MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha.

BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Effects of hyperoxia on alveolar and pulmonary vascular development in germ-free mice.

Airway microbial dysbiosis is associated with subsequent bronchopulmonary dysplasia (BPD) development in very preterm infants. However, the relationship of airway microbiome in normal pulmonary development has not been defined. To better understand the role of the airway microbiome, we compared normal and abnormal alveolar and pulmonary vascular development in mice with or without a microbiome. We hypothesized that the lungs of germ-free (GF) mice would have an exaggerated phenotypic response to hyperoxia compared with non-germ-free (NGF) mice. With the use of a novel gnotobiotic hyperoxia chamber, GF and NGF mice were exposed to either normoxia or hyperoxia. Alveolar morphometry, pulmonary mechanics, echocardiograms, inflammatory markers, and measures of pulmonary hypertension were studied. GF and NGF mice in normoxia showed no difference, whereas GF mice in hyperoxia showed protected lung structure and mechanics and decreased markers of inflammation compared with NGF mice. We speculate that an increase in abundance of pathogenic bacteria in NGF mice may play a role in BPD pathogenesis by regulating the proinflammatory signaling and neutrophilic inflammation in lungs. Manipulation of the airway microbiome may be a potential therapeutic intervention in BPD and other lung diseases.

Airway Microbiome and Development of Bronchopulmonary Dysplasia in Preterm Infants: A Systematic Review.

OBJECTIVES: To summarize evidence regarding microbial dysbiosis of the airway associated with bronchopulmonary dysplasia (BPD) and to explore heterogeneity among studies. STUDY DESIGN: We included studies that evaluated the airway microbiome in preterm infants who developed BPD using culture-independent molecular techniques and reported alpha- and beta-diversity metrics and microbial profiles. RESULTS: The 6 included studies had substantial clinical and methodological heterogeneity. Most studies reported the presence of an airway microbiome early after birth and an evolution in the first weeks of life with increasing bacterial loads. The early airway microbiome was dominated by Staphylococcus and Ureaplasma spp. Two studies reported differences in alpha- and beta- diversity indices in preterm infants with BPD compared with those who did not develop BPD. Increased microbial community turnover, changes in the relative abundance of Proteobacteria and Firmicutes, and decreased Lactobacilli were reported with BPD progression. Most included infants were born by cesarean delivery, and a majority were exposed to postnatal antibiotics. No data regarding feeding human milk or correlations with the development of gut microbiota (gut-lung axis) were available. CONCLUSIONS: Microbial dysbiosis may be associated with BPD progression and severity, and further study of microbiome optimization in preterm infants at risk for BPD is warranted.

Iloprost attenuates hyperoxia-mediated impairment of lung development in newborn mice.

Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg-1·day-1, nimesulide (selective COX-2 antagonist) 100 mg·kg-1·day-1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1ß and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.

Early airway microbial metagenomic and metabolomic signatures are associated with development of severe bronchopulmonary dysplasia.

The pathogenesis of bronchopulmonary dysplasia (BPD) is not well understood. We previously identified differences in the airway microbiome at birth between preterm infants who were BPD predisposed versus those who were BPD resistant. In this study, we attempted to identify mechanisms by which the airway microbiome could modify the risk for BPD. We used a software-based method to predict the metagenome of the tracheal aspirate (TA) microbiome from 16S rRNA sequencing data in preterm infants and to identify functional ortholog genes that were differentially abundant in BPD-predisposed and BPD-resistant infants. We also identified metabolites that were differentially enriched in these samples by use of untargeted mass spectrometry and mummichog to identify the metabolic pathways involved. Microbial metagenome analysis identified specific pathways that were less abundant in the functional metagenome of the microbiota of BPD-predisposed infants compared with BPD-resistant infants. The airway metabolome of BPD-predisposed infants was enriched for metabolites involved in fatty acid activation and androgen and estrogen biosynthesis compared with BPD-resistant infants. These findings suggest that in extremely preterm infants the early airway microbiome may alter the metabolome, thereby modifying the risk of BPD. The differential enrichment of sex steroid metabolic pathways supports previous studies suggesting a role for sexual dimorphism in BPD risk. This study also suggests a role for metabolomic and metagenomic profiles to serve as early biomarkers of BPD risk.

