Advertisement

Maternal phthalate exposure promotes allergic airway inflammation over 2 generations through epigenetic modifications

  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Susanne Jahreis
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany

    Department of Dermatology, Venerology and Allergology, Leipzig University Medical Center, Leipzig, Germany

    Infections in Hematology/Oncology, Leibniz Institute for Natural Product Research and Infection Biology–Hans Knöll Institute, Jena, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Saskia Trump
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany

    Institute of Pharmacy and Molecular Biotechnology, and Bioquant Center, University of Heidelberg, Heidelberg, Germany
    Search for articles by this author
  • Mario Bauer
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Tobias Bauer
    Affiliations
    Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany
    Search for articles by this author
  • Loreen Thürmann
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Ralph Feltens
    Affiliations
    Department Molecular Systems Biology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Qi Wang
    Affiliations
    Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany
    Search for articles by this author
  • Lei Gu
    Affiliations
    Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Department of Cell Biology, Harvard Medical School, Boston
    Search for articles by this author
  • Konrad Grützmann
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Stefan Röder
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Marco Averbeck
    Affiliations
    Department of Dermatology, Venerology and Allergology, Leipzig University Medical Center, Leipzig, Germany
    Search for articles by this author
  • Dieter Weichenhan
    Affiliations
    Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany
    Search for articles by this author
  • Christoph Plass
    Affiliations
    Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany
    Search for articles by this author
  • Ulrich Sack
    Affiliations
    Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany
    Search for articles by this author
  • Michael Borte
    Affiliations
    Municipal Hospital “St Georg” Children's Hospital, Leipzig, Germany
    Search for articles by this author
  • Virginie Dubourg
    Affiliations
    Young Investigators Group Bioinformatics and Transcriptomics, Department Molecular Systems Biology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Gerrit Schüürmann
    Affiliations
    Department of Ecological Chemistry, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany

    Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Freiberg, Germany
    Search for articles by this author
  • Jan C. Simon
    Affiliations
    Department of Dermatology, Venerology and Allergology, Leipzig University Medical Center, Leipzig, Germany
    Search for articles by this author
  • Martin von Bergen
    Affiliations
    Department Molecular Systems Biology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany

    Institute of Biochemistry, Faculty of Bioscience, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany

    Department of Chemistry and Bioscience, University of Aalborg, Aalborg, Denmark
    Search for articles by this author
  • Jörg Hackermüller
    Affiliations
    Young Investigators Group Bioinformatics and Transcriptomics, Department Molecular Systems Biology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Roland Eils
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Institute of Pharmacy and Molecular Biotechnology, and Bioquant Center, University of Heidelberg, Heidelberg, Germany

    Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Irina Lehmann
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Tobias Polte
    Correspondence
    Corresponding author: Tobias Polte, PhD, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Helmholtz University Research Group Experimental Allergy and Immunology, Permoserstr. 15, Leipzig, Germany.
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Environmental Immunology, UFZ–Helmholtz Centre for Environmental Research Leipzig-Halle, Leipzig, Germany

    Department of Dermatology, Venerology and Allergology, Leipzig University Medical Center, Leipzig, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.
Open AccessPublished:April 06, 2017DOI:https://doi.org/10.1016/j.jaci.2017.03.017

      Background

      Prenatal and early postnatal exposures to environmental factors are considered responsible for the increasing prevalence of allergic diseases. Although there is some evidence for allergy-promoting effects in children because of exposure to plasticizers, such as phthalates, findings of previous studies are inconsistent and lack mechanistic information.

      Objective

      We investigated the effect of maternal phthalate exposure on asthma development in subsequent generations and their underlying mechanisms, including epigenetic alterations.

      Methods

      Phthalate metabolites were measured within the prospective mother-child cohort Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk (LINA) and correlated with asthma development in the children. A murine transgenerational asthma model was used to identify involved pathways.

      Results

      In LINA maternal urinary concentrations of mono-n-butyl phthalate, a metabolite of butyl benzyl phthalate (BBP), were associated with an increased asthma risk in the children. Using a murine transgenerational asthma model, we demonstrate a direct effect of BBP on asthma severity in the offspring with a persistently increased airway inflammation up to the F2 generation. This disease-promoting effect was mediated by BBP-induced global DNA hypermethylation in CD4+ T cells of the offspring because treatment with a DNA-demethylating agent alleviated exacerbation of allergic airway inflammation. Thirteen transcriptionally downregulated genes linked to promoter or enhancer hypermethylation were identified. Among these, the GATA-3 repressor zinc finger protein 1 (Zfpm1) emerged as a potential mediator of the enhanced susceptibility for TH2-driven allergic asthma.

      Conclusion

      These data provide strong evidence that maternal BBP exposure increases the risk for allergic airway inflammation in the offspring by modulating the expression of genes involved in TH2 differentiation through epigenetic alterations.

      Key words

      Abbreviations used:

