Pharmacological Improvement of Cystic Fibrosis Transmembrane Conductance Regulator Function Rescues Airway Epithelial Homeostasis and Host Defense in Children with Cystic Fibrosis.

Rationale: Pharmacological improvement of cystic fibrosis transmembrane conductance regulator (CFTR) function with elexacaftor/tezacaftor/ivacaftor (ETI) provides unprecedented improvements in lung function and other clinical outcomes in patients with cystic fibrosis (CF). However, ETI effects on impaired mucosal homeostasis and host defense at the molecular and cellular levels in the airways of patients with CF remain unknown. Objectives: To investigate effects of ETI on the transcriptome of nasal epithelial and immune cells from children with CF at the single-cell level. Methods: Nasal swabs from 13 children with CF and at least one F508del allele aged 6 to 11 years were collected at baseline and 3 months after initiation of ETI, subjected to single-cell RNA sequencing, and compared with swabs from 12 age-matched healthy children. Measurements and Main Results: Proportions of CFTR-positive cells were decreased in epithelial basal, club, and goblet cells, but not in ionocytes, from children with CF at baseline and were restored by ETI therapy to nearly healthy levels. Single-cell transcriptomics revealed an impaired IFN signaling and reduced expression of major histocompatibility complex classes I and II encoding genes in epithelial cells of children with CF at baseline, which was partially restored by ETI. In addition, ETI therapy markedly reduced the inflammatory phenotype of immune cells, particularly of neutrophils and macrophages. Conclusions: Pharmacological improvement of CFTR function improves innate mucosal immunity and reduces immune cell inflammatory responses in the upper airways of children with CF at the single-cell level, highlighting the potential to restore epithelial homeostasis and host defense in CF airways by early initiation of ETI therapy.

Global hypomethylation in childhood asthma identified by genome-wide DNA-methylation sequencing preferentially affects enhancer regions.

BACKGROUND: Childhood asthma is a result of a complex interaction of genetic and environmental components causing epigenetic and immune dysregulation, airway inflammation and impaired lung function. Although different microarray based EWAS studies have been conducted, the impact of epigenetic regulation in asthma development is still widely unknown. We have therefore applied unbiased whole genome bisulfite sequencing (WGBS) to characterize global DNA-methylation profiles of asthmatic children compared to healthy controls. METHODS: Peripheral blood samples of 40 asthmatic and 42 control children aged 5-15 years from three birth cohorts were sequenced together with paired cord blood samples. Identified differentially methylated regions (DMRs) were categorized in genotype-associated, cell-type-dependent, or prenatally primed. Network analysis and subsequent natural language processing of DMR-associated genes was complemented by targeted analysis of functional translation of epigenetic regulation on the transcriptional and protein level. RESULTS: In total, 158 DMRs were identified in asthmatic children compared to controls of which 37% were related to the eosinophil content. A global hypomethylation was identified affecting predominantly enhancer regions and regulating key immune genes such as IL4, IL5RA, and EPX. These DMRs were confirmed in n = 267 samples and could be linked to aberrant gene expression. Out of the 158 DMRs identified in the established phenotype, 56 were perturbed already at birth and linked, at least in part, to prenatal influences such as tobacco smoke exposure or phthalate exposure. CONCLUSION: This is the first epigenetic study based on whole genome sequencing to identify marked dysregulation of enhancer regions as a hallmark of childhood asthma.

Prenatal paraben exposure and atopic dermatitis-related outcomes among children.