The neutrophil chemoattractant peptide proline-glycine-proline is associated with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by unrelenting polymorphonuclear neutrophil (PMN) inflammation and vascular permeability. The matrikine proline-glycine-proline (PGP) and acetylated PGP (Ac-PGP) have been shown to induce PMN inflammation and endothelial permeability in vitro and in vivo. In this study, we investigated the presence and role of airway PGP peptides in acute lung injury (ALI)/ARDS. Pseudomonas aeruginosa-derived lipopolysaccharide (LPS) was instilled intratracheally in mice to induce ALI, and increased Ac-PGP with neutrophil inflammation was noted. The PGP inhibitory peptide, arginine-threonine-arginine (RTR), was administered (it) 30 min before or 6 h after LPS injection. Lung injury was evaluated by detecting neutrophil infiltration and permeability changes in the lung. Pre- and posttreatment with RTR significantly inhibited LPS-induced ALI by attenuating lung neutrophil infiltration, pulmonary permeability, and parenchymal inflammation. To evaluate the role of PGP levels in ARDS, minibronchoalveolar lavage was collected from nine ARDS, four cardiogenic edema, and five nonlung disease ventilated patients. PGP levels were measured and correlated with Acute Physiology and Chronic Health Evaluation (APACHE) score, PaO2 to FIO2 (P/F), and ventilator days. PGP levels in subjects with ARDS were significantly higher than cardiogenic edema and nonlung disease ventilated patients. Preliminary examination in both ARDS and non-ARDS populations demonstrated PGP levels significantly correlated with P/F ratio, APACHE score, and duration on ventilator. These results demonstrate an increased burden of PGP peptides in ARDS and suggest the need for future studies in ARDS cohorts to examine correlation with key clinical parameters.

Vascular mediators in chronic lung disease of infancy: role of endothelial monocyte activating polypeptide II (EMAP II).

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Over the years, the BPD phenotype has evolved, but despite various advances in neonatal management approaches, the reduction in the BPD burden is minimal. With the advent of surfactant, glucocorticoids, and new ventilation strategies, BPD has evolved from a disease of structural injury into a new BPD, marked by an arrest in alveolar growth in the lungs of extremely premature infants. This deficient alveolar growth has been associated with a diminution of pulmonary vasculature. Several investigators have described the epithelial / vascular co-dependency and the significant role of crosstalk between vessel formation, alveologenesis, and lung dysplasia's; hence identification and study of factors that regulate pulmonary vascular emergence and inflammation has become crucial in devising effective therapeutic approaches for this debilitating condition. The potent antiangiogenic and proinflammatory protein Endothelial Monocyte Activating Polypeptide II (EMAP II) has been described as a mediator of pulmonary vascular and alveolar formation and its expression is inversely related to the periods of vascularization and alveolarization in the developing lung. Hence the study of EMAP II could play a vital role in studying and devising appropriate therapeutics for diseases of aberrant lung development, such as BPD. Herein, we review the vascular contribution to lung development and the implications that vascular mediators such as EMAP II have in distal lung formation during the vulnerable stage of alveolar genesis.

Clinical trial of a probiotic and herbal supplement for lung health.

Introduction: Dysbiosis of the gut microbiome may augment lung disease via the gut-lung axis. Proteobacteria may contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic inflammation. To study the effects of probiotics across the gut-lung axis, we sought to determine if a Lactobacillus probiotic and herbal blend was safe and well-tolerated in healthy volunteers and asthmatic patients. Methods: We conducted a 1-month randomized, open-label clinical trial in Cork, Ireland with healthy and asthmatic patients who took the blend twice a day. The primary endpoint was safety with exploratory endpoints including quality of life, lung function, gut microbiome ecology, and inflammatory biomarkers. Results: All subjects tolerated the blend without adverse events. Asthmatic subjects who took the blend showed significant improvements in lung function as measured by forced expiratory volume and serum short chain fatty acid levels from baseline to Week 4. The gut microbiome of asthmatic subjects differed significantly from controls, with the most prominent difference in the relative abundance of the proteobacteria Escherichia coli. Administration of the probiotic maintained overall microbial community architecture with the only significant difference being an increase in absolute abundance of the probiotic strains measured by strain-specific PCR. Conclusion: This study supports the safety and efficacy potential of a Lactobacillus probiotic plus herbal blend to act on the gut-lung axis. However, due to the lack of a control group, a longer blinded, placebo-controlled study will be warranted to confirm the efficacy improvements observed in this trial. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05173168.