      AHR (Airway hyperreactivity), Aza (5-Aza-2′-deoxycytidine), BBP (Butyl benzyl phthalate), DEG (Differentially expressed gene), DMR (Differentially methylated region), Fads1 (Fatty acid desaturase 1), Fanca (Fanconi anemia complementation group A), Foxp3 (Forkhead box p3), LINA (Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk), MBzP (Monobenzyl phthalate), MnBP (Mono-n-butyl phthalate), OVA (Ovalbumin), WGBS (Whole-genome bisulfite sequencing), Zfpm1 (Zinc finger protein 1)
      Allergic asthma is caused by a TH2 cell–mediated immune response to common environmental allergens and characterized by airway inflammation with pulmonary eosinophilia, airway hyperreactivity (AHR), and increased serum IgE levels.
      • Umetsu D.T.
      • McIntire J.J.
      • Akbari O.
      • Macaubas C.
      • DeKruyff R.H.
      Asthma: an epidemic of dysregulated immunity.
      This inflammatory airway disease has a strong genetic background, but also, a multitude of environmental trigger factors have been described.
      • Pearce N.
      • Douwes J.
      The global epidemiology of asthma in children.
      • Tachdjian R.
      • Mathias C.
      • Al Khatib S.
      • Bryce P.J.
      • Kim H.S.
      • Blaeser F.
      • et al.
      Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma.
      Epidemiologic and experimental studies demonstrated that exposure to environmental pollutants, such as diesel exhaust particles or environmental tobacco smoke, or chemicals, such as volatile organic compounds, can lead to exacerbation of respiratory symptoms and asthma.
      • Dick S.
      • Friend A.
      • Dynes K.
      • AlKandari F.
      • Doust E.
      • Cowie H.
      • et al.
      A systematic review of associations between environmental exposures and development of asthma in children aged up to 9 years.
      • Alessandrini F.
      • Schulz H.
      • Takenaka S.
      • Lentner B.
      • Karg E.
      • Behrendt H.
      • et al.
      Effects of ultrafine carbon particle inhalation on allergic inflammation of the lung.
      • Bönisch U.
      • Böhme A.
      • Kohajda T.
      • Mögel I.
      • Schütze N.
      • von Bergen M.
      • et al.
      Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model.
      • Franck U.
      • Weller A.
      • Roder S.W.
      • Herberth G.
      • Junge K.M.
      • Kohajda T.
      • et al.
      Prenatal VOC exposure and redecoration are related to wheezing in early infancy.
      In particular, the prenatal and early postnatal periods appear critical to environmental exposures, probably interfering with the developmental programming of the immune system and thereby altering the disease risk in later life.
      • Martino D.
      • Prescott S.
      Epigenetics and prenatal influences on asthma and allergic airways disease.
      Epigenetic changes, such as modified DNA methylation, have been described as mediators between exposure and disease development.
      • Martino D.
      • Prescott S.
      Epigenetics and prenatal influences on asthma and allergic airways disease.
      • Kabesch M.
      Epigenetics in asthma and allergy.
      This has been shown for allergy-preventing effects induced by upbringing in a farming environment or exposure to microbial components, as well as for asthma-promoting influences, such as maternal exposure to tobacco smoke during pregnancy.
      • Kabesch M.
      Epigenetics in asthma and allergy.
      • Reiprich M.
      • Rudzok S.
      • Schutze N.
      • Simon J.C.
      • Lehmann I.
      • Trump S.
      • et al.
      Inhibition of endotoxin-induced perinatal asthma protection by pollutants in an experimental mouse model.
      • Conrad M.L.
      • Ferstl R.
      • Teich R.
      • Brand S.
      • Blumer N.
      • Yildirim A.O.
      • et al.
      Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78.
      These findings emphasize that the developing immune system is especially susceptible to perturbations by external factors.
      Here, we focused on phthalates, a group of chemicals commonly used as plasticizers in large quantities worldwide. These chemicals are present in a wide range of consumer products, such as cosmetics, plastics, floor coverings, building materials, toys, and cleaning products.
      • Dodson R.E.
      • Nishioka M.
      • Standley L.J.
      • Perovich L.J.
      • Brody J.G.
      • Rudel R.A.
      Endocrine disruptors and asthma-associated chemicals in consumer products.
      • Braun J.M.
      • Sathyanarayana S.
      • Hauser R.
      Phthalate exposure and children's health.
      Human subjects are exposed to phthalates mainly through ingestion or inhalation throughout life, starting in utero.
      • Adibi J.J.
      • Whyatt R.M.
      • Williams P.L.
      • Calafat A.M.
      • Camann D.
      • Herrick R.
      • et al.
      Characterization of phthalate exposure among pregnant women assessed by repeat air and urine samples.
      • Wormuth M.
      • Scheringer M.
      • Vollenweider M.
      • Hungerbuhler K.
      What are the sources of exposure to eight frequently used phthalic acid esters in Europeans?.
      Although there is some evidence of allergy-promoting effects in children by maternal phthalate exposure, findings of previous studies are conflicting regarding the effect direction, the investigated exposure conditions, and their assessment.
      • Bornehag C.G.
      • Sundell J.
      • Weschler C.J.
      • Sigsgaard T.
      • Lundgren B.
      • Hasselgren M.
      • et al.
      The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study.
      • Bertelsen R.J.
      • Carlsen K.C.
      • Calafat A.M.
      • Hoppin J.A.
      • Haland G.
      • Mowinckel P.
      • et al.
      Urinary biomarkers for phthalates associated with asthma in Norwegian children.
      • Robinson L.
      • Miller R.
      The impact of bisphenol A and phthalates on allergy, asthma, and immune function: a review of latest findings.
      Furthermore, little is known about the mechanisms of maternal phthalate exposure, leading to an altered disease risk in children's later life.
      In the present study we report a significant association between maternal urinary concentrations of the phthalate metabolite mono-n-butyl phthalate (MnBP) and an increased asthma risk in children of our prospective mother-child cohort Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk (LINA).
      • Franck U.
      • Weller A.
      • Roder S.W.
      • Herberth G.
      • Junge K.M.
      • Kohajda T.
      • et al.
      Prenatal VOC exposure and redecoration are related to wheezing in early infancy.
      Using a murine transgenerational asthma model, we demonstrate that maternal exposure to butyl benzyl phthalate (BBP), a parent compound of MnBP, has a direct effect on severity of allergic airway inflammation in the offspring, observing an enhanced airway inflammation, even in the F2 generation. This asthma-promoting effect was mediated by BBP-induced DNA hypermethylation, as shown by diminished BBP effects after treating mice with a hypomethylation-inducing compound and a subsequently reduced expression of genes involved in the differentiation of TH2 cells, such as the GATA-3 repressor zinc finger protein 1 (Zfpm1), offering an explanation for the increased allergic immune response.

      Methods

      For more detailed information, please see the Methods section in this article's Online Repository at www.jacionline.org.

       LINA study design and sample collection

      The LINA cohort study recruited 629 mother-child pairs between May 2006 and December 2008 in Leipzig, Germany, to investigate how environmental factors in the prenatal and postnatal period influence immune system development and the resulting disease risks.
      • Hinz D.
      • Simon J.C.
      • Maier-Simon C.
      • Milkova L.
      • Roder S.
      • Sack U.
      • et al.
      Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood.
      • Weisse 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.
      • Herberth G.
      • Bauer M.
      • Gasch M.
      • Hinz D.
      • Roder S.
      • Olek S.
      • et al.
      Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.

       Urine samples and sample preparation

      Phthalate metabolite quantification was carried out for 540 early morning maternal urine samples, as described previously.
      • Feltens R.
      • Roeder S.
      • Otto W.
      • Borte M.
      • Lehmann I.
      • von Bergen M.
      • et al.
      Evaluation of population and individual variances of urinary phthalate metabolites in terms of epidemiological studies.

       Exposure to BBP and ovalbumin immunization

      BALB/c mice were exposed to BBP (3 μg/mL) exclusively through drinking water (custom-built glass bottles) for different time periods, as described in detail in the Methods section in this article's Online Repository.

       Measurement of airway responsiveness

      Lung resistance was measured by using invasive plethysmography in response to inhaled methacholine, as described previously
      • Polte T.
      • Petzold S.
      • Bertrand J.
      • Schutze N.
      • Hinz D.
      • Simon J.C.
      • et al.
      Critical role for syndecan-4 in dendritic cell migration during development of allergic airway inflammation.
      • Polte T.
      • Foell J.
      • Werner C.
      • Hoymann H.G.
      • Braun A.
      • Burdach S.
      • et al.
      CD137-mediated immunotherapy for allergic asthma.
      • Schutze N.
      • Lehmann I.
      • Bonisch U.
      • Simon J.C.
      • Polte T.
      Exposure to mycotoxins increases the allergic immune response in a murine asthma model.
      and in more detail in the Methods section in this article's Online Repository.

       Whole-genome bisulfite sequencing, differentially methylated region calling, and annotation

      Whole-genome bisulfite sequencing (WGBS) library construction and sequencing was performed, as previously described,
      • Wang Q.
      • Gu L.
      • Adey A.
      • Radlwimmer B.
      • Wang W.
      • Hovestadt V.
      • et al.
      Tagmentation-based whole-genome bisulfite sequencing.
      by applying a tagmentation-based, ultralow-input library preparation protocol with a quantity of 20 ng of input DNA. Sequencing and alignment (mm10) were carried out, as described previously.
      • Bauer T.
      • Trump S.
      • Ishaque N.
      • Thurmann L.
      • Gu L.
      • Bauer M.
      • et al.
      Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.
      Differentially methylated region (DMR) calling was performed, as described earlier.
      • Bauer T.
      • Trump S.
      • Ishaque N.
      • Thurmann L.
      • Gu L.
      • Bauer M.
      • et al.
      Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

       RNA sequencing

      Total RNA was isolated, and RNA sequencing was performed, as described previously.
      • Hackermuller J.
      • Reiche K.
      • Otto C.
      • Hosler N.
      • Blumert C.
      • Brocke-Heidrich K.
      • et al.
      Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs.
      • Martin M.
      Cutadapt removes adapter sequences from high-throughput sequencing reads.
      • Hoffmann S.
      • Otto C.
      • Doose G.
      • Tanzer A.
      • Langenberger D.
      • Christ S.
      • et al.
      A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection.

