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Rationale Identifying neonatal and post-discharge exposures among extremely low gestational age newborns (ELGANs) that drive increased pulmonary morbidity and abnormal lung function at 1 year of age proves challenging. Objective The NIH-sponsored Prematurity and Respiratory Outcomes Program (PROP), evaluated infant pulmonary function tests (iPFTs) at one year corrected age to determine which demographic and clinical factors are associated with abnormal lung function. Methods Infant pulmonary function tests were performed on a PROP subcohort of 135 participants following IRB- approved written consent. Demographic data, Neonatal Intensive Care Unit (NICU) clinical care and post-NICU exposures were analyzed for association with iPFTs. Main results A significant decrease in forced expiratory volume at 0.5 seconds (FEV0.5 ) and/or forced expiratory flows at 75% of forced vital capacity (FEF75 ), were associated with male sex and African American race. Clinical factors including longer duration of ventilatory support, exposure to systemic steroids, and weight less than the 10th percentile at 36 weeks post-menstrual age were also associated with airflow obstruction, whereas supplemental oxygen requirement and bronchopulmonary dysplasia were not. Additionally, the need for respiratory medications, technology or hospitalizations during the first year, ascertained by a quarterly survey, were the only post- NICU factors associated with decreased FEV0.5 and FEF75 . Only 7% of infants had reversible airflow obstruction. Conclusions Neonatal demographic factors, respiratory support in the NICU, and a history of greater post-NICU medical utilization for respiratory disease had the strongest association with lower lung function at one year in ELGANs. This article is protected by copyright. All rights reserved.

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.

Rationale Bronchopulmonary dysplasia (BPD) is associated with post-prematurity respiratory disease (PRD) in survivors of extreme preterm birth. Identifying early biomarkers that correlate with later development of BPD and PRD may provide insights for intervention. In a preterm baboon model, elevated gastrin-releasing peptide (GRP) is associated with BPD, and GRP inhibition mitigates BPD occurrence. Objective We performed a prospective cohort study to investigate whether urine GRP levels obtained in the first postnatal week were associated with BPD, PRD, and other urinary biomarkers of oxidative stress. Methods Extremely low gestational age infants (23-28 completed weeks) were enrolled in a US multicenter observational study, The Prematurity and Respiratory Outcomes Program (http://clinicaltrials.gov/ct2/show/NCT01435187). We used multivariable logistic regression to examine the association between urine GRP in the first postnatal week and multiple respiratory outcomes: BPD, defined as supplemental oxygen use at 36 + 0 weeks postmenstrual age, and post-PRD, defined by positive quarterly surveys for increased medical utilization over the first year (PRD score). Results A total of 109 of 257 (42%) infants had BPD, and 120 of 217 (55%) had PRD. On adjusted analysis, GRP level more than 80 was associated with BPD (adjusted odds ratio [aOR], 1.83; 95% confidence interval [CI], 1.03-3.25) and positive PRD score (aOR, 2.46; 95% CI, 1.35-4.48). Urine GRP levels correlated with duration of NICU ventilatory and oxygen support and with biomarkers of oxidative stress: allantoin and 8-hydroxydeoxyguanosine. Conclusions Urine GRP in the first postnatal week was associated with concurrent urine biomarkers of oxidative stress and with later diagnoses of BPD and PRD.

