Maternal allergen exposure reprograms the developmental lung transcriptome in atopic and normoresponsive rat pups.

The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.

Genetic influences on asthma susceptibility in the developing lung.

Asthma is the leading serious pediatric chronic illness in the United States, affecting 7.1 million children. The prevalence of asthma in children under 4 years of age has increased dramatically in the last 2 decades. Existing evidence suggests that this increase in prevalence derives from early environmental exposures acting on a pre-existing asthma-susceptible genotype. We studied the origins of asthma susceptibility in developing lung in rat strains that model the distinct phenotypes of airway hyperresponsiveness (Fisher rats) and atopy (brown Norway [BN] rats). Postnatal BN rat lungs showed increased epithelial proliferation and tracheal goblet cell hyperplasia. Fisher pups showed increased lung resistance at age 2 weeks, with elevated neutrophils throughout the postnatal period. Diverse transcriptomic signatures characterized the distinct respiratory phenotypes of developing lung in both rat models. Linear regression across age and strain identified developmental variation in expression of 1,376 genes, and confirmed both strain and temporal regulation of lung gene expression. Biological processes that were heavily represented included growth and development (including the T Box 1 transcription factor [Tbx5], the epidermal growth factor receptor [Egfr], the transforming growth factor beta-1-induced transcript 1 [Tgfbr1i1]), extracellular matrix and cell adhesion (including collagen and integrin genes), and immune function (including lymphocyte antigen 6 (Ly6) subunits, IL-17b, Toll-interacting protein, and Ficolin B). Genes validated by quantitative RT-PCR and protein analysis included collagen III alpha 1 Col3a1, Ly6b, glucocorticoid receptor and Importin-13 (specific to the BN rat lung), and Serpina1 and Ficolin B (specific to the Fisher lung). Innate differences in patterns of gene expression in developing lung that contribute to individual variation in respiratory phenotype are likely to contribute to the pathogenesis of asthma.

Modulation of Lgl1 by steroid, retinoic acid, and vitamin D models complex transcriptional regulation during alveolarization.

Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA), and vitamin D (VitD). Bronchopulmonary dysplasia, a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of bronchopulmonary dysplasia, reduced levels of late gestation lung 1 (Lgl1) normalize during recovery. We show that GC (100 nM) stimulates (7- to 115-fold) and VitD (100 microM) suppresses (twofold) Lgl1 expression. RA (all-trans/9-cis, 10 microM) effects are biphasic. From postnatal days 7-10, RA was stimulatory (twofold) at 24 h, after which effects were inhibitory (3- to 15-fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared with 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.

Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice.

BACKGROUND: Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. METHODS: An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation. RESULTS: Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics. CONCLUSION: Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.

Impact of the loss of Hoxa5 function on lung alveogenesis.

The involvement of genes controlling embryonic processes in the etiology of diseases often escapes attention because of the focus given to their inherent developmental role. Hoxa5 belongs to the Hox gene family encoding transcription factors known for their role in skeletal patterning. Hoxa5 is required for embryonic respiratory tract morphogenesis. We now show that the loss of Hoxa5 function has severe repercussions on postnatal lung development. Hoxa5-/- lungs present an emphysema-like morphology because of impaired alveogenesis. Chronic inflammation characteristics, including goblet cell hyperplasia, mucus hypersecretion, and recruitment of inflammatory cells, were also observed. Altered cell specification during lung morphogenesis triggered goblet cell anomalies. In addition, the defective motility of alveolar myofibroblast precursors in the embryonic lung led to the mispositioning of the alveolar myofibroblasts and to abnormal elastin deposition postnatally. Both goblet cell hyperplasia and elastic fiber abnormalities contributed to the chronic physiopathological features of Hoxa5-/- lungs. They constituted an attractive stimulus to recruit activated macrophages that in turn generated a positive feedback loop that perpetuated macrophage accumulation in the lung. The present work corroborates the notion that altered Hox gene expression may predispose to lung pathologies.

Respiratory adaptations to lung morphological defects in adult mice lacking Hoxa5 gene function.

The Hoxa5 mutation is associated with a high perinatal mortality rate caused by a severe obstruction of the laryngotracheal airways, pulmonary dysmorphogenesis, and a decreased production of surfactant proteins. Surviving Hoxa5(-/-) mutant mice also display lung anomalies with deficient alveolar septation and areas of collapsed tissue, thus demonstrating the importance of Hoxa5 throughout lung development and maturation. Here, we address the functional consequences of the Hoxa5 mutation on respiration and chemoreflexes by comparing the breathing pattern of Hoxa5(-/-) mice to that of wild-type animals under resting conditions and during exposure to moderate ventilatory stimuli such as hypoxia and hypercapnia. Resting Hoxa5(-/-) mice present a higher breathing frequency and overall minute ventilation that likely compensate for their reduced lung alveolar surface available for gas exchange and their increased upper airway resistance. When exposed to ventilatory stimuli, Hoxa5(-/-) mice maintain the higher minute ventilation by adapting the tidal volume and/or the breathing frequency. The minute ventilation increase seen during hypoxia was similar for both groups of mice; however, the dynamics of the frequency response was genotype-dependent. The hypercapnic ventilatory response did not differ between genotypes. These findings reveal the strategies allowing survival of Hoxa5(-/-) mice facing morphologic anomalies leading to a significant deficit in gas exchange capacity.