       Mapping of DMRs to target genes

      DMRs were mapped on gene bodies, promoters, and putative enhancers of the differentially expressed genes (DEGs), as described in detail in the Methods section in this article's Online Repository.

       MassARRAY methylation analysis

      MassARRAY analysis was performed, as described previously.
      • Bauer T.
      • Trump S.
      • Ishaque N.
      • Thurmann L.
      • Gu L.
      • Bauer M.
      • et al.
      Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

       Statistical analysis

      Equal parameter distribution was tested by using χ2 tests. Logistic regression models were implemented, adjusting for known confounding factors of lung disease or atopy in early childhood, to assess the contribution of phthalate exposure on risk increase for asthma development or increased IgE levels in the children.
      Nonparametric tests were applied for all nonnormally distributed parameters (eg, Mann-Whitney U test [MWU] and Spearman correlations). All calculations were performed in STATISTICA software for Windows (Version 10; Statsoft, Tulsa, Okla). Data were expressed as means ± SEMs, and P values of less than .05 were considered significant.

      Results

       Association between maternal phthalate metabolites and the development of allergic asthma in children (LINA)

      For the LINA mother-child study, 629 mother-child pairs were recruited between 2006 and 2008. The 6-year follow-up investigation included 420 (66.8%) of the families. Regarding asthma development, only physician-diagnosed asthma was considered for this study. For 24 of the LINA children, an asthma diagnosis was reported until the age of 6 years. General characteristics of the study participants are shown in Table E1 in this article's Online Repository at www.jacionline.org. Apart from a slightly lower number of tobacco smoke–exposed children participating until the age of 6 years, there were no differences between the analyzed subcohort (n = 371) and the total LINA cohort (n = 629).
      The concentration of 10 phthalate metabolites was determined in a total of 540 maternal urine samples of the 34th week of gestation. However, only measurements of the 371 children who participated in the study until age 6 were considered for our analysis (see Table E2 in this article's Online Repository at www.jacionline.org). Interestingly, an increased risk of asthma symptoms until age 6 was observed only in children from mothers with increased urinary MnBP levels (Fig 1, A and B), whereas all other metabolites showed no significant association (see Table E3 and E5 in this article's Online Repository at www.jacionline.org). In addition to the lung phenotype, high maternal MnBP concentrations were also associated with a higher risk for allergic sensitization against inhalant allergens in the children (Fig 1, C and D, and see Table E4 and E5 in this article's Online Repository at www.jacionline.org).
      Figure thumbnail gr1
      Fig 1Maternal phthalate exposure and children's asthma and allergic sensitization. A and C, Maternal urine MnBP concentrations (34th gestational week) for children with asthma until age 6 (Fig 1, A) and sensitization against inhalant allergens (Fig 1, C) compared with control subjects (medians and interquartile ranges). B and D, Frequency and risk (adjusted odds ratio per 100 ng/mg MnBP increase) for asthma (Fig 1, B) and sensitization against inhalant allergens (Fig 1, D) according to maternal MnBP concentrations (high = greater than median).

       Prenatal and perinatal exposure to BBP increased allergic airway inflammation in a murine asthma model

      Next to monobenzyl phthalate (MBzP), MnBP is the major metabolite of BBP.
      • Eigenberg D.A.
      • Bozigian H.P.
      • Carter D.E.
      • Sipes I.G.
      Distribution, excretion, and metabolism of butylbenzyl phthalate in the rat.
      To further investigate whether the observed increase in asthma risk in the human study cohort might be directly caused by maternal exposure to BBP, we used a well-established transgenerational ovalbumin (OVA)–induced asthma mouse model.
      • Reiprich M.
      • Rudzok S.
      • Schutze N.
      • Simon J.C.
      • Lehmann I.
      • Trump S.
      • et al.
      Inhibition of endotoxin-induced perinatal asthma protection by pollutants in an experimental mouse model.
      • Polte T.
      • Petzold S.
      • Bertrand J.
      • Schutze N.
      • Hinz D.
      • Simon J.C.
      • et al.
      Critical role for syndecan-4 in dendritic cell migration during development of allergic airway inflammation.
      • Polte T.
      • Hennig C.
      • Hansen G.
      Allergy prevention starts before conception: maternofetal transfer of tolerance protects against the development of asthma.
      • Petzold S.
      • Averbeck M.
      • Simon J.C.
      • Lehmann I.
      • Polte T.
      Lifetime-dependent effects of bisphenol A on asthma development in an experimental mouse model.
      During the exposure period, BALB/c mice received 3 μg/mL BBP through drinking water, a concentration relevant for human exposure. MnBP levels measured in the urine of BBP-exposed dams (see Table E6 in this article's Online Repository at www.jacionline.org) were comparable with those detected in highly exposed LINA mothers (12.47-6894.87 ng/mL in maternal urine). Dams were exposed to BBP during pregnancy (prenatal) or during pregnancy and breast-feeding (perinatal) to analyze the effect of maternal BBP exposure on allergic airway inflammation. Grown-up offspring were then subjected to antigen sensitization without being exposed further to BBP (Fig 2, A). In another treatment regimen, adult mice were exposed to BBP starting 1 week before OVA sensitization until the end of the asthma protocol (Fig 2, B).
      Figure thumbnail gr2
      Fig 2Phthalate exposure and induction of an asthma-like phenotype. A, Dams were exposed to BBP during pregnancy (prenatal) or until weaning when pups were 3 weeks old (perinatal exposure). B, Exposure of adult mice to BBP started 1 week before the first allergen injection and was continued until the end of the asthma induction protocol. C, Female F1 mice from exposed or control dams were mated with unexposed male mice. i.n., Intranasal; i.p., intraperitoneal.
      Although exposure of adult mice to BBP had no effect on the asthma-like phenotype, prenatal and perinatal BBP exposure significantly increased the number of eosinophils within the bronchoalveolar lavage fluid compared with the offspring from unexposed dams (Fig 3, A). Maternal BBP exposure led to substantially more inflammatory infiltrates in the airways, as demonstrated by means of histologic examination of hematoxylin and eosin–stained lung sections (Fig 3, B), as confirmed by means of software-based image analysis (Fig 3, C). Accordingly, AHR measured as lung resistance was enhanced in the offspring from BBP-exposed dams (Fig 3, D).
      Figure thumbnail gr3
      Fig 3Prenatal and perinatal exposure to BBP increases allergic airway inflammation in a murine asthma model. Total cell numbers in bronchoalveolar lavage (BAL) fluid (A), airway inflammation examined by lung histology (hematoxylin and eosin, magnification ×100; B) quantified by an investigator-independent computer-based analysis (C), and lung resistance (D) were examined in offspring from BBP-exposed dams and in BBP-exposed adult mice. Data are expressed as means ± SEMs (n ≥ 9 animals per group in the offspring [from ≥5 dams] and adult mice from 4 independent experiments). *P < .05.
      Furthermore, OVA-specific IgE levels were increased in prenatally and perinatally exposed offspring compared with control mice (Fig 4, A). Moreover, OVA-restimulated spleen and lymph node cells from offspring of BBP-exposed dams produced significantly higher amounts of the TH2 cytokines IL-5 and IL-13, whereas there was no significant effect on the TH1 cytokine IFN-γ (Fig 4, B and C).
      Figure thumbnail gr4
      Fig 4Prenatal and perinatal exposure to BBP enhances allergen-specific IgE levels and TH2 cytokine production. OVA-specific IgE serum levels (A) and cytokine production of restimulated splenocytes (B) and lymph node cells (C) were analyzed in OVA-immunized offspring from BBP-exposed dams and in BBP-exposed adult mice. Data are expressed as means ± SEMs (n ≥ 9 animals per group in the offspring [from ≥5 dams] and adult groups from 4 independent experiments). *P < .05 and **P < .01.
      To evaluate which BBP metabolite promotes the occurrence of an asthma-like phenotype, we perinatally exposed mice to MnBP or MBzP and subsequently characterized the asthma phenotype in the offspring. Interestingly, neither MnBP nor MBzP significantly increased the allergic airway inflammation (see Fig E1 in this article's Online Repository at www.jacionline.org).

