Genes, environment, and developmental timing: New insights from translational approaches to understand early origins of respiratory diseases

Abstract

Over the past decade, “omics” approaches have advanced our understanding of the molecular programming of the airways in humans. Several studies have identified potential molecular mechanisms that contribute to early life epigenetic reprogramming, including DNA methylation, histone modifications, microRNAs, and the homeostasis of the respiratory mucosa (epithelial function and microbiota). Current evidence supports the notion that early infancy is characterized by heightened susceptibility to airway genetic reprogramming in response to the first exposures in life, some of which can have life-long consequences. Here, we summarize and analyze the latest insights from studies that support a novel epigenetic paradigm centered on human maturational and developmental programs including three cardinal elements: genes, environment, and developmental timing. The combination of these factors is likely responsible for the functional trajectory of the respiratory system at the molecular, functional, and clinical levels.

1 |. INTRODUCTION: EARLY LIFE AS THE CRITICAL DEVELOPMENTAL PERIOD FOR GENE–ENVIRONMENTAL INTERACTIONS IN THE RESPIRATORY SYSTEM

Studies over the past three decades have established that early life environmental influences define the individual risk for respiratory diseases.^1–5 For instance, severe viral respiratory infections taking place in early life are strongly associated with an increased susceptibility for asthma and chronic obstructive pulmonary disease (COPD).^1,6 Longitudinal birth cohorts have provided conclusive evidence that the molecular phenotype and microenvironment of the respiratory mucosa (e.g., nasal microbial commensals and concomitant inflammation) are predictive of future respiratory health and disease.^7–10 Some studies also suggest that the determinants of respiratory disease may begin during intrauterine life.^11,12 Among these, determinants, maternal obesity seems to have a dramatic impact on the development of respiratory illnesses in the offspring, during infancy, and beyond.^13–15

The impact of these studies indicates that the traditional “gene–environmental interaction model” must be expanded to include the developmental timing of the human airways. Early life represents a critical time point in which perturbations (e.g., environmental exposures) can have a dramatic impact on the developmental trajectory of a given individual and consequently lead to a life-long risk for respiratory disorders. An example of the complex interplay between genes, environment, and developmental timing can be seen in the relationship between early viral infections and the risk of respiratory diseases. Several studies have shown that genetic variants at the 17q21 locus are strongly associated with asthma risk in children who had rhinovirus wheezing illnesses.¹⁶ However, among young children exposed to animal barns in the first year of life, these genetic variants are protective.¹⁷ These findings indicate that a precise combination of genetic background and early life events may shape the individual life-long risk for respiratory diseases.

In this review, we summarize and analyze the latest insights from studies that have increased our understanding of the mechanisms of early life epigenetic programming of the respiratory system. From the molecular perspective, we are taking a broad approach to include different epigenetic mechanisms with complex interactions, including DNA modifications (e.g. methylation), posttranscriptional changes (e.g., microRNAs [miRNAs]), and the biology of the mucosal barrier as a critical interface for genetic–environmental interactions during early life.

2 |. MOLECULAR EVIDENCE OF A STEREOTYPICAL DEVELOPMENTAL PROGRAM AND A “WINDOW OF SUSCEPTIBILITY” FOR GENETIC REPROGRAMMING IN HUMANS

Birth cohorts have shown that the epigenetic influences on human development begin in utero.¹⁸ Results from the Boston birth cohort, one of the largest birth cohorts in the U.S. (>3500 maternal–infant dyads in active follow-up) showed that DNA methylation patterns are largely established in utero as cord blood signatures are more than 99% identical than those observed at 2 years of age.¹⁸ A remarkable exception to this pattern is the DNA methylation of immune genes, which continues to evolve during early infancy showing substantial variability during the first years of life.^18,19 The finding that the developmental program of the immune system is shaped by the environment in early life has been further illustrated by a longitudinal study that included a comprehensive immune analysis of newborns (n = 100) with high resolution transcriptomic and functional prospective signatures of infant immune cell populations.²⁰ This study demonstrated that (1) cord blood signatures are not representative of postnatal immunity, and (2) stereotypical postnatal developmental gene expression trajectories, which seem to be associated with specific microbial interactions, are established during the first weeks of life.²⁰ Collectively, these studies provide solid evidence that during early life, environmental influences shape the developmental molecular program of the immune system in humans. However, little is still known about the molecular reprogramming of the respiratory system in newborns and human infants. Filling this critical gap may provide unique insights into how the earliest environmental airway exposures can modify stereotypical developmental programs and define individual life-long susceptibility and/or resilience to respiratory disorders during and beyond childhood.