Neutrophil extracellular traps increase cystic fibrosis (CF) airway inflammation. We hypothesized that macrophage exposure to neutrophil elastase (NE) would trigger the release of macrophage extracellular traps (METs), a novel mechanism to augment NE-induced airway inflammation in CF. To test whether human blood monocyte derived macrophages (hBMDM) from CF and non-CF subjects take up proteolytically active NE resulting in clipping of chromatin binding proteins and the release of METs. Human BMDM from CF and non-CF subjects were treated with FITC-NE to determine NE localization. Intracellular NE activity was determined by DQ-elastin assay. MET DNA release was detected by Pico-green for hBMDM, and visualized by confocal microscopy for hBMDM, and for alveolar macrophages harvested from intratracheal NE-exposed Cftr-null and wild-type littermate mice. Immunofluorescence assays for histone citrullination and western analyses for histone clipping were performed. FITC-NE was localized to cytoplasmic and nuclear domains, and NE retained proteolytic activity in hBMDM. NE (100 to 500 nM) significantly increased extracellular DNA release from hBMDM. NE activated MET release by confocal microscopy in hBMDM, and in alveolar macrophages from Cftr-null and Cftr wild-type mice. NE-triggered MET release was associated with H3 citrullination and partial cleavage of Histone H3 but not H4. Exposure to NE caused release of METs from both CF and non-CF hBMDM in vitro and murine alveolar macrophages in vivo. MET release was associated with NE-activated H3 clipping, a mechanism associated with chromatin decondensation, a prerequisite for METs.

Cystic fibrosis (CF) lung disease is marked by high concentrations of neutrophil elastase (NE) and DNA polymers; both factors contribute to airway disease. Although inhaled recombinant human dornase alfa reduces the frequency of CF pulmonary exacerbations, it also increases free NE activity in the sputum. There are no approved anti-NE therapies for patients with CF. We investigated whether synthetic, low–molecular weight polysulfated hyaluronan GlycoMira-1111 (GM-1111) would be effective as an anti-NE drug using ex vivo CF sputum. Anti-NE activity of GM-1111 was tested in CF sputum in the presence or absence of dornase alfa and/or hypertonic saline using a spectrophotometric assay specific for human NE and was compared with unfractionated heparin. We tested whether GM-1111 disaggregated DNA from CF sputum (using gel electrophoresis analysis) or modified CF sputum viscoelastic properties (using a dynamic rheometer). GM-1111 and unfractionated heparin had near equivalent anti-NE activity in CF sputum in the presence of dornase alfa. Both GM-1111 and unfractionated heparin retained anti-NE activity in hypertonic saline but with decreased activity. GM-1111 increased the release of soluble DNA in CF sputum, resulting in improved depolymerization efficacy of dornase alfa. GM-1111 decreased CF sputum elasticity. GM-1111 inhibited NE activity, enhanced DNA depolymerization by deoxyribonuclease, and decreased viscoelastic properties of CF sputum, similar to effects reported previously for unfractionated heparin. Unlike heparins, GM-1111 is synthetic, with minimal anticoagulant activity, and is not derived from animal products. These key attributes provide advantages over unfractionated heparin as a potential therapeutic for CF.

Unprecedented opportunities and daunting difficulties are anticipated in the future of pediatric pulmonary medicine. To address these issues and optimize pediatric pulmonary training, a group of faculty from various institutions met in 2019 and proposed specific, long-term solutions to the emerging problems in the field. Input on these ideas was then solicited more broadly from faculty with relevant expertise and from recent trainees. This proposal is a synthesis of these ideas. Pediatric pulmonology was among the first pediatric specialties to be grounded deliberately in science, requiring its fellows to demonstrate expertise in scientific inquiry (1). In the future, we will need more training in science, not less. Specifically, the scope of scientific inquiry will need to be broader. The proposal outlined below is designed to help optimize the practices of current providers and to prepare the next generation to be leaders in pediatric care in the future. We are optimistic that this can be accomplished. Our broad objectives are (a) to meet the pediatric subspecialty workforce demand by increasing interest and participation in pediatric pulmonary training; (b) to modernize training to ensure that future pediatric pulmonologists will be prepared clinically and scientifically for the future of the field; (c) to train pediatric pulmonologists who will add value in the future of pediatric healthcare, complemented by advanced practice providers and artificial intelligence systems that are well-informed to optimize quality healthcare delivery; and (d) to decrease the cost and improve the quality of care provided to children with respiratory diseases.