       Maternal BBP exposure induces global hypermethylation mediating the asthma-promoting effect

      Early environmental influences have been shown to perturb epigenetic changes, such as DNA methylation, that might contribute to programming of disease risks.
      • Martino D.
      • Prescott S.
      Epigenetics and prenatal influences on asthma and allergic airways disease.
      To evaluate whether maternal (perinatal) BBP exposure alters the global DNA methylation pattern in the F1 generation, we subjected splenic CD4+ T cells isolated from dams and their 3-week-old offspring to WGBS. DMRs were called with a difference in the methylation level of greater than 5% and a P value of less than .05 between the exposed group (F0 or F1) versus nonexposed control mice of the same generation (n = 3, median false discovery rate: F0, 0.056; F1, 0.036).
      Our data show that maternal BBP exposure induced global DNA hypermethylation in the F0 generation (63% hypermethylated regions among identified DMRs) and to an even higher extent in the offspring (83%; Fig 5, A, data set 1). Similar to the F0 generation, hypermethylation in the F1 generation was not restricted to any particular genomic region but occurred genome wide (Fig 5, B).
      Figure thumbnail gr5
      Fig 5Maternal BBP exposure induces global hypermethylation, which mediates the asthma-promoting effect. A, DMRs were called from dams and their offspring between the exposed (F0 or F1) and nonexposed groups. B, Numbers of DMRs in different DNA regions of the F1 generation. C-G, Total cell numbers in bronchoalveolar lavage (BAL) fluid (Fig 5, C), airway inflammation (Fig 5, D), lung resistance (Fig 5, E), OVA-specific IgE levels (Fig 5, F), and cytokine production in spleen and lymph node cells (Fig 5, G) were examined in offspring from BBP-exposed dams compared with F1 mice treated with the DNA methyltransferase inhibitor Aza. Data are expressed as means ± SEMs (n ≥ 9 animals per group in the offspring [from ≥6 dams) from 6 independent experiments). *P < .05, OVA versus OVA + BBP. #P < .05, OVA + BBP versus OVA + BBP + Aza.
      One-week-old pups from BBP-exposed dams were treated with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) for 2 weeks until weaning to evaluate whether BBP-induced hypermethylation is linked to the severe allergic airway inflammation observed in the offspring.
      • Brand S.
      • Kesper D.A.
      • Teich R.
      • Kilic-Niebergall E.
      • Pinkenburg O.
      • Bothur E.
      • et al.
      DNA methylation of TH1/TH2 cytokine genes affects sensitization and progress of experimental asthma.
      Treatment of the offspring with Aza reversed enhanced airway inflammation caused by maternal BBP exposure, as demonstrated by the reduced number of eosinophilic granulocytes in bronchoalveolar lavage fluid (Fig 5, C), diminished lung inflammation (Fig 5, D), and attenuated OVA-specific lung resistance (Fig 5, E), reduced IgE levels (Fig 5, F), and IL-5/IL-13 levels in splenocytes and IL-4/IL-5 levels in lymph node cells (Fig 5, G). Aza treatment of pups from unexposed dams had no effect on the asthma-like phenotype in the offspring.

       BBP-induced hypermethylation leads to downregulation of certain target genes

      To characterize the functional consequences of BBP-induced hypermethylation on transcription, we assessed gene expression using RNA sequencing in CD4+ T cells from 3-week-old offspring alongside with our WGBS analysis. At a false discovery rate of less than 0.01, we detected 1138 differential expressed genes (DEGs) in the offspring of BBP-exposed dams in comparison with those of nonexposed mice. Treated versus nontreated mice clustered with respect to their genome-wide RNA expression by means of unsupervised clustering (Fig 6, A, data set 2). Two hundred forty-two of the obtained DEGs could be linked to 346 DMRs (Fig 6, B). Those target genes most likely mediating the BBP-related asthma promotion should fulfill certain criteria (see Fig E2 in this article's Online Repository at www.jacionline.org and data set 3). As already mentioned, BBP-induced hypermethylation was correlated with increased airway inflammation, which was alleviated by treatment with the DNA-demethylating agent Aza. Therefore, in a first step, we concentrated on those DEGs related to hypermethylated DMRs (201 DEGs) located in functionally translating regulatory elements (promoter/enhancer; 33 DEGs remaining). Because DNA hypermethylation is a key mechanism to gene silencing, we focused only on those transcriptionally repressed genes, leaving us with 13 potential candidates (data set 3). Among those, 3 genes were previously described to play a role in TH2-driven allergic immune diseases and were subsequently evaluated in more detail.
      Figure thumbnail gr6
      Fig 6BBP-induced hypermethylation leads to downregulation of selected target genes. A, Heat map displays DEGs from pups of BBP-exposed dams versus control mice. B, Two hundred forty-two of 1338 DEGs in the offspring from BBP-exposed dams can be linked to 346 DMRs. C, RNA expression of selected genes in dams (n = 5), pups, or adult mice (n ≥ 6 from ≥4 dams) was verified by using quantitative PCR. D, Mice from BBP-exposed dams were treated with Aza, as described in the section, and RNA expression was measured in mature offspring (n ≥ 6 from ≥2 dams). Data are expressed as means ± SEMs from at least 2 independent experiments. *P < .05, CON versus BBP. #P < .05, BBP versus BBP + Aza.
      Fatty acid desaturase 1 (Fads1) is an enzyme that regulates desaturation of fatty acids and is involved in the synthesis of long-chain polyunsaturated fatty acid. It has been shown that downregulation of the Fads gene is associated with lower polyunsaturated fatty acid levels and atopic eczema.
      • Chisaguano A.M.
      • Montes R.
      • Perez-Berezo T.
      • Castellote A.I.
      • Guerendiain M.
      • Bustamante M.
      • et al.
      Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.
      The second gene, Fanconi anemia complementation group A (Fanca), regulates the activity of regulatory T cells. Deletion of Fanca leads to a decreased efficiency to suppress effector T-cell function.
      • Du W.
      • Erden O.
      • Wilson A.
      • Sipple J.M.
      • Schick J.
      • Mehta P.
      • et al.
      Deletion of Fanca or Fancd2 dysregulates Treg in mice.
      Third, we focused on Zfpm1, also known as friend of GATA protein 1 (Fog1), a gene that acts as a repressor of GATA-3–mediated TH2 cell development.
      • Kurata H.
      • Lee H.J.
      • McClanahan T.
      • Coffman R.L.
      • O'Garra A.
      • Arai N.
      Friend of GATA is expressed in naive Th cells and functions as a repressor of GATA-3-mediated Th2 cell development.
      • Kitamura N.
      • Mori A.
      • Tatsumi H.
      • Nemoto S.
      • Hiroi T.
      • Kaminuma O.
      Zinc finger protein, multitype 1, suppresses human Th2 development via downregulation of IL-4.
      • Zhou M.
      • Ouyang W.
      • Gong Q.
      • Katz S.G.
      • White J.M.
      • Orkin S.H.
      • et al.
      Friend of GATA-1 represses GATA-3-dependent activity in CD4+ T cells.
      Therefore downregulation of Zfpm1 could promote a TH2-driven immune response. Validation of our RNA-sequencing results by using real-time PCR confirmed a reduced expression of all 3 genes in CD4+ T cells from 3-week-old offspring of BBP-exposed dams compared with control animals (Fig 6, C). In contrast, in BBP-exposed F0 mice expression of Fads1, Fanca, and Zfpm1 was not altered (Fig 6, C). Furthermore, downregulation of Fanca and Zfpm1 was also seen in full-grown offspring of BBP-exposed dams (8 to 12 weeks old), whereas the effect of maternal BBP exposure on Fads1 expression in adult mice was not significant (Fig 6, C). Aza treatment of BBP-treated pups not only alleviated the asthma-promoting effect, as described earlier, but also abrogated downregulation of Fanca and Zfpm1 in the mature F1 generation (Fig 6, D).
      To evaluate whether the potential mediators of phenotype development identified in our mouse model might also play a role in human subjects, we performed real-time PCR analysis in whole-blood samples of 4-year-old children of the LINA cohort. Interestingly, high urinary maternal MnBP concentrations (ie, MnBP concentrations greater than the median) were associated with a significant decrease in ZFPM1 expression in the children (MWU: P = .025; see Fig E3, A, in this article's Online Repository at www.jacionline.org) in accordance with our observations in the mouse model, no significant decrease of FADS1 or FANCA expression in prenatally MnBP-exposed children was observed (data not shown). Concomitant with a decrease in ZFPM1 expression, asthmatic children showed a significant increase in methylation at a CpG site (chromosome 16: 88564360, R = −0.87, P = .001) located in an intronic human enhancer region corresponding to the region observed in mice, which also in human subjects appears to regulate its host gene ZFPM1 (see Fig E3, B and C).