3 |. EPIGENETIC MECHANISMS AND EVIDENCE LINKING GENE–ENVIRONMENTAL INTERACTIONS WITH PEDIATRIC RESPIRATORY DISORDERS

Epigenetic information is essential for organogenesis, developmental processes, and postnatal differentiation and maturation.^21–23 Epigenetic changes have been mapped in multiple lineages and developmental windows in which they are essential. For instance, following fertilization, the parental genomes undergo extensive histone modifications and global demethylation, to allow for initial cell totipotency and facilitate the establishment of embryonic patterns.^24,25 Later, embryogenesis and fetal development are characterized by cell lineage-specific epigenetic remodeling that is responsible for cell differentiation.²⁵ The epigenome is influenced by the environment and undergoes dynamic changes during development and aging.^26–30 Thus, it is important to keep in mind that epigenetic mechanisms are not only referring to “gene–environmental interactions” but also to normal developmental and maturational processes. From the molecular biology perspective, epigenetics has mostly been centered on heritable alterations that are not due to changes in DNA sequence.^21,23 Classical epigenetic mechanisms include histone modifications and DNA methylation,²⁹ which regulate gene expression in response to the environment, particularly during early development.^31,32 DNA methylation is almost exclusively found in CpG dinucleotides in mammals,^22,33 and is typically associated with gene repression,^22,33 although it has also been connected to other regulatory gene expression effects such as the regulation of tissue-specific gene expression, promoter use, and alternative splicing in humans.^22,34

4 |. PRENATAL EXPOSURES AND EPIGENETICS IN PEDIATRIC RESPIRATORY DISEASES

Fetal epigenetic regulation is modifiable by the maternal environment and has been proposed as a mechanism by which prenatal environmental stimuli contribute to the development of respiratory disorders.³⁵ In support of this notion, mechanistic animal studies have demonstrated that maternal microbial exposures, through DNA methylation and histone modifications, may modulate the expression of important immune factors such as interleukin (IL-4) and interferon-γ genes in the offspring’s lungs.^36,37 Increases in histone H3 acetylation linked to an asthma phenotype have been also identified in mice pups after maternal nicotine exposure.³⁸ In humans, epigenetic modifications (e.g., SMAD3 methylation) have been proposed as a mechanism mediating the intergenerational risk of asthma in children from asthmatic mothers.³⁹ Furthermore, epigenetic modifications may also mediate the effect of noninheritable prenatal factors on respiratory outcomes. For example, maternal stress is associated with differential methylation patterns linked to subsequent risk of wheeze or asthma.⁴⁰ Maternal smoking, a recognized risk factor for the development of asthma has been linked to epigenome-wide and gene-specific methylation changes.^41,42 For example, Neilsen et al.⁴² showed that the effects of maternal smoking on asthma may be mediated by DNA methylation modifications in the AHHR gene, a mediator for cell growth and differentiation and Gao et al.⁴³ showed associations between prenatal smoking, higher methylation level in the AXL gene body and increased risk of childhood wheezing, in two independent populations including 1391 children. Furthermore, methylation patterns linked to subsequent risk of wheeze or asthma have been associated with environmental stimuli including, air pollutants, allergens, viruses, and other environmental factors such as season of birth and inner-city living.^6,44,45 Passive smoking exposure may also cause histone acetylation modifications (H3 and H4) in alveolar macrophages of asthmatic children.⁴⁶ Conversely, prenatal farm living, has a protective effect on the development of childhood asthma and allergies. The farm environment also affects the epigenome and is linked to differential DNA methylation in genes related to IgE regulation and Th2 differentiation (e.g., ORMDL1, STAT6, RAD50, and IL13)⁴⁷ prompting questions about whether these changes may explain, at least partly, the protective effect of farm living on the development of childhood asthma and allergies. In summary, these studies suggest that gene-environmental interactions may start in-utero and epigenetic mechanisms may mediate some of the described effects of prenatal environmental exposures on respiratory disease.