Cystic fibrosis (CF) is a multifactorial disease in which dysfunction of protease–antiprotease balance plays a key role. The current CF therapy relies on dornase α, hypertonic saline, and antibiotics and does not address the high neutrophil elastase (NE) activity observed in the lung and sputum of CF patients. Our hypothesis is that variants of heparin, which potently inhibit NE but are not anticoagulant, would help restore the protease–antiprotease balance in CF. To realize this concept, we studied molecular principles governing the effectiveness of different heparins, especially 2-O,3-O-desulfated heparin (ODSH), in the presence of sputum components and therapeutic agents. Using sputa from CF patients and an NE activity assay, we found that heparins are ineffective if used in the absence of dornase. This is true even when mucolytics, such as DTT or N-acetylcysteine, were used. Computational modeling suggested that ODSH and DNA compete for binding to an overlapping allosteric site on NE, which reduces the anti-NE potential of ODSH. NE inhibition of both DNA and ODSH is chain length–dependent, but ODSH chains exhibit higher potency per unit residue length. Likewise, ODSH chains exhibit higher NE inhibition potential compared with DNA chains in the presence of saline. These studies suggest fundamental differences in DNA and ODSH recognition and inhibition of NE despite engaging overlapping sites and offer unique insights into molecular principles that could be used in developing antiprotease agents in the presence of current treatments, such as dornase and hypertonic saline.

OBJECTIVE Vascular endothelial growth factor (VEGF) and sphingolipid metabolites, sphingosine 1-phosphate (S1P), and ceramides are important to lung development and repair. We hypothesized specific sphingolipid and VEGF alterations would be associated with BPD development and aimed to investigate the early tracheal aspirate (TA) VEGF and S1P relationship with later diagnosis of preterm infant bronchopulmonary dysplasia, BPD. DESIGN TA VEGF and lipidomics were measured in TA from Infants

Introduction Sphingolipids are associated with the regulation of pulmonary inflammation. Although sphingolipids have been investigated in the context of cystic fibrosis (CF), the focus has been on loss of CF transmembrane conductance regulator (CFTR) function in mice, and in CF human lung epithelial cell lines. The sphingolipid content of CF sputum and the potential link between ceramide and airway inflammation in CF remain relatively unexplored. Methods Fifteen patients with CF provided two spontaneously expectorated sputum samples, one collected during a hospitalization for an acute pulmonary exacerbation and one from an outpatient visit at a time of clinical stability. Sputum was processed, and the supernatant assessed for active neutrophil elastase (NE) using a chromogenic microplate assay and sphingolipid content using reverse phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Relevant demographic data including age, sex, CF genotype, FEV1 % predicted, and sputum bacteriology were assessed as possible modifying factors that could influence the correlation between NE and sputum sphingolipids. Data were analyzed for linear correlation, with statistical significance pre-defined as P Results There was a significant association between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA), FEV1 % predicted, and female gender further strengthened the association of NE and sphingolipids, but Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. Conclusions These data suggest that NE may increase pro-inflammatory sphingolipid signaling, and the association is strengthened in female patients and patients with MRSA.

Cystic fibrosis (CF) is a disease of dysregulated salt and fluid homeostasis that results in massive accumulation of neutrophil elastase resulting in lung degradation and death. The current CF therapy, which relies on mucus modifying agents, inhaled deoxyribonuclease, and hypertonic saline, improves quality of life but does not target neutrophil elastase, a key mediator of chronic lung inflammation and an established biomarker of disease progression. It is highly challenging to identify small anti-neutrophil elastase agents that would work in sync with current therapy. In fact, several orthosteric inhibitors, including some in clinical trials, have suffered setbacks. We reasoned that small, synthetic, allosteric agents that target the heparin-binding site of neutrophil elastase without being affected by components of current therapy would offer a new therapeutic paradigm. Screening a focused library of non-saccharide glycosaminoglycan mimetics (NSGMs), we discovered a group of 23 promising inhibitors of neutrophil elastase. We developed a protocol based on fundamental computational, biochemical, mechanistic, and adverse effects studies to identify one promising NSGM that worked simultaneously with current anti-CF agents. This NSGM fully neutralized neutrophil elastase present in the sputum of six CF patients only in the presence of deoxyribonuclease with sustained activity under high salt conditions. Our work develops the process for discovering potent heparin-binding site-based allosteric anti-CF agents and presents a novel molecule for further development in animal models of CF with disease manifestations similar to humans.