       Altered function of CD4+ T cells in the offspring of BBP-exposed mice

      Both Fanca and Zfpm1 are described to play a crucial role in T-cell differentiation and function.
      • Du W.
      • Erden O.
      • Wilson A.
      • Sipple J.M.
      • Schick J.
      • Mehta P.
      • et al.
      Deletion of Fanca or Fancd2 dysregulates Treg in mice.
      • Kurata H.
      • Lee H.J.
      • McClanahan T.
      • Coffman R.L.
      • O'Garra A.
      • Arai N.
      Friend of GATA is expressed in naive Th cells and functions as a repressor of GATA-3-mediated Th2 cell development.
      To investigate a possible effect of maternal BBP exposure on both of these end points in the next generation, we isolated splenic CD4+ effector T cells from 8-week-old offspring and stimulated the cells with anti-CD3/CD28 antibodies. Although proliferation of T cells was not affected (data not shown), CD4+ T cells from BBP-exposed offspring appeared more activatable than T cells from nonexposed control mice, as shown by expression of the activation marker CD25 (Fig 7, A). Interestingly, we detected significantly more IL-4–producing CD4+ T cells in the F1 generation from exposed mice, whereas the increased number of IL-13–producing cells reached borderline significance and the number of IFN-γ–producing cells was unaffected (Fig 7, B). Furthermore, we found a reduced number of forkhead box p3 (Foxp3)–positive regulatory T cells in stimulated CD4+CD25+ T cells from BBP-exposed offspring compared with cells derived from control mice (Fig 7, C). However, the described differences did not reach statistical significance. Furthermore, the frequency of Foxp3-positive regulatory T cells in the blood and in unstimulated splenocytes of BBP-exposed offspring was not affected (see Fig E4 in this article's Online Repository at www.jacionline.org). Accordingly, the efficiency of CD4+CD25+ regulatory T cells to suppress responder T cells was slightly but not significantly impaired by using cells from BBP-exposed mice compared with regulatory T cells from control animals (Fig 7, D).
      Figure thumbnail gr7
      Fig 7Altered function of CD4+ T cells in offspring of BBP-exposed mice. A-C, Numbers of CD4+CD25+ T cells (Fig 7, A), percentages of cytokine-producing T cells (Fig 7, B), and Foxp3+CD4+CD25+ T cells (Fig 7, C) are shown. D, Efficiency of CD4+CD25+ regulatory T (Treg) cells to suppress proliferation of responder T cells. Data are expressed as means ± SEMs (n ≥ 5 from 3 independent experiments). *P < .05.

       Maternal BBP exposure affects airway inflammation in the F2 generation

      To investigate whether the asthma-promoting effect of perinatal BBP exposure persists up to the F2 generation, we mated perinatally exposed female with nonexposed male mice and induced an asthma-like phenotype in the F2 generation (Fig 2, C). Surprisingly, exposure to BBP during pregnancy and breast-feeding enhanced the eosinophilic airway inflammation (Fig 8, A and B) and AHR (Fig 8, C) but did not alter OVA-specific IgE levels in the F2 generation (Fig 8, D). Because treatment of F2 pups with Aza reduced their increased airway inflammation in relation to BBP exposure of their grandmother, an involvement of epigenetic mechanisms, including DNA hypermethylation, in promoting this immune response is highly probable (Fig 8, A and C). However, we did not observe changes in DNA methylation related to BBP exposure in F2 pups in Fads1, Fanca (both promoters), or Zfpm1 DMRs (enhancer) identified in F1 pups, and these genes were not significantly differentially expressed (see Fig E5 in this article's Online Repository at www.jacionline.org).
      Figure thumbnail gr8
      Fig 8Maternal BBP exposure affects airway inflammation in the F2 generation. Total cell numbers in bronchoalveolar lavage (BAL) fluid (A), airway inflammation (B), lung resistance (C), and OVA-specific IgE levels (D) were examined in OVA-immunized F2 generation from prenatally BBP-exposed F1 dams compared with control mice. Data are expressed as means ± SEMs (n ≥ 9 animals per group in the F2 offspring [from ≥6 dams] from 6 independent experiments). *P < .05, OVA versus OVA + BBP. #P < .05, OVA + BBP versus OVA + BBP + Aza.

