for other known key regulators of vitamin D metabolism. To further filter the 508 significant DMRs assessed by whole genome bisulfite sequencing, we used WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) with all predicted target genes (see Table E3) for pathway analyses. As a preliminary result, 103 significantly affected pathways (P < .01) were identified (see Table E4 in this article's Online Repository at www.jacionline.org), including pathways with a potential link to immune system dysfunction or allergy development: "immune system disease," "lung disease/obstructive lung disease," and "skin and connective tissue disease." In total, 3 genes involved in all these 3 pathways were identified: thymic stromal lymphopoietin (TSLP), IL17F, and MBL2. IL17F and MBL2 are involved in inflammation and infection, whereas for TSLP links have already been shown to both allergic diseases and vitamin D. TSLP was selected for further validation because of its known functional role in allergic diseases. For TSLP, a DMR spanning 5 CpGs 113kbp upstream of the TSLP TSS was identified (Δmethylation, 24%; Fig E3, C, and Table E3). Further analysis of the region flanking this DMR revealed a broader, significantly regulated region that includes this DMR together with an annotated enhancer region located 100bp downstream (Chr5:110.292.001-110.293.600; P = .0058; Δmethylation, 12.2%). Although this broader region was at a remarkable distance from the TSLP TSS, chromatin interaction data indicate that this enhancer targets TSLP (Fig 1, A). We validated the loss of methylation in this region in relation to high cord blood 25(OH)D3 levels by MassARRAY-based analysis in the entire LINA cohort (see Fig E4, A, in this article's Online Repository at www.jacionline.org). No overall correlation was found in all children or those with low 25(OH)D3 levels. However, in children with high cord blood 25(OH)D3 levels, the methylation level of this region correlated significantly with 25(OH)D3 concentrations. This association was observed for 2 CpGs (Chr5:110.292.389-392) located in the TSLP enhancer (P = .026; R = −0.236; Fig 1, B) as well as for 1 further CpG (Chr5:110.292.306) located in the TSLP DMR (P = .024; R = −0.233; see Fig E6 in this article's Online Repository at www.jacionline.org). The methylation level of the TSLP enhancer region was stable from birth until age 3 years (Fig E4, B and C). On analyzing histone modifications in the TSLP-associated DMR and the adjacent enhancer, we found repressive marks (H3K9me3 and/or H3K27me3) in this region in blood cells of children with low 25(OH)D3 levels whereas children with high 25(OH)D3 levels were deprived of repressive histone marks. Neither the TSLP DMR nor the TSLP enhancer region has meQTL-single nucleotide polymorphisms in its neighborhood. Thus, we conclude that differences in TSLP methylation levels are not linked to genetic variation. Altogether, the concomitant loss of DNA methylation and repressive chromatin marks strongly suggest a gain of expression of the target gene TSLP. In fact, a negative correlation between methylation in the enhancer region and TSLP mRNA expression was observed (Fig 1, C). The TSLP mRNA expression was significantly elevated at birth (P = .048) and age 1 year (P = .003) in the high vitamin D group (Fig 1, D and E). Finally, we found that children who suffered from wheezing symptoms later in life had significantly enhanced TSLP mRNA expression at age 1 year (Fig 1, F). Furthermore, a link between wheezing symptoms and reduced methylation at CpG Chr5:110.292.315 located in the TSLP DMR region could be shown (Δmethylation, 2%; P = .045; n = 309 controls vs n = 66 wheezing children). TSLP plays a critical role in allergic diseases by inducing an inflammatory TH2 response via conditioning dendritic cell maturation.6Liu Y.J. Soumelis V. Watanabe N. Ito T. Wang Y.H. Malefyt R.D. et al.TSLP: an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation.Annu Rev Immunol. 2007; 25: 193-219Crossref PubMed Scopus (547) Google Scholar, 7Takai T. TSLP expression: cellular sources, triggers, and regulatory mechanisms.Allergol Int. 2012; 61: 3-17Abstract Full Text PDF PubMed Scopus (200) Google Scholar TSLP mRNA as well as protein levels were shown to correlate with asthma severity,8Ying S. O'Connor B. Ratoff J. Meng Q. Mallett K. Cousins D. et al.Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of TH2-attracting chemokines and disease severity.J Immunol. 2005; 174: 8183-8190Crossref PubMed Scopus (709) Google Scholar while treatment of patients with asthma with an anti-TSLP antibody reduced airway inflammation before and after allergen challenge.9Gauvreau G.M. O'Byrne P.M. Boulet L.P. Wang Y. Cockcroft D. Bigler J. et al.Effects of an anti-TSLP antibody on allergen-induced asthmatic responses.N Engl J Med. 2014; 370: 2102-2110Crossref PubMed Scopus (620) Google Scholar Here, we demonstrated that high cord blood vitamin D levels were associated with epigenetic regulation of an enhancer region shown to interact with the TSLP promoter. Children with higher 25(OH)D3 levels at birth showed a lower DNA-methylation level and a loss of repressive histone marks in this TSLP enhancer region, resulting in a higher TSLP mRNA expression. Furthermore, a link between enhanced TSLP expression and wheezing was found. Our result provides evidence that epigenetic deregulation of TSLP could be involved in the vitamin D–related programming for allergic diseases. However, this result does not exclude that vitamin D may act in comparable manner via other genes. We thank Stephan Wolf and Nicole Diessl at the German Cancer Research Center (DKFZ) Genomics and Proteomics Core Facility for the excellent technical support and expertise. Furthermore, we are grateful to Marion Bähr and Monika Helf who provided support in MassARRAY validation. We cordially thank the participants of the LINA study and Beate Fink, Anne Hain, Livia Sztraka, and Melanie Nowak for their excellent technical assistance and fieldwork. We are grateful to Martin von Bergen and Ulrike Rolle-Kampczyk for providing urine cotinine concentrations. 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