5 |. EPIGENETIC MECHANISMS AND EARLY LIFE RESPIRATORY DISORDERS

Most of the human-based evidence linking epigenetic mechanisms to the development of early life respiratory disorders has been derived from genome-wide DNA methylation profiles utilizing blood specimens of children with asthma.⁴⁵ Interestingly, in an epigenome-wide meta-analysis,⁴⁸ Xu et al.⁴⁸ examined specific blood cell-specific methylation patterns and demonstrated that hypomethylation of 14 CpG sites in eosinophils is associated with childhood asthma. Thus, the presence of cell-specific methylation patterns may affect the interpretation of most blood methylation studies published to date. An alternative approach to address this issue is the use of emergent single-cell technologies and computational referenced-based and reference-free deconvolution methods to examine DNA methylation at the single-cell level.^49–51 However, a more fundamental problem of the studies using only blood DNA methylation is that epigenetic information, including DNA methylation, is tissue-specific²⁴ and circulating blood cells may not reflect the diverse gene-environmental and developmental processes of the airways.⁵² Thus, epigenetic studies ideally should include airway cell types since the premise of environmentally driven genetic reprogramming of the respiratory system implicates the presence of specific DNA methylation and transcriptomic marks on the airway epithelial barrier. Given that airway epithelial cells are in direct contact with the environment, the analysis of these cell types may reflect better functionally relevant epigenetic effects in the human airways.

6 |. THE HUMAN AIRWAY EPITHELIUM TO DEFINE SIGNATURES OF DEVELOPMENT AND GENE–ENVIRONMENTAL INTERACTIONS

An elegant proof of concept study conducted by Nicodemus-Johnson et al.⁵³ demonstrated that the airway epithelial barrier can be epigenetically “reprogrammed” by environmental stimuli relevant to respiratory diseases.⁵³ In that study, the investigators used an in vitro model of methylation and gene expression response to IL-13 in airway epithelial cells to demonstrate that this pro-asthmatic type 2 cytokine selectively induces long-lasting DNA methylation changes that mirror those found in vivo in the asthmatic airway epithelial cells.^53,54 Recently, Cardenas et al.⁵⁵ also demonstrated epigenome-wide associations of asthma and allergic phenotypes with lower DNAm of genes driving Th2 and eosinophilic responses (e.g., EPX, IL4, and IL13) in nasal epithelial cells from 547 children.⁵⁵ Interestingly, in this study, the epigenetic age of the nasal epithelium was also accelerated in children with asthma. Additional epigenetic studies in airway epithelial cells of asthmatic individuals have shown differential methylation in the STAT5A transcription factor leading to downregulation of STAT5A expression,⁵⁶ and hypomethylation of IL-6 and nitric oxide synthase 2 (NOS2) associated with increased FeNO.⁵⁷ In African American children, arachidonate 15-lipoxygenase (ALOX15) and periostin (POSTN), two genes involved in Type 2 immune responses, were differentially methylated in the nasal epithelium.⁵⁸ Moreover, a large study showed that methylation profiles in the airway epithelium of children with atopy included genes involved in epithelial barrier function (e.g., CDHR3 and CDH26), and airway epithelial integrity and immune regulation (FBXL7, NTRK1, and SLC9A3).⁵⁹ This study also demonstrated that a methylation-based classifier in the nasal epithelium is able to discriminate atopy and atopic asthma in Hispanic, African American, and European children,⁵⁹ suggesting that a methylation signature in the nasal epithelium has potential implications in the pathobiology of wheezing and asthma in children.⁵⁹ Notably, methylation signatures in the airways may be imprinted by interactions with local factors such as the local microbiota. For example, Morin et al demonstrated lasting effects of the infantile microbiota on DNA methylation patterns in lysosomal and antimicrobial gene pathways in the nasal epithelium of 562 children associated with the development of allergic rhinitis.⁶⁰ Importantly, the human neonatal and infant airway epithelium also shows that specific DNA methylation profiles in airway epithelial cells have important functional effects in gene expression during early life.⁶¹ Thus, all this accumulated human-based evidence provides a strong scientific rationale to conduct longitudinal studies in newborns and infants to define the dynamics of the epigenetic reprogramming of the human airway epithelium and the potential impact on long-term gene expression and individual susceptibility to respiratory conditions during infancy and beyond early childhood.