In this review, we discuss the impact of environmental tobacco smoke and particulate and gaseous air pollutants derived from fossil fuel combustion on a particularly vulnerable population, infants and children. Indoor and outdoor air pollutants exacerbate chronic respiratory diseases and lower respiratory tract infections. However, there is an even more alarming impact of antenatal air pollution exposures. There are several reports in rodents and monkeys that maternal exposure to tobacco smoke or fossil fuel-generated air pollutants causes in utero growth retardation, lung remodeling, and immune cell activation which increase the risk for asthma or the risk of morbidity with respiratory infections. Importantly, epidemiologic studies confirm that maternal exposure to air pollutants decreases lung function in infants and children which may persist to young adulthood. Thus, environmental air pollutants contribute to childhood origins of chronic obstructive lung disease by changing the capacity for normal lung development and repair, by promoting early lung inflammation which increases the susceptibility to pollution-triggered symptomatic lung disease in adulthood, and by limiting the capacity for later adaptive/repair responses to environmental and infectious insults.

OBJECTIVE: To determine if daily respiratory status improved more in extremely low gestational age premature infants after diuretic exposure compared with those not exposed in modern neonatal intensive care units (NICU). STUDY DESIGN: The Prematurity and Respiratory Outcomes Program (PROP) was a multi-center observational cohort study of 835 extremely premature infants, gestational ages 23 0/7–28 6/7 weeks, enrolled in the first week of life from 13 U.S. tertiary NICUs. We analyzed the PROP study daily medication and respiratory support records of infants up to 34 weeks postmenstrual age. We determined whether there was a temporal association between administration of diuretics and an acute change in respiratory status in premature infants in the NICU, using an ordered categorical ranking of respiratory status. RESULTS: Infants in the diuretic exposed group of PROP were of lower mean gestational age and lower mean birth weight (p 1) for each day after the initial day of diuretic exposure. CONCLUSIONS: Our analysis did not support the ability of diuretics to substantially improve the extremely premature infant’s respiratory status. Further study of both safety and efficacy of diuretics in this setting are warranted. TRIAL REGISTRATION: Clinicaltrials.gov NCT01435187

Background Cystic fibrosis (CF) airway secretions are abnormal, contributing to decreased clearance and a cycle of infection and inflammation. CF sputum properties may predict disease progression. We hypothesized that sputum viscoelasticity and clearance abnormalities would inversely correlate with pulmonary function during exacerbation and that sputum properties would return to baseline after therapy. Methods We collected sputa longitudinally from 13 subjects with CF with moderate to severe lung disease during both clinical stability and exacerbation. Dynamic rheology was analyzed, using a cone-and-plate rheometer. Mucociliary clearability was measured on mucus-depleted frog palate, cough clearability in a simulated cough machine, and sputum hydration as percent solids was measured following lyophilization. Results Elastic modulus G ′ and viscous modulus G ′′ increased during exacerbation and returned to baseline levels with recovery ( P P 1 % predicted was inversely correlated with G ′ and G ′′ ( P G ′ and G ′′ vs FEV 1 % predicted was statistically homogeneous among subjects (estimated common slope m = –3.84, P P Conclusions Among these subjects with CF, there is a striking identity of the slope defining the relationship between ln G ′ or ln G ′′ and FEV 1 . There are dramatic increases in dynamic viscosity and elasticity during a pulmonary exacerbation with return to baseline at recovery. This suggests that sputum viscoelastic properties are tightly associated with lung function and disease status.

Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high-mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long-chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long-chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by qRT-PCR and Western analyses, respectively. NE increased lung SPT long-chain subunit 2 (SPTLC2) protein levels but not SPTLC1 and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2 h before NE administration. Myriocin decreased BAL d18:1/22:0 and d18:1/24:1 ceramide, KC, and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.