Efficacy of a Probiotic and Herbal Supplement in Models of Lung Inflammation.

BACKGROUND: Gut microbiome dysbiosis is associated with lung disease through the gut-lung axis. Abundant proteobacteria increase MMP-9 and contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic lung disease. We sought to determine if a scientifically formulated probiotic and herbal supplement could attenuate neutrophilic inflammation and improve lung structure and function in models of lung inflammation. METHODS: For in vitro experiments, epithelial cells exposed to proteobacteria were treated with resB-a blend of three probiotic Lactobacillus strains and turmeric, holy basil, and vasaka herbal extracts. For in vivo experimentation, mice exposed to pulmonary proteobacteria-derived lipopolysaccharide were treated by gavage with resB. RESULTS: In vitro, the bacterial and herbal components of resB decreased activity of the MMP-9 pathway. Mice exposed to LPS and pre- and post-treated with resB had decreased neutrophil recruitment and inflammatory biomarkers in bronchoalveolar lavage fluid, serum, and lung tissue compared to untreated mice. CONCLUSIONS: This study describes the mechanisms and efficacy of probiotic and herbal blend in pre-clinical models of lung injury and inflammation.

Hospital and Neurodevelopmental Outcomes in Nano-Preterm Infants Receiving Invasive vs Noninvasive Ventilation at Birth.

Importance: Infants with gestational age between 22 0/7 and 23 6/7 weeks (referred to as nano-preterm infants) are at very high risk of adverse outcomes. Noninvasive respiratory support at birth improves outcomes in infants born at 24 0/7 to 27 6/7 weeks' gestational age. Evidence is limited on whether similar benefits of non-invasive respiratory support at birth extend to nano-preterm infants. Objective: To evaluate the hypothesis that intubation at 10 minutes or earlier after birth is associated with a higher incidence of bronchopulmonary dysplasia (BPD) or death by 36 weeks' postmenstrual age (PMA) in nano-preterm infants. Design, Setting, and Participants: This observational cohort study included all nano-preterm infants at a level IV neonatal intensive care unit who were delivered from January 1, 2014, to June 30, 2021. Infants receiving palliative or comfort care at birth were excluded. Exposures: Infants were grouped based on first intubation attempt timing after birth (>10 minutes after birth and ≤10 minutes as noninvasive and invasive respiratory support at birth groups, respectively). Main Outcomes and Measures: The primary outcome was the composite outcome of BPD (physiological definition) or death by 36 weeks' PMA. Results: All 230 consecutively born, eligible nano-preterm infants were included, of whom 88 (median [IQR] gestational age, 23.6 [23.4-23.7] weeks; 45 [51.1%] female; 54 [62.1%] Black) were in the noninvasive respiratory support at birth group and 142 (median [IQR] gestational age, 23.0 [22.4-23.3] weeks; 71 [50.0%] female; 94 [66.2%] Black) were in the invasive respiratory support at birth group. The incidence of BPD or death by 36 weeks' PMA did not differ between the noninvasive and invasive respiratory support groups (83 of 88 [94.3%] in the noninvasive group vs 129 of 142 [90.9%] in the invasive group; adjusted odds ratio, 2.09; 95% CI, 0.60-7.25; P = .24). Severe intraventricular hemorrhage or death by 36 weeks' PMA was lower in the invasive respiratory support at birth group (adjusted odds ratio, 2.20; 95% CI, 1.07-4.51; P = .03). Conclusions and Relevance: This cohort study's findings suggest that noninvasive respiratory support in the first 10 minutes after birth is feasible but is not associated with a decrease in the risk of BPD or death compared with intubation and early surfactant delivery in nano-preterm infants.

Predicting BPD: Lessons Learned From the Airway Microbiome of Preterm Infants.