7 |. MECHANISTIC EVIDENCE OF EPIGENETIC REPROGRAMMING AT THE AIRWAY MUCOSAL BARRIER

Notwithstanding the importance of associating specific airway DNA methylation profiles with respiratory disease in children, there is still a need to conduct mechanistic studies showing that specific environmental clues can induce gene reprogramming in the airways. To date, this has been primarily examined in animal models.^62,63 For instance, a recent study in mice showed that environmental clues regulate epigenetic reprogramming of airway-resident memory CD8+ T cells.⁶² Specifically, the combination of transcriptome and chromatin accessibility analyses using Assay for Transposase-accessible Chromatin revealed an enrichment of genes in airway immune tissue-resident cells associated with stress-related programs (e.g., amino acid starvation pathway) in response to changes in the airway microenvironment.⁶² Additional animal studies have shown that environmental manipulation can cause early life epigenetic reprogramming of mucosal barriers in an age-specific manner.⁶⁴ Pan et al.⁶⁴ examined the transcriptomic and epigenetic profiles of epithelial gut cells from mice representing the infant, juvenile, and adult stages in the presence or absence of a germ-free environment.⁶⁴ This study showed the presence of microbiota-dependent DNA methylation differences early after birth in genes linked to immune pathways and metabolic processes.⁶⁴ Taken together, these mechanistic animal studies demonstrate that there are environment-responsive transcripts in early life that shape stage-specific cellular programs during postnatal development.

8 |. EXPANDING THE UMBRELLA OF ENVIRONMENTALLY DRIVEN GENETIC REPROGRAMMING IN THE AIRWAYS: NONCODING RNA AND POSTTRANSLATIONAL MODIFICATIONS

Progress in molecular biology has expanded the umbrella of environmentally driven genetic reprogramming to include noncoding RNAs involved in the regulation of cellular functions.^65–69 Noncoding RNAs (ncRNAs) can be classified by size into long ncRNAs (>200 nucleotides) and small RNAs (≤200 nucleotides).⁷⁰ The most studied family of small noncoding RNAs involved in genetic reprogramming are the miRNAs (≈22 nucleotides). miRNAs are highly evolutionary conserved molecules that play essential roles in RNA silencing and posttranscriptional regulation of gene expression.^71,72 miRNAs bind to recognition elements within the 3ʹ-untranslated region (3ʹ–UTR) of target messenger RNAs (mRNAs).⁷³ Several studies have confirmed that miRNAs regulate gene expression via base-pairing of complementary mRNA sequences in the 3ʹ-UTR, leading to posttranscriptional silencing and mRNA decay.^71,73 In addition to these intracellular effects, there is also evidence that some miRNAs may be selectively exported in protective extracellular vesicles (exosomes) to induce genetic reprogramming between cells.^74–76 Exosomes containing miRNAs have been isolated from immune cells,^77,78 as well as from body fluids such as nasal and pulmonary secretions.^79,80 Recent evidence also indicates that exosomal miRNAs are taken up by airway epithelial cells and modify their gene expression.⁸¹