BACKGROUND: Parabens, widely used as preservatives in cosmetics, foods, and other consumer products, are suspected of contributing to allergy susceptibility. The detection of parabens in the placenta or amniotic fluid raised concerns about potential health consequences for the child. Recently, an increased asthma risk following prenatal exposure has been reported. Here, we investigated whether prenatal paraben exposure can influence the risk for atopic dermatitis (AD). METHODS: 261 mother-child pairs of the German mother-child study LINA were included in this analysis. Eight paraben species were quantified in maternal urine obtained at gestational week 34. According to the parental report of physician-diagnosed AD from age 1 to 8 years, disease onset, and persistence, childhood AD was classified into four different phenotypes. RESULTS: 4.6% (n = 12) and 12.3% (n = 32) of the children were classified as having very early-onset AD (until age two) either with or without remission, 11.9% (n = 31) as early-onset (after age two), and 3.1% (n = 8) as childhood-onset AD (after age six). Exposure to ethylparaben and n-butylparaben was associated with an increased risk to develop very early-onset AD without remission (EtP: adj.OR/95% CI:1.44/1.04-2.00,nBuP:adj.OR/95% CI:1.95/1.22-3.12). The effects of both parabens were predominant in children without a history of maternal AD and independent of children's sex. CONCLUSION: Prenatal EtP or nBuP exposure may increase children's susceptibility for persistent AD with disease onset at very early age. This association was particularly pronounced in children without a history of maternal AD, indicating that children without a genetic predisposition are more susceptible to paraben exposure.

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

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.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Undiagnosed Pediatric Elevated Blood Pressure Is Characterized by Induction of Proinflammatory and Cytotoxic Mediators.

BACKGROUND: Inflammatory processes have been suggested as a culprit of vascular damage in pediatric hypertension. We aimed to investigate transcriptional changes of immune modulators and determine their association with office blood pressure in adolescents who were not diagnosed with hypertension at the time of the study visit. METHODS: Office blood pressure measurements and blood samples were taken from adolescents of 2 German birth cohorts, GINIplus (The German Infant Study on the Influence of Nutrition Intervention Plus Air Pollution and Genetics on Allergy Development; discovery cohort, n=1219) and LISA (Influences of Lifestyle-related factors on the Immune System and the Development of Allergies in Childhood; validation cohort, n=809), during the 15-year follow-up visit and categorized based on the European Society of Hypertension Guideline. Hs-CRP (high-sensitivity C-reactive protein) and expression of 51 genes encoding cytokines/receptors and transcription factors were analyzed. RESULTS: The prevalence of elevated systolic blood pressure (overweight/obese) was 14.0% (5.1%) and 16.4% (5.2%) in the discovery and validation cohorts, respectively. An enhanced cytotoxic (GZMB, PRF1, IL2RB) and proinflammatory (FOS, IL1B, hs-CRP) immune profile was observed in association with the hypertension class in both cohorts. Expression of hs-CRP and IL1B was driven by overweight with IL1B being identified as a mediator between body mass index and elevated systolic blood pressure (adj.ß/95% CI, 0.01/0.0002-0.02). The association of GZMB (adjusted odds ratio/95% CI, 1.67/1.26-2.21; P=0.0004) and PRF1 (adjusted odds ratio/95% CI, 1.70/1.26-2.29; P=0.0005) in the hypertension class remained significant in normal-weight individuals without parental predisposition. These effects were confirmed in LISA. CONCLUSIONS: Adolescent hypertension is not limited to known risk groups. As adolescents in the hypertension class show an inflammatory profile similar to that of established hypertension in adults, blood pressure monitoring at a young age is critical to ensure early intervention and prevention of adverse sequelae.

Effects of exposure to single and multiple parabens on asthma development in an experimental mouse model and a prospective cohort study.

Parabens are widely used preservatives present in consumer products like cosmetics and food. Although several epidemiological studies suggest that early-life exposure to parabens might alter the immune response and allergy risk in childhood, the evidence with respect to asthma is not clear. Therefore, we investigated the effect of paraben exposure on asthma development in mice and humans. Using a murine asthma model the experimental data show both, an asthma-reducing effect after direct exposure of adult mice to n-butyl paraben (nBuP) as well as an asthma-promoting effect after maternal exposure to ethyl paraben (EtP) in the female offspring. Interestingly, exposure of mice to a mixture of EtP and nBuP starting prenatally until the end of asthma induction in the adult offspring was without effect on allergic airway inflammation. In addition, parabens were determined within the German prospective mother-child cohort LINA and their single and mixture effect on asthma development in children within the first 10 years of life was estimated by logistic and Bayesian kernel machine regression (BKMR). Both approaches revealed no adverse effects of parabens on children's asthma development, neither when stratified for being at risk due to a positive family history of atopy nor when analysed separately for sex specificity. Therefore, we conclude that although single parabens might differentially impact asthma development, an adverse effect could not be seen in a multiple paraben exposure setting. Consequently, not only the time point of exposure but also multiple exposure scenarios to parabens should be considered in the evaluation of individuals' specific disease risk.

Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19.

In coronavirus disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying causes and the effects of the main anti-hypertensive therapies-angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)-remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical COVID-19 progression. ACEI treatment was associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic antiviral responses, whereas ARB treatment related to enhanced epithelial-immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.

Maternal paraben exposure triggers childhood overweight development.

Parabens are preservatives widely used in consumer products including cosmetics and food. Whether low-dose paraben exposure may cause adverse health effects has been discussed controversially in recent years. Here we investigate the effect of prenatal paraben exposure on childhood overweight by combining epidemiological data from a mother-child cohort with experimental approaches. Mothers reporting the use of paraben-containing cosmetic products have elevated urinary paraben concentrations. For butyl paraben (BuP) a positive association is observed to overweight within the first eight years of life with a stronger trend in girls. Consistently, maternal BuP exposure of mice induces a higher food intake and weight gain in female offspring. The effect is accompanied by an epigenetic modification in the neuronal Pro-opiomelanocortin (POMC) enhancer 1 leading to a reduced hypothalamic POMC expression. Here we report that maternal paraben exposure may contribute to childhood overweight development by altered POMC-mediated neuronal appetite regulation.

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis.

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.

Elevated Gestational IL-13 During Fetal Development Is Associated With Hyperactivity and Inattention in Eight-Year-Old Children.

Maternal immune activation (MIA) during fetal development leads to behavioral and psychological disorders in the offspring. Concomitantly, insufficient supply of polyunsaturated fatty acids (PUFAs) is suspected to contribute to early neuronal maldevelopment due to the immune modulatory capabilities of PUFAs. However, human data are missing considering both of these aspects and their impact on children's behavioral outcomes. In line, this study aimed to elucidate the influence of gestational cytokines and PUFA-containing lipids during late pregnancy on behavioral sequelae in childhood, particularly focusing on an immune activation shaped by a history of maternal atopic diseases instead of a pathogen-mediated immune response. Based on the prospective mother-child cohort LINA we assessed the unstimulated blood cytokine profiles and concentrations of PUFA-containing lipids of 293 mothers at the 34th week of pregnancy. Maternal history of atopic diseases was obtained from questionnaires and behavior in eight-year-old children was assessed by the standardized Strength and Difficulties Questionnaires (SDQ) generating scores for hyperactivity/inattention, emotional symptoms, conduct problems, and peer relationship problems. Elevated IL-13 increased the risk for the child to show behavioral difficulties, in particular, hyperactive/inattentive behavior [adj. OR (95% CI): 2.47 (1.51-4.02), n = 255 vs. 38] at the age of eight years. Although the presence of maternal atopic dermatitis (AD) was associated with increased gestational IL-13 concentrations [adj. MR (95% CI): 1.17 (1.04-1.32)], no effect on children's behavioral difficulties was observed. However, a decrease in the PUFA containing lipid species PC aa C38:6 was not only associated with an increased gestational IL-13 concentration but also mediated the indirect effect of low PC aa C38:6 concentrations on children's abnormal behavior independent of maternal AD. We additionally assessed whether maternal IL-13 and PC aa C38:6 concentrations translate their effect by altering children's cord blood PC aa C38:6 and IL-13. While also the children's cord blood IL-13 was related to children's behavior, no effect of children's PC aa C38:6 was observed. This is the first study demonstrating that elevated gestational IL-13 increases the risk for children to develop behavioral difficulties. Analyses suggest that a reduced supply of gestational PC aa C38:6 contributes to elevated gestational IL-13 leading to behavioral sequelae in the offspring.

MEST mediates the impact of prenatal bisphenol A exposure on long-term body weight development.