      Discussion

      The role of phthalate exposure in the increasing prevalence of atopic diseases has been a topic of great interest in recent years. However, from current studies, no clear conclusion can be drawn about the role of phthalates in asthma development or about the potential mechanisms involved.
      • Robinson L.
      • Miller R.
      The impact of bisphenol A and phthalates on allergy, asthma, and immune function: a review of latest findings.
      In the present study we showed that higher concentrations of MnBP in maternal urine during pregnancy were associated with an increased risk of asthma in children up to age 6, whereas none of the other phthalate metabolites analyzed were associated with this disease outcome. These results are in line with findings of a recent study by Whyatt et al,
      • Whyatt R.M.
      • Perzanowski M.S.
      • Just A.C.
      • Rundle A.G.
      • Donohue K.M.
      • Calafat A.M.
      • et al.
      Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort.
      who found a positive association between the maternal urine phthalate metabolites MnBP and MBzP and asthma risk in children between the ages of 5 and 11,
      • Whyatt R.M.
      • Perzanowski M.S.
      • Just A.C.
      • Rundle A.G.
      • Donohue K.M.
      • Calafat A.M.
      • et al.
      Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort.
      whereas other studies only found such a correlation for MBzP but not MnBP.
      • Gascon M.
      • Casas M.
      • Morales E.
      • Valvi D.
      • Ballesteros-Gomez A.
      • Luque N.
      • et al.
      Prenatal exposure to bisphenol A and phthalates and childhood respiratory tract infections and allergy.
      These discrepancies might in part be due to differences in study size but might also be related to regional differences in exposure pattern to the different members of the phthalate family. Interestingly, beyond the increased risk of asthma development by MnBP, we also observed a link between maternal MnBP concentrations and the risk for sensitization against inhalant allergens in the children, suggesting involvement of an atopic mechanism.
      MBzP and MnBP are both described as breakdown products of BBP,
      • Whyatt R.M.
      • Perzanowski M.S.
      • Just A.C.
      • Rundle A.G.
      • Donohue K.M.
      • Calafat A.M.
      • et al.
      Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort.
      with MnBP being the major metabolite of BBP and appearing in larger quantities in urine compared with MBzP.
      • Eigenberg D.A.
      • Bozigian H.P.
      • Carter D.E.
      • Sipes I.G.
      Distribution, excretion, and metabolism of butylbenzyl phthalate in the rat.
      • Nativelle C.
      • Picard K.
      • Valentin I.
      • Lhuguenot J.C.
      • Chagnon M.C.
      Metabolism of n-butyl benzyl phthalate in the female Wistar rat. Identification of new metabolites.
      Thus we investigated the effect of maternal BBP exposure on the asthma-like phenotype in our murine transgenerational model.
      Applied BBP exposure concentrations were based on the tolerable daily intake of BBP in human subjects of 0.5 mg/kg body weight per day,
      European Food Safety Initiative
      Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Butylbenzylphthalate (BBP) for use in food contact materials.
      which corresponds to an estimated uptake of 0.48 to 0.6 mg BBP/kg body weight per day in our mouse experiments (assuming 4-5 mL/d drinking water intake containing 3 μg/mL BBP). The resulting urinary MnBP concentrations in our murine model were similar to those observed in highly exposed mothers of the LINA cohort, suggesting that BBP concentrations investigated here are representative for the real exposure situation in human subjects.
      Although prenatal and perinatal exposure to BBP increased asthma severity in the offspring, exposure of adult mice did not affect the allergic immune response, further corroborating the notion that the most vulnerable time period for phthalate exposure is during pregnancy and shortly after birth.
      • Robinson L.
      • Miller R.
      The impact of bisphenol A and phthalates on allergy, asthma, and immune function: a review of latest findings.
      Another interesting finding was the inability of the BBP metabolites MnBP or MBzP to significantly increase allergic airway inflammation in the offspring when applied perinatally to the dams. These data point to a direct effect of the parent compound BBP, even without biotransformation. However, further insight into the BBP-mediated initial molecular events in the dams or the fetus has to be explored in future studies.
      There is growing evidence that prenatal and perinatal exposure to diverse environmental chemicals might dysregulate the fetal or neonatal epigenome, with potential consequences for diseases manifesting in childhood.
      • Perera F.
      • Herbstman J.
      Prenatal environmental exposures, epigenetics, and disease.
      Because the asthma-promoting effects by BBP in the present study were only detectable in the offspring from exposed dams, we investigated the effects of BBP on the epigenetic landscape. Previous studies already showed an effect on DNA methylation by certain phthalates but only based on targeted approaches restricted to preselected genes, such as glucose transporter type 4 (Glut4) or proinflammatory cytokines (TNF-α).
      • Wang I.J.
      • Karmaus W.J.
      • Chen S.L.
      • Holloway J.W.
      • Ewart S.
      Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
      • Sekaran S.
      • Jagadeesan A.
      In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny.
      • Rajesh P.
      • Balasubramanian K.
      Phthalate exposure in utero causes epigenetic changes and impairs insulin signalling.
      In the current study we subjected samples from our transgenerational mouse model to a genome-wide evaluation of DNA methylation changes related to BBP. This analysis was centered on the CD4+ T-cell subset because these cells have a central role in initiation and maintenance of the allergic immune response.
      • Larche M.
      • Robinson D.S.
      • Kay A.B.
      The role of T lymphocytes in the pathogenesis of asthma.
      Maternal BBP exposure led to global dysregulation in DNA methylation in the offspring dominated by DNA hypermethylation. In contrast to our findings, Wang et al
      • Wang I.J.
      • Karmaus W.J.
      • Chen S.L.
      • Holloway J.W.
      • Ewart S.
      Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
      described a phthalate-induced DNA hypomethylation to be responsible for their observed increased asthma risk. However, the study was based on methylation analysis of 21 selected genes measured in human blood samples compared with our genome-wide approach in a T-cell subset. Nevertheless, we also observed phthalate-induced hypomethylation, such as in the promoter of IL-4 (chromosome 11: 53620267-53620300) similar to what has been described by Wang et al
      • Wang I.J.
      • Karmaus W.J.
      • Chen S.L.
      • Holloway J.W.
      • Ewart S.
      Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
      in human subjects (data set 1). Counteracting the global DNA hypermethylation in our study by treating pups with the DNA methyltransferase inhibitor Aza prevented BBP-related exacerbation of allergic airway inflammation, suggesting a direct link between DNA hypermethylation and phenotype development.
      Although recent findings indicated a wide variety of relationships between DNA methylation and gene expression, high promotor/enhancer methylation is classically associated with low levels of gene expression.
      • Vinson C.
      • Chatterjee R.
      CG methylation.
      • Wagner J.R.
      • Busche S.
      • Ge B.
      • Kwan T.
      • Pastinen T.
      • Blanchette M.
      The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts.
      Therefore we focused on significantly repressed genes with a promoter or enhancer hypermethylation to further elucidate the functional translation of the BBP associated hypermethylation. In the offspring of BBP-exposed dams, we identified 13 genes fulfilling these criteria, including 3 interesting candidates involved in immune processes contributing to TH2 differentiation and therefore resulting in a higher susceptibility to experience allergic diseases.
      • Chisaguano A.M.
      • Montes R.
      • Perez-Berezo T.
      • Castellote A.I.
      • Guerendiain M.
      • Bustamante M.
      • et al.
      Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.
      • Du W.
      • Erden O.
      • Wilson A.
      • Sipple J.M.
      • Schick J.
      • Mehta P.
      • et al.
      Deletion of Fanca or Fancd2 dysregulates Treg in mice.
      • Kitamura N.
      • Mori A.
      • Tatsumi H.
      • Nemoto S.
      • Hiroi T.
      • Kaminuma O.
      Zinc finger protein, multitype 1, suppresses human Th2 development via downregulation of IL-4.
      Considering the longitudinal stability of differential RNA expression induced by maternal BBP exposure from 3-week-old pups to the adult offspring, Fanca and Zfpm1 appeared to be the most promising genes. Characterizing the CD4+ T-cell function from offspring of BBP-exposed dams primarily revealed an increased susceptibility for a TH2-driven immune response, as expected from downregulation of the GATA-3 repressor Zfpm1.
      • Kurata H.
      • Lee H.J.
      • McClanahan T.
      • Coffman R.L.
      • O'Garra A.
      • Arai N.
      Friend of GATA is expressed in naive Th cells and functions as a repressor of GATA-3-mediated Th2 cell development.
      In contrast, we could not show significant perturbations in regulatory T-cell function, which would have been one possible consequence of suppressed Fanca expression. Most interestingly, we could corroborate a possible mediating role of Zfpm1 in the phthalate effect by transferring the results of the mouse model to our LINA cohort. ZFPM1 mRNA expression was similarly decreased in children prenatally exposed to high levels of MnBP. This decrease in gene expression was correlated with an increase in ZFPM1 methylation in the same region identified as a differentially methylated enhancer in our mouse model. The importance of reduced expression of the GATA-3 repressor Zfpm1 by maternal BBP exposure is supported by various studies therapeutically targeting GATA-3 to attenuate the TH2-regulated inflammatory response.
      • Krug N.
      • Hohlfeld J.M.
      • Kirsten A.M.
      • Kornmann O.
      • Beeh K.M.
      • Kappeler D.
      • et al.
      Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme.
      • Homburg U.
      • Renz H.
      • Timmer W.
      • Hohlfeld J.M.
      • Seitz F.
      • Luer K.
      • et al.
      Safety and tolerability of a novel inhaled GATA3 mRNA targeting DNAzyme in patients with TH2-driven asthma.
      To investigate whether the BBP-induced asthma-promoting effect functions also in the absence of direct environmental exposures through germline transmission of an altered epigenome,
      • Rissman E.F.
      • Adli M.
      Minireview: transgenerational epigenetic inheritance: focus on endocrine disrupting compounds.
      we characterized asthma severity in the grandmotherly BBP-exposed F2 generation. Although we observed increased airway inflammation, the phenotype compared with the F1 generation was slightly different, showing no effect on IgE serum levels. Furthermore, neither DNA methylation nor RNA expression of Fads1, Fanca, and Zfpm1 were affected in the F2 generation, suggesting that the underlying mechanisms of an exacerbated asthma-like phenotype in the F1 and F2 generations are different.
      In summary, data from our study strongly suggest that early exposure to specific phthalates increases susceptibility to the development of allergic asthma in the offspring and even contributes to airway inflammation in the F2 generation. The asthma-promoting effect was mediated by epigenetic changes, leading to altered expression of genes that play a crucial role in immune regulation. Therefore chemical exposure of the developing immune system leads to an altered epigenetic prenatal programming, contributing not only to asthma development in later life but potentially also to other immune-regulated diseases.
      Key messages
      • Maternal MnBP levels associate with an increased asthma risk in children.
      • In mice maternal exposure to BBP enhances airway inflammation in the F1 and F2 generations through epigenetic alterations.
      We thank Marita Reiprich, Michaela Loschinski, Beate Fink, Stephan Schreiber, Marion Bähr, Monika Helf, Oliver Mücke, and Melanie Bänsch for excellent technical assistance and all participants of the LINA study. We thank the DKFZ Genomics and Proteomics Core Facility for the excellent technical support and expertise.