High mobility group box 1 (HMGB1) is an alarmin released from macrophages after infection or inflammation and is a biomarker of lung disease progression in patients with cystic fibrosis. We reported that 2-O, 3-O desulfated heparin (ODSH) inhibits the release of HMGB1 from murine macrophages triggered by neutrophil elastase both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this study, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase that has been linked to HMGB1 acetylation and release. ODSH inhibited both neutrophil elastase and LPS-triggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration-dependent manner. Fluorescein-labeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus, suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro, resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognizes a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (dissociation constant ∼1 nM) in a highly cooperative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.

Neutrophil elastase (NE) is a major inflammatory mediator in cystic fibrosis (CF) that is a robust predictor of lung disease progression. NE directly causes airway injury via protease activity, and propagates persistent neutrophilic inflammation by up-regulation of neutrophil chemokine expression. Despite its key role in the pathogenesis of CF lung disease, there are currently no effective antiprotease therapies available to patients with CF. Although heparin is an effective antiprotease and anti-inflammatory agent, its anticoagulant activity prohibits its use in CF, due to risk of pulmonary hemorrhage. In this report, we demonstrate the efficacy of a 2-O, 3-O-desulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity, to inhibit NE activity and to block NE-induced airway inflammation. Using an established murine model of intratracheal NE-induced airway inflammation, we tested the efficacy of intratracheal ODSH to block NE-generated neutrophil chemoattractants and NE-triggered airway neutrophilic inflammation. ODSH inhibited NE-induced keratinocyte-derived chemoattractant and high-mobility group box 1 release in bronchoalveolar lavage. ODSH also blocked NE-stimulated high-mobility group box 1 release from murine macrophages in vitro, and inhibited NE activity in functional assays consistent with prior reports of antiprotease activity. In summary, this report suggests that ODSH is a promising antiprotease and anti-inflammatory agent that may be useful as an airway therapy in CF.

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.

OBJECTIVE: To determine patterns of respiratory medications used in neonatal intensive care unit (NICU) graduates. STUDY DESIGN: The Prematurity Respiratory Outcomes Program enrolled 835 babies

To use a large current prospective cohort of infants29 weeks to compare bronchopulmonary dysplasia (BPD) rates in black and white infants.The Prematurity and Respiratory Outcome Program (PROP) enrolled 835 infants born in 2011-2013 at29 weeks of gestation; 728 black or white infants survived to 36 weeks postmenstrual age (PMA). Logistic regression was used to compare BPD outcomes (defined as supplemental oxygen requirement at 36 weeks PMA) between the races, adjusted for gestational age (GA), antenatal steroid use, intubation at birth, and surfactant use at birth.Of 707 black or white infants with available BPD outcomes, BPD was lower in black infants (38% vs 45%), even though they were of significantly lower GA. At every GA, BPD was more common in white infants. The aOR for BPD was 0.60 (95% CI, 0.42-0.85; P = .004) for black infants compared with white infants after adjusting for GA. Despite the lower rate of BPD, black infants had a higher rate of first-year post-prematurity respiratory disease (black, 79%; white, 63%).In this large cohort of recently born preterm infants at29 weeks GA, compared with white infants, black infants had a lower risk of BPD but an increased risk of persistent respiratory morbidity.

Neutrophilic inflammation is associated with chronic airway diseases. It has been observed that human neutrophil elastase (HNE), which is secreted by active neutrophils during inflammation, induces both mucin overproduction and goblet cell metaplasia. Several in vitro studies suggest that tumor necrosis factor-α converting enzyme (TACE) regulates the signaling axis that mediates HNE-induced mucin overproduction; however, it is unknown whether TACE performs a similar function in HNE-induced goblet cell metaplasia in vivo. We conducted this study to determine whether the inactivation of Tace gene expression attenuates HNE-induced goblet cell metaplasia in mice. Deletion of Tace is lethal shortly after birth in mice; therefore, we utilized Tace flox/flox R26CreER +/− mice and induced conditional deletion of Tace using a tamoxifen injection. Wild-type mice were given tamoxifen to control for its effect. Tace conditional deletion mice and wild-type mice were exposed to HNE via nasal instillation three times at 3-day intervals, and the lungs were harvested on day 11 after initial HNE exposure. Using periodic acid-Schiff staining and MUC5AC immunohistochemical staining to visualize goblet cells in the lungs, we found that HNE induced goblet cell metaplasia in the wild-type mice and that HNE-induced goblet cell metaplasia was significantly attenuated in the Tace conditional deletion mice. These findings suggest that TACE could be a potential target in the treatment of goblet cell metaplasia in patients with chronic airway diseases.