miRNAs share important features with classical epigenetic mechanisms (e.g., DNA methylation), including (1) essential roles regulating gene expression during organogenesis and the development and maturation of the lungs,⁸² and (2) responsiveness to environmental clues relevant to the pathogenesis of respiratory disorders.^69,83 Indeed, Solberg et al.,⁸⁴ conducted a seminal study using an in vitro model to define miRNA and gene expression responses to IL-13 in airway epithelial cells form healthy and asthmatic individuals. In this study, IL-13 stimulation mirrored changes in many differentially expressed miRNAs observed in asthmatic airways, including repression of miR-34/449 family members,⁸⁴ which are molecules with critical roles in airway epithelial cell differentiation.^85–87 In addition, Najrana et al.⁸⁸ recently showed that mechanical stretch regulates the expression of specific miRNA in extracellular vesicles released from lung epithelial cells. Other studies have reported additional miRNAs profiles linked to respiratory disorders.^83,89–92 The airway production of miR-155 has been linked to the pathogenesis of cigarette smoke-induced lung inflammation in COPD⁹³ and the regulation of airway antiviral and pro-inflammatory responses.^92,94 miR-155 has also been associated with TH2 allergic responses in several cellular components, including eosinophils,⁹⁵ macrophages,⁹⁶ innate lymphoid cells type 2,^97,98 dendritic cells,⁹⁹ and mast cells.¹⁰⁰ Several other miRNAs such a miR-126, miR-21, miR-146a, miR-221, and miR-222 have been implicated in the regulation of airway inflammatory responses in different animal models and cell systems.^82,101–104

In contrast to DNA methylation, and despite the strong evidence demonstrating that miRNAs play essential roles in lung development,¹⁰² only a few studies have explored the potential role of the miRNA-driven genetic programming of the human airways during early life. Lal et al.¹⁰⁵ demonstrated that premature newborns with severe bronchopulmonary dysplasia have reduced levels of airway exosomal miR 876–3p. Complementary studies showed that miR-876–3p treatment led to reduced alveolar hypoplasia and neutrophilic inflammation in mice after exposure to hyperoxia and LPS, environ mental challenges previously linked to the pathogenesis of bronchopulmonary dysplasia.¹⁰⁵ Davis et al.¹⁰⁶ examined serum samples obtained at randomization in 160 children aged 5–12 years from the Childhood Asthma Management Program cohort and identified that eight serum miRNAs were associated with airway hyperresponsiveness to methacholine challenge (PC20) in asthmatic children. Studies in human infants and young children have also reported specific miRNA signatures during viral respiratory infections.^92,94 Hasegawa et al.¹⁰⁷ identified that in infants with rhinovirus and RSV infections had different nasal airway miRNA profiles associated with NF-κB signaling. A separate study demonstrated that in vivo and in vitro differentiated human airway epithelial cells⁹² show a baseline “airway secretory exosomal miRNAome” that primarily encompasses the production of miR-630, miR-302d-3p, miR-320e, and miR-612.⁹² Furthermore, this study identified abundant nasal airway production of exosomes containing miR-155 in response to rhinovirus infection in young children.⁹² These findings were recently expanded in a larger study (n = 150 young children) that confirmed that miR-155 is strongly associated with the presence of TH1 inflammation against different viruses, including RSV.⁹⁴ An independent study reported that miR-155 is highly upregulated in the nasal mucosa of infants with RSV infection.¹⁰⁸ The latter study also showed that miRNA expression in nasal epithelium of RSV-positive infants exhibits a distinct profile of miRNAs, including the repression of miR-34/449 family members, as previously described in the asthmatic condition.¹⁰⁸

In summary, current evidence demonstrates that in human infants, there are airway miRNA responses to environmental cues (e.g., premature birth or viral infections). Thus, translational and systems biology studies are critically needed to (1) establish specific early human life miRNA profiles and corresponding transcriptomic signatures in different airway cell types as well as in response to various environmental stimuli, and (2) provide mechanistic evidence of miRNA-driven genetic programming of the human airways during early life.

9 |. FUTURE DIRECTIONS

Over the past decade, transdisciplinary “omic” approaches have advanced our understanding of the molecular programming of the airways in humans. Several studies have identified potential molecular mechanisms that contribute to early life epigenetic reprogramming, including DNA methylation, histone modifications, miRNAs, and the homeostasis of the respiratory mucosa (epithelial function and microbiota). Future studies must utilize the progress in systems biology and methodologies to obtain and examine samples in human newborns and infants^20,26,109 to conduct prospective and mechanistic studies that define the normal development and maturation of the human airways along with the effect of the environment on this fundamental process. This novel epigenetic paradigm centered on human maturational and developmental programs must include three cardinal elements: genes, environment, and developmental timing in early life. The combination of these factors is likely what really determines the functional trajectory of the respiratory system at the molecular, functional, and clinical levels.