      Supplementary data

      Figure thumbnail figs1
      Figure thumbnail figs2
      Figure thumbnail figs3
      Figure thumbnail figs4
      Figure thumbnail figs5

      References

        • Umetsu D.T.
        • McIntire J.J.
        • Akbari O.
        • Macaubas C.
        • DeKruyff R.H.
        Asthma: an epidemic of dysregulated immunity.
        Nat Immunol. 2002; 3: 715-720
        • Pearce N.
        • Douwes J.
        The global epidemiology of asthma in children.
        Int J Tuberc Lung Dis. 2006; 10: 125-132
        • Tachdjian R.
        • Mathias C.
        • Al Khatib S.
        • Bryce P.J.
        • Kim H.S.
        • Blaeser F.
        • et al.
        Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma.
        J Exp Med. 2009; 206: 2191-2204
        • Dick S.
        • Friend A.
        • Dynes K.
        • AlKandari F.
        • Doust E.
        • Cowie H.
        • et al.
        A systematic review of associations between environmental exposures and development of asthma in children aged up to 9 years.
        BMJ Open. 2014; 4: e006554
        • Alessandrini F.
        • Schulz H.
        • Takenaka S.
        • Lentner B.
        • Karg E.
        • Behrendt H.
        • et al.
        Effects of ultrafine carbon particle inhalation on allergic inflammation of the lung.
        J Allergy Clin Immunol. 2006; 117: 824-830
        • Bönisch U.
        • Böhme A.
        • Kohajda T.
        • Mögel I.
        • Schütze N.
        • von Bergen M.
        • et al.
        Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model.
        PLoS One. 2012; 7: e39817
        • Franck U.
        • Weller A.
        • Roder S.W.
        • Herberth G.
        • Junge K.M.
        • Kohajda T.
        • et al.
        Prenatal VOC exposure and redecoration are related to wheezing in early infancy.
        Environ Int. 2014; 73: 393-401
        • Martino D.
        • Prescott S.
        Epigenetics and prenatal influences on asthma and allergic airways disease.
        Chest. 2011; 139: 640-647
        • Kabesch M.
        Epigenetics in asthma and allergy.
        Curr Opin Allergy Clin Immunol. 2014; 14: 62-68
        • Reiprich M.
        • Rudzok S.
        • Schutze N.
        • Simon J.C.
        • Lehmann I.
        • Trump S.
        • et al.
        Inhibition of endotoxin-induced perinatal asthma protection by pollutants in an experimental mouse model.
        Allergy. 2013; 68: 481-489
        • Conrad M.L.
        • Ferstl R.
        • Teich R.
        • Brand S.
        • Blumer N.
        • Yildirim A.O.
        • et al.
        Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78.
        J Exp Med. 2009; 206: 2869-2877
        • Dodson R.E.
        • Nishioka M.
        • Standley L.J.
        • Perovich L.J.
        • Brody J.G.
        • Rudel R.A.
        Endocrine disruptors and asthma-associated chemicals in consumer products.
        Environ Health Perspect. 2012; 120: 935-943
        • Braun J.M.
        • Sathyanarayana S.
        • Hauser R.
        Phthalate exposure and children's health.
        Curr Opin Pediatr. 2013; 25: 247-254
        • Adibi J.J.
        • Whyatt R.M.
        • Williams P.L.
        • Calafat A.M.
        • Camann D.
        • Herrick R.
        • et al.
        Characterization of phthalate exposure among pregnant women assessed by repeat air and urine samples.
        Environ Health Perspect. 2008; 116: 467-473
        • Wormuth M.
        • Scheringer M.
        • Vollenweider M.
        • Hungerbuhler K.
        What are the sources of exposure to eight frequently used phthalic acid esters in Europeans?.
        Risk Anal. 2006; 26: 803-824
        • Bornehag C.G.
        • Sundell J.
        • Weschler C.J.
        • Sigsgaard T.
        • Lundgren B.
        • Hasselgren M.
        • et al.
        The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study.
        Environ Health Perspect. 2004; 112: 1393-1397
        • Bertelsen R.J.
        • Carlsen K.C.
        • Calafat A.M.
        • Hoppin J.A.
        • Haland G.
        • Mowinckel P.
        • et al.
        Urinary biomarkers for phthalates associated with asthma in Norwegian children.
        Environ Health Perspect. 2013; 121: 251-256
        • Robinson L.
        • Miller R.
        The impact of bisphenol A and phthalates on allergy, asthma, and immune function: a review of latest findings.
        Curr Environ Health Rep. 2015; 2: 379-387
        • Hinz D.
        • Simon J.C.
        • Maier-Simon C.
        • Milkova L.
        • Roder S.
        • Sack U.
        • et al.
        Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood.
        Clin Exp Allergy. 2010; 40: 419-426
        • Weisse 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: 220-228
        • Herberth G.
        • Bauer M.
        • Gasch M.
        • Hinz D.
        • Roder S.
        • Olek S.
        • et al.
        Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.
        J Allergy Clin Immunol. 2014; 133: 543-550
        • Feltens R.
        • Roeder S.
        • Otto W.
        • Borte M.
        • Lehmann I.
        • von Bergen M.
        • et al.
        Evaluation of population and individual variances of urinary phthalate metabolites in terms of epidemiological studies.
        J Chromatogr Sep Tech. 2015; 6: 290
        • Polte T.
        • Petzold S.
        • Bertrand J.
        • Schutze N.
        • Hinz D.
        • Simon J.C.
        • et al.
        Critical role for syndecan-4 in dendritic cell migration during development of allergic airway inflammation.
        Nat Commun. 2015; 6: 7554
        • Polte T.
        • Foell J.
        • Werner C.
        • Hoymann H.G.
        • Braun A.
        • Burdach S.
        • et al.
        CD137-mediated immunotherapy for allergic asthma.
        J Clin Invest. 2006; 116: 1025-1036
        • Schutze N.
        • Lehmann I.
        • Bonisch U.
        • Simon J.C.
        • Polte T.
        Exposure to mycotoxins increases the allergic immune response in a murine asthma model.
        Am J Respir Crit Care Med. 2010; 181: 1188-1199
        • Wang Q.
        • Gu L.
        • Adey A.
        • Radlwimmer B.
        • Wang W.
        • Hovestadt V.
        • et al.
        Tagmentation-based whole-genome bisulfite sequencing.