NADPH quinone oxidoreductase 1 (NQO1) is recognized as a major susceptibility gene for ozone-induced pulmonary toxicity. In the absence of NQO1 as can occur by genetic mutation, the human airway is protected from harmful effects of ozone. We recently reported that NQO1-null mice are protected from airway hyperresponsiveness and pulmonary inflammation following ozone exposure. However, NQO1 regenerates intracellular antioxidants and therefore should protect the individual from oxidative stress. To explain this paradox, we tested whether in the absence of NQO1 ozone exposure results in increased generation of A2-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D2- and E2-isoprostanes, the precursors of J2- and A2-isoprostanes, both at base line and following ozone exposure compared with congenic wild-type mice. We confirmed in primary cultures of normal human bronchial epithelial cells that A2-isoprostane inhibited ozone-induced NF-κB activation and IL-8 regulation. Furthermore, we determined that A2-isoprostane covalently modified the active Cys179 domain in inhibitory κB kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A2-isoprostane inhibition of NF-κB. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A2-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone. Background: NQO1 regulates pulmonary susceptibility to ozone. Results: In NQO1-null mice, ozone exposure generates precursors of A2-isoprostane in the lung. A2-isoprostane suppresses ozone-induced IL-8 expression, inhibits NF-κB, and modifies Cys179 in IKK. Conclusion: A2-isoprostane inhibits ozone-induced NF-κB activation via IKK inhibition. Significance: This molecular mechanism explains the paradoxical observation that loss of NQO1 protects from ozone toxicity.

To assess the utility of clinical predictors of persistent respiratory morbidity in extremely low gestational age newborns (ELGANs).We enrolled ELGANs (29 weeks' gestation) at ≤7 postnatal days and collected antenatal and neonatal clinical data through 36 weeks' postmenstrual age. We surveyed caregivers at 3, 6, 9, and 12 months' corrected age to identify postdischarge respiratory morbidity, defined as hospitalization, home support (oxygen, tracheostomy, ventilation), medications, or symptoms (cough/wheeze). Infants were classified as having postprematurity respiratory disease (PRD, the primary study outcome) if respiratory morbidity persisted over ≥2 questionnaires. Infants were classified with severe respiratory morbidity if there were multiple hospitalizations, exposure to systemic steroids or pulmonary vasodilators, home oxygen after 3 months or mechanical ventilation, or symptoms despite inhaled corticosteroids. Mixed-effects models generated with data available at 1 day (perinatal) and 36 weeks' postmenstrual age were assessed for predictive accuracy.Of 724 infants (918 ± 234 g, 26.7 ± 1.4 weeks' gestational age) classified for the primary outcome, 68.6% had PRD; 245 of 704 (34.8%) were classified as severe. Male sex, intrauterine growth restriction, maternal smoking, race/ethnicity, intubation at birth, and public insurance were retained in perinatal and 36-week models for both PRD and respiratory morbidity severity. The perinatal model accurately predicted PRD (c-statistic 0.858). Neither the 36-week model nor the addition of bronchopulmonary dysplasia to the perinatal model improved accuracy (0.856, 0.860); c-statistic for BPD alone was 0.907.Both bronchopulmonary dysplasia and perinatal clinical data accurately identify ELGANs at risk for persistent and severe respiratory morbidity at 1 year.ClinicalTrials.gov: NCT01435187.

