Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04–0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.

Previous studies have identified genetic variants associated with bronchopulmonary dysplasia (BPD) in extremely preterm infants. However, findings with genome-wide significance have been rare, and not replicated. We hypothesized that whole exome sequencing (WES) of premature subjects with extremely divergent phenotypic outcomes could facilitate the identification of genetic variants or gene networks contributing disease risk. The Prematurity and Respiratory Outcomes Program (PROP) recruited a cohort of > 765 extremely preterm infants for the identification of markers of respiratory morbidity. We completed WES on 146 PROP subjects (85 affected, 61 unaffected) representing extreme phenotypes of early respiratory morbidity. We tested for association between disease status and individual common variants, screened for rare variants exclusive to either affected or unaffected subjects, and tested the combined association of variants across gene loci. Pathway analysis was performed and disease-related expression patterns were assessed. Marginal association with BPD was observed for numerous common and rare variants. We identified 345 genes with variants unique to BPD-affected preterm subjects, and 292 genes with variants unique to our unaffected preterm subjects. Of these unique variants, 28 (19 in the affected cohort and 9 in unaffected cohort) replicate a prior WES study of BPD-associated variants. Pathway analysis of sets of variants, informed by disease-related gene expression, implicated protein kinase A, MAPK and Neuregulin/epidermal growth factor receptor signaling. We identified novel genes and associated pathways that may play an important role in susceptibility/resilience for the development of lung disease in preterm infants.

Background: Extremely premature infants are at risk for circulatory collapse or respiratory failure that are often treated with hydrocortisone (HC); however, there is no information on the metabolic consequences of this therapy. Methods: Longitudinal urine samples from infants <28 weeks gestation in the Trial of Late Surfactant were analyzed by untargeted UHPLC:MS/MS. Fourteen infants who received a tapering course of HC beginning at 3 mg/kg/day for ≥9 days were compared to 14 matched control infants. A secondary cross-sectional analysis by logistic regression used urines from 314 infants. Results: Of 1145 urinary metabolites detected, abundance of 219, representing all the major biochemical pathways, changed at p < 0.05 in the HC-treated group with 90% decreasing; 3 cortisol derivatives increased ~2-fold with HC therapy. Only 11% of regulated metabolites remained responsive at the lowest HC dose. Regulated metabolites included two steroids and thiamin that are associated with lung inflammation in infants. HC responsiveness was confirmed in 57% of metabolites by cross-sectional analysis. Conclusions: HC treatment of premature infants influenced in a dose-dependent manner abundance of 19% of identified urinary metabolites of diverse biochemical systems, primarily reducing concentrations. These findings indicate that exposure to HC reversibly impacts the nutritional status of premature infants. Impact: Hydrocortisone treatment of premature infants with respiratory failure or circulatory collapse alters levels of a subset of urinary metabolites representing all major biochemical pathways. This is the first description of the scope, magnitude, timing and reversibility of metabolomic changes in infants in response to hydrocortisone, and it confirms corticosteroid regulation of three biochemicals that are associated with lung inflammatory status. The findings indicate a dose-dependency of hydrocortisone for metabolomic and anti-inflammatory effects, that prolonged therapy may lower the supply of many nutrients, and that monitoring concentrations of cortisol and inflammation markers may be a useful clinical approach during corticosteroid therapy.

Objective Inhaled nitric oxide (iNO) has been tested to prevent bronchopulmonary dysplasia (BPD) in premature infants, however, the role of cyclic guanosine monophosphate (cGMP) is not known. We hypothesized that levels of NO metabolites (NOx) and cGMP in urine, as a noninvasive source for biospecimen collection, would reflect the dose of iNO and relate to pulmonary outcome. Study Design Studies were performed on 125 infants who required mechanical ventilation at 7 to 14 days and received 24 days of iNO at 20–2 ppm. A control group of 19 infants did not receive iNO. Results In NO-treated infants there was a dose-dependent increase of both NOx and cGMP per creatinine (maximal 3.1- and 2-fold, respectively, at 10–20 ppm iNO) compared with off iNO. NOx and cGMP concentrations at both 2 ppm and off iNO were inversely related to severity of lung disease during the 1st month, and the NOx levels were lower in infants who died or developed BPD at term. NOx was higher in Caucasian compared with other infants at all iNO doses. Conclusion Urinary NOx and cGMP are biomarkers of endogenous NO production and lung uptake of iNO, and some levels reflect the severity of lung disease. These results support a role of the NO–cGMP pathway in lung development. Other contributing TOLSURF Pilot and TOLSURF investigators: Elizabeth Rogers, MD (University of California San Francisco, San Francisco CA); Anne Marie Reynolds, MD (Women and Children's Hospital of Buffalo, Buffalo, NY); Aruna Parekh, MD (Stonybrook University Hospital, Stonybrook, NY); Dennis Mayock, MD (University of Washington, Seattle, WA); Mark Hudak, MD (University of Florida College of Medicine at Jacksonville, UF Health and Wolfson Children's Hospital, Jacksonville, FL); Beatrice Stefanescu, MD, Jennifer B. Helderman, MD, T. Michael O'Shea, MD (Wake Forest School of Medicine and Forsyth Medical Center, Winston Salem, NC); Michael Georgieff, MD, Catherine M. Bendel, MD (University of Minnesota Amplatz Children's Hospital, Minneapolis, MN); Andrea Lampland, MD (Department of Pediatrics, Children's Hospital and Clinics of Minnesota, St. Paul, MN); Ellen Bendel-Stenzel, MD, Neil Mulrooney (Children's Hospitals and Clinics of Minnesota, Minneapolis, MN); Frances Koch, MD (Medical University of South Carolina, Charleston, SC); Reddy Dhanireddy, MD (University of Tennessee-Memphis, Memphis TN); Victor McKay, MD (All Children's Hospital, St Petersburg, FL); Rajan Wadhawan, MD (Florida Hospital for Children, Orlando FL); Nicolas Porta, MD (Northwestern Memorial Hospital, Chicago, IL)