Background: Exposure to endocrine-disrupting chemicals can alter normal physiology and increase susceptibility to non-communicable diseases like obesity. Especially the prenatal and early postnatal period is highly vulnerable to adverse effects by environmental exposure, promoting developmental reprogramming by epigenetic alterations. To obtain a deeper insight into the role of prenatal bisphenol A (BPA) exposure in children's overweight development, we combine epidemiological data with experimental models and BPA-dependent DNA methylation changes. Methods: BPA concentrations were measured in maternal urine samples of the LINA mother-child-study obtained during pregnancy (n = 552), and BPA-associated changes in cord blood DNA methylation were analyzed by Illumina Infinium HumanMethylation450 BeadChip arrays (n = 472). Methylation changes were verified by targeted MassARRAY analyses, assessed for their functional translation by qPCR and correlated with children's body mass index (BMI) z scores at the age of 1 and 6 years. Further, female BALB/c mice were exposed to BPA from 1 week before mating until delivery, and weight development of their pups was monitored (n ≥ 8/group). Additionally, human adipose-derived mesenchymal stem cells were treated with BPA during the adipocyte differentiation period and assessed for exposure-related epigenetic, transcriptional and morphological changes (n = 4). Results: In prenatally BPA-exposed children two CpG sites with deviating cord blood DNA-methylation profiles were identified, among them a hypo-methylated CpG in the promoter of the obesity-associated mesoderm-specific transcript (MEST). A mediator analysis suggested that prenatal BPA exposure was connected to cord blood MEST promoter methylation and MEST expression as well as BMI z scores in early infancy. This effect could be confirmed in mice in which prenatal BPA exposure altered Mest promoter methylation and transcription with a concomitant increase in the body weight of the juvenile offspring. An experimental model of in vitro differentiated human mesenchymal stem cells also revealed an epigenetically induced MEST expression and enhanced adipogenesis following BPA exposure. Conclusions: Our study provides evidence that MEST mediates the impact of prenatal BPA exposure on long-term body weight development in offspring by triggering adipocyte differentiation.

Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer.

Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Prenatal maternal stress and wheeze in children: novel insights into epigenetic regulation.

Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children's life.

Tobacco smoking differently influences cell types of the innate and adaptive immune system-indications from CpG site methylation.

BACKGROUND: Tobacco smoke is worldwide one of the main preventable lifestyle inhalative pollutants causing severe adverse health effects. Epidemiological studies revealed association of tobacco smoking with epigenetic changes at single CpGs in blood. However, the biological relevance of the often only marginal methylation changes remains unclear. RESULTS: Comparing genome-wide changes in CpG methylation of three recently reported epidemiological datasets, two obtained on whole blood and one on peripheral blood mononuclear cells (PBMCs), it becomes evident that the majority of methylation changes (86.7 and 93.3 %) in whole blood account for changes in granulocytes. Analyzing, in more detail, seven highly significant reported smoking-induced methylation changes at single CpGs in different blood cell types of healthy volunteers (n = 32), we confirmatively found a strong cell-type specificity. Two CpGs in GFI1 and F2RL3 were significantly hypomethylated in granulocytes (-11.3 %, p = 0.001; -8.7 %, p = 0.001, respectively) but not in PBMCs of smokers while two CpGs in CPOX and GPR15 were found to be hypomethylated in PBMC (-4.3 %, p = 0.003; -4.2 %, P = 0.009, respectively) and their subtypes of GPR15 non-expressing (-3.2 %, p = 0.027; -2.5 %, p = 0.032, respectively) and smoking-evoked GPR15 expressing T cells (-15.8 %, p < 0.001; -13.8 %, p = 0.018, respectively) but not in granulocytes. In contrast, cg05575921 within AHRR was hypomethylated in every analyzed cell type of smokers, but with a different degree. Both, hypomethylation at cg05575921 in granulocytes (-55.2 % methylation change in smokers, p < 0.001) and the frequency of GPR15+ T cells (9.8-37.1 % in smokers), possessing a specific hypomethylation at cg19859270, were strongly associated with smoking behavior at individual level and could therefore serve as valuable biomarkers indicating a disturbed homeostasis in smokers. In contrast to the reported long-term persistent methylation changes in adult smokers after cessation, the hypomethylation at cg05575921 in prenatally tobacco smoke-exposed children (n = 13) from our LINA cohort was less stable and disappeared already within 2 years after birth. CONCLUSIONS: Studying cell type-specific methylation changes provides helpful information regarding the biological relevance of epigenetic modifications. Here, we could show that smoking differently affects both cells of the innate and adaptive immune systems.