Bronchopulmonary dysplasia (BPD) is the major cause of chronic lung disease and morbidity in preterm infants. Since it was first described fifty years ago, the epidemiology, pathogenesis, and treatment for BPD has changed dramatically. This review summarizes these changes and the clinical outcomes for infants diagnosed with BPD.

This editorial summarizes the challenges of exhaled breath biomarker research particularly for nasal NO. We also introduce a new focus for Pediatric Pulmonology, a section on Translational Medicine.

Alpha-1-antitrypsin (A1AT) deficiency is characterized by increased neutrophil elastase (NE) activity and oxidative stress in the lung. We hypothesized that NE exposure generates reactive oxygen species by increasing lung non-heme iron. To test this hypothesis, we measured bronchoalveolar lavage (BAL) iron and ferritin levels, using inductively coupled plasma (ICP) optical emission spectroscopy and an ELISA respectively, in A1AT-deficient patients and healthy subjects. To confirm the role of NE in regulating lung iron homeostasis, we administered intratracheally NE or control buffer to rats and measured BAL and lung iron and ferritin. Our results demonstrated that A1AT-deficient patients and rats post-elastase exposure have elevated levels of iron and ferritin in the BAL. To investigate the mechanism of NE-induced increased iron levels, we exposed normal human airway epithelial cells to either NE or control vehicle in the presence or absence of ferritin, and quantified intracellular iron uptake using calcein fluorescence and ICP mass spectroscopy. We also tested whether NE degraded ferritin in vitro using ELISA and western analysis. We demonstrated in vitro that NE increased intracellular non-heme iron levels and degraded ferritin. Our results suggest that NE digests ferritin increasing the extracellular iron pool available for cellular uptake.

The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/ progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/ progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.

Cystic fibrosis is the most common, inherited fatal disease in Caucasians. The major cause of morbidity and mortality is chronic lung disease due to infection and inflammation in the airways leading to bronchiectasis and respiratory failure. The signature pathologic features of CF lung disease including abnormal mucus obstructing airways, chronic infection with Staphylococcus aureus, Pseudomonas aeruginosa and other gram negative bacteria, and a robust neutrophil-dominant airway inflammation, are exacerbated by unopposed proteases present at high concentrations in the ASL. There is strong evidence that proteases, particularly neutrophil elastase, contribute to the pathology of CF by impairing mucociliary clearance, interfering with innate immune functions, and perpetuating neutrophilic inflammation. The mechanisms employed by proteases to impact airway function in CF will be reviewed.

Neutrophil elastase (NE), a serine protease present in high concentrations in the airways of cystic fibrosis patients, injures the airway epithelium. We examined the epithelial response to NE-mediated proteolytic injury. We have previously reported that NE treatment of airway epithelial cells causes a marked decrease in epithelial DNA synthesis and proliferation. We hypothesized that NE inhibits DNA synthesis by arresting cell cycle progression. Progression through the cell cycle is positively regulated by cyclin complexes and negatively regulated by cyclin-dependent kinase inhibitors (CKI). To test whether NE arrests cell cycle progression, we treated normal human bronchial epithelial (NHBE) cells with NE (50 nM) or control vehicle for 24 h and assessed the effect of treatment on the cell cycle by flow cytometry. NE treatment resulted in G1 arrest. Arrest in G1 phase may be the result of CKI inhibition of the cyclin E complex; therefore, we evaluated whether NE upregulated CKI expression and/or affected the interaction of CKIs with the cyclin E complex. Following NE or control vehicle treatment, expression of p27Kip1, a member of the Cip/Kip family, was evaluated. NE increased p27Kip1 gene and protein expression. NE increased the coimmunoprecipitation of p27Kip1 with cyclin E complex, suggesting that p27Kip1 inhibited cyclin E complex activity. Our results demonstrate that p27 is regulated by NE and is critical for NE-induced cell cycle arrest.