        Nat Protoc. 2013; 8: 2022-2032
        • Bauer T.
        • Trump S.
        • Ishaque N.
        • Thurmann L.
        • Gu L.
        • Bauer M.
        • et al.
        Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.
        Mol Syst Biol. 2016; 12: 861
        • Hackermuller J.
        • Reiche K.
        • Otto C.
        • Hosler N.
        • Blumert C.
        • Brocke-Heidrich K.
        • et al.
        Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs.
        Genome Biol. 2014; 15: R48
        • Martin M.
        Cutadapt removes adapter sequences from high-throughput sequencing reads.
        EMBnet.journal. 2011; 17: 10-12
        • Hoffmann S.
        • Otto C.
        • Doose G.
        • Tanzer A.
        • Langenberger D.
        • Christ S.
        • et al.
        A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection.
        Genome Biol. 2014; 15: R34
        • Eigenberg D.A.
        • Bozigian H.P.
        • Carter D.E.
        • Sipes I.G.
        Distribution, excretion, and metabolism of butylbenzyl phthalate in the rat.
        J Toxicol Environ Health. 1986; 17: 445-456
        • Polte T.
        • Hennig C.
        • Hansen G.
        Allergy prevention starts before conception: maternofetal transfer of tolerance protects against the development of asthma.
        J Allergy Clin Immunol. 2008; 122: 1022-1030.e5
        • Petzold S.
        • Averbeck M.
        • Simon J.C.
        • Lehmann I.
        • Polte T.
        Lifetime-dependent effects of bisphenol A on asthma development in an experimental mouse model.
        PLoS One. 2014; 9: e100468
        • Brand S.
        • Kesper D.A.
        • Teich R.
        • Kilic-Niebergall E.
        • Pinkenburg O.
        • Bothur E.
        • et al.
        DNA methylation of TH1/TH2 cytokine genes affects sensitization and progress of experimental asthma.
        J Allergy Clin Immunol. 2012; 129: 1602-1610.e6
        • Chisaguano A.M.
        • Montes R.
        • Perez-Berezo T.
        • Castellote A.I.
        • Guerendiain M.
        • Bustamante M.
        • et al.
        Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.
        PLoS One. 2013; 8: e78245
        • Du W.
        • Erden O.
        • Wilson A.
        • Sipple J.M.
        • Schick J.
        • Mehta P.
        • et al.
        Deletion of Fanca or Fancd2 dysregulates Treg in mice.
        Blood. 2014; 123: 1938-1947
        • Kurata H.
        • Lee H.J.
        • McClanahan T.
        • Coffman R.L.
        • O'Garra A.
        • Arai N.
        Friend of GATA is expressed in naive Th cells and functions as a repressor of GATA-3-mediated Th2 cell development.
        J Immunol. 2002; 168: 4538-4545
        • Kitamura N.
        • Mori A.
        • Tatsumi H.
        • Nemoto S.
        • Hiroi T.
        • Kaminuma O.
        Zinc finger protein, multitype 1, suppresses human Th2 development via downregulation of IL-4.
        Int Arch Allergy Immunol. 2011; 155: 53-56
        • Zhou M.
        • Ouyang W.
        • Gong Q.
        • Katz S.G.
        • White J.M.
        • Orkin S.H.
        • et al.
        Friend of GATA-1 represses GATA-3-dependent activity in CD4+ T cells.
        J Exp Med. 2001; 194: 1461-1471
        • Whyatt R.M.
        • Perzanowski M.S.
        • Just A.C.
        • Rundle A.G.
        • Donohue K.M.
        • Calafat A.M.
        • et al.
        Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort.
        Environ Health Perspect. 2014; 122: 1141-1146
        • Gascon M.
        • Casas M.
        • Morales E.
        • Valvi D.
        • Ballesteros-Gomez A.
        • Luque N.
        • et al.
        Prenatal exposure to bisphenol A and phthalates and childhood respiratory tract infections and allergy.
        J Allergy Clin Immunol. 2015; 135: 370-378
        • Nativelle C.
        • Picard K.
        • Valentin I.
        • Lhuguenot J.C.
        • Chagnon M.C.
        Metabolism of n-butyl benzyl phthalate in the female Wistar rat. Identification of new metabolites.
        Food Chem Toxicol. 1999; 37: 905-917
        • European Food Safety Initiative
        Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Butylbenzylphthalate (BBP) for use in food contact materials.
        European Food Safety Initiative Food Ingredients and Packaging, Parma (Italy)2005
        • Perera F.
        • Herbstman J.
        Prenatal environmental exposures, epigenetics, and disease.
        Reprod Toxicol. 2011; 31: 363-373
        • Wang I.J.
        • Karmaus W.J.
        • Chen S.L.
        • Holloway J.W.
        • Ewart S.
        Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
        Clin Epigenetics. 2015; 7: 27
        • Sekaran S.
        • Jagadeesan A.
        In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny.
        J Cell Biochem. 2015; 116: 1466-1477
        • Rajesh P.
        • Balasubramanian K.
        Phthalate exposure in utero causes epigenetic changes and impairs insulin signalling.
        J Endocrinol. 2014; 223: 47-66
        • Larche M.
        • Robinson D.S.
        • Kay A.B.
        The role of T lymphocytes in the pathogenesis of asthma.
        J Allergy Clin Immunol. 2003; 111: 450-464
        • Vinson C.
        • Chatterjee R.
        CG methylation.
        Epigenomics. 2012; 4: 655-663
        • Wagner J.R.
        • Busche S.
        • Ge B.
        • Kwan T.
        • Pastinen T.
        • Blanchette M.
        The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts.
        Genome Biol. 2014; 15: R37
        • Krug N.
        • Hohlfeld J.M.
        • Kirsten A.M.
        • Kornmann O.
        • Beeh K.M.
        • Kappeler D.
        • et al.
        Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme.
        N Engl J Med. 2015; 372: 1987-1995
        • Homburg U.
        • Renz H.
        • Timmer W.
        • Hohlfeld J.M.
        • Seitz F.
        • Luer K.
        • et al.
        Safety and tolerability of a novel inhaled GATA3 mRNA targeting DNAzyme in patients with TH2-driven asthma.
        J Allergy Clin Immunol. 2015; 136: 797-800
        • Rissman E.F.
        • Adli M.
        Minireview: transgenerational epigenetic inheritance: focus on endocrine disrupting compounds.
        Endocrinology. 2014; 155: 2770-2780