Bronchopulmonary dysplasia (BPD) is the chronic lung disease of prematurity with an operational definition, various different clinical phenotypes, and a complex, multifactorial etiology. Newer unbiased systems biology approaches have identified various "omic" factors associated with the pathogenesis and prediction of BPD. Recent microbi "omic" studies have discovered that airways of newborns harbor a low biomass but distinct microbiome signature as early as at the time of birth. This early airway microbiome may serve to prime the host immune system and may play a role in modulating the infant's future susceptibility to severe BPD development. Temporal changes are observed in airway microbiome of preterm infants from birth to the diagnosis of BPD, with an overall decrease in bacterial diversity, and development of a relative dysbiosis marked by increased Gammaproteobacteria and decreased Lactobacilli abundance. This review will summarize previous investigations of the airway microbiome in preterm infants, appraise the utility of using the airway microbiome to predict BPD development, discuss possible molecular mechanisms involved, and speculate on future microbiome-mediated therapeutics for BPD.

Genomics, microbiomics, proteomics, and metabolomics in bronchopulmonary dysplasia.

Bronchopulmonary Dysplasia (BPD) is a disorder with a multifactorial etiology and highly variable clinical phenotype. Several traditional biomarkers have been identified, but due to the complex disease phenotype, these biomarkers have low predictive accuracy for BPD. In recent years, newer technologies have facilitated the in-depth and unbiased analysis of 'big data' in delineating the diagnosis, pathogenesis, and mechanisms of diseases. Novel systems-biology based 'omic' approaches, including but not limited to genomics, microbiomics, proteomics, and metabolomics may help define the multiple cellular and humoral interactions that regulate normal as well as abnormal lung development and response to injury that are the hallmarks of BPD.

Early-Onset Neonatal Sepsis with Extended Spectrum Beta-Lactamase Producing Escherichia Coli in Infants Born to South and South East Asian Immigrants: A Case Series.

Extended-spectrum ß -lactamase (ESBL)-producing Enterobacteriaceae represent a major worldwide threat. We present three cases of early onset ESBL Escherichia coli sepsis in infants born to families from South and Southeast Asia to inform the practitioner community about this emerging threat. Infants with suspected sepsis, whose mother is from Asia or Southeast Asia, should be suspected of having an infection with an ESBL-producing organism, and practitioners should strongly consider adding a carbapenem to their usual initial antibiotic regimen.

Supraphysiological Levels of Oxygen Exposure During the Neonatal Period Impairs Signaling Pathways Required for Learning and Memory.

Preterm infants often require prolonged oxygen supplementation and are at high risk of neurodevelopmental impairment. We recently reported that adult mice exposed to neonatal hyperoxia (postnatal day [P] 2 to 14) had spatial navigation memory deficits associated with hippocampal shrinkage. The mechanisms by which early oxidative stress impair neurodevelopment are not known. Our objective was to identify early hyperoxia-induced alterations in hippocampal receptors and signaling pathways necessary for memory formation. We evaluated C57BL/6 mouse pups at P14, exposed to either 85% oxygen or air from P2 to 14. We performed targeted analysis of hippocampal ligand-gated ion channels and proteins necessary for memory formation, and global bioinformatic analysis of differentially expressed hippocampal genes and proteins. Hyperoxia decreased hippocampal mGLU7, TrkB, AKT, ERK2, mTORC1, RPS6, and EIF4E and increased α3, α5, and ɤ2 subunits of GABAA receptor and PTEN proteins, although changes in gene expression were not always concordant. Bioinformatic analysis indicated dysfunction in mitochondria and global protein synthesis and translational processes. In conclusion, supraphysiological oxygen exposure reduced proteins necessary for hippocampus-dependent memory formation and may adversely impact hippocampal mitochondrial function and global protein synthesis. These early hippocampal changes may account for memory deficits seen in preterm survivors following prolonged oxygen supplementation.

Exosomal microRNA predicts and protects against severe bronchopulmonary dysplasia in extremely premature infants.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Cellular and humoral biomarkers of Bronchopulmonary Dysplasia.

The pathogenesis of Bronchopulmonary Dysplasia (BPD) is multifactorial and the clinical phenotype of BPD is extremely variable. Predicting BPD is difficult, as it is a disease with a clinical operational definition but many clinical phenotypes and endotypes. Most biomarkers studied over the years have low predictive accuracy, and none are currently used in routine clinical care or shown to be useful for predicting longer-term respiratory outcome. Targeted cellular and humoral biomarkers and novel systems biology 'omic' based approaches including genomic and microbiomic analyses are described in this review.