Neutrophil elastase (NE), a potent neutrophil inflammatory mediator, increases MUC5AC mucin gene expression through undefined pathways involving reactive oxygen species. To determine the source of NE-generated reactive oxygen species, we used pharmacologic inhibitors of oxidoreductases to test whether they blocked NE-regulated MUC5AC mRNA expression. We found that dicumarol, an inhibitor of the NADP(H) quinone oxidoreductase 1 (NQO1), inhibited MUC5AC mRNA expression in A549 lung adenocarcinoma cells and primary normal human bronchial epithelial (NHBE) cells. We further tested the role of NQO1 in mediating NE-induced MUC5AC expression by inhibiting NQO1 expression using siRNA. Transfection with short interfering RNA (siRNA) specific for NQO1 suppressed NQO1 expression and significantly abrogated MUC5AC mRNA expression. NE treatment caused lipid peroxidation in A549 cells; this effect was inhibited by pretreatment with dicumarol, suggesting that NQO1 also regulates oxidant stress in A549 cells following NE exposure. NE exposure increased NQO1 protein and activity levels; NQO1 expression and activity were limited to the cytosol and did not translocate to the plasma membrane. Our results indicate that NQO1 has an important role as a key mediator of NE-regulated oxidant stress and MUC5AC mucin gene expression.

In this review, we summarize work over the past 15 years on mucin gene expression and regulation in the lung, as well as how mucin gene expression is altered in chronic lung diseases. This field owes a great debt to Carol Basbaum for her pioneering work in dissecting signaling pathways regulating mucin gene expression and for her tremendous energy in promoting the importance of understanding the basic pathogenic mechanisms that drive mucus overproduction in cystic fibrosis, chronic obstructive pulmonary disease, and asthma.

This review focuses on the role and regulation of mucin glycoproteins (mucins) in airway health and disease. Mucins are highly glycosylated macromolecules (≥50% carbohydrate, wt/wt). MUC protein backbones are characterized by numerous tandem repeats that contain proline and are high in serine and/or threonine residues, the sites of O-glycosylation. Secretory and membrane-tethered mucins contribute to mucociliary defense, an innate immune defense system that protects the airways against pathogens and environmental toxins. Inflammatory/immune response mediators and the overproduction of mucus characterize chronic airway diseases: asthma, chronic obstructive pulmonary diseases (COPD), or cystic fibrosis (CF). Specific inflammatory/immune response mediators can activate mucin gene regulation and airway remodeling, including goblet cell hyperplasia (GCH). These processes sustain airway mucin overproduction and contribute to airway obstruction by mucus and therefore to the high morbidity and mortality associated with these diseases. Importantly, mucin overproduction and GCH, although linked, are not synonymous and may follow from different signaling and gene regulatory pathways. In section i, structure, expression, and localization of the 18 human MUC genes and MUC gene products having tandem repeat domains and the specificity and application of MUC-specific antibodies that identify mucin gene products in airway tissues, cells, and secretions are overviewed. Mucin overproduction in chronic airway diseases and secretory cell metaplasia in animal model systems are reviewed in section ii and addressed in disease-specific subsections on asthma, COPD, and CF. Information on regulation of mucin genes by inflammatory/immune response mediators is summarized in section iii. In section iv, deficiencies in understanding the functional roles of mucins at the molecular level are identified as areas for further investigations that will impact on airway health and disease. The underlying premise is that understanding the pathways and processes that lead to mucus overproduction in specific airway diseases will allow circumvention or amelioration of these processes.

In chronic obstructive pulmonary diseases, the airway epithelium is chronically exposed to neutrophil elastase, an inflammatory protease. The cellular response to neutrophil elastase dictates the balance between epithelial injury and repair. Key regulators of epithelial migration and proliferation are the ErbB receptor tyrosine kinases, including the epidermal growth factor receptor. In this context, we investigated whether neutrophil elastase may regulate expression of MUC4, a membrane-tethered mucin that has recently been identified as a ligand for ErbB2, the major heterodimerization partner of the epidermal growth factor receptor. In normal human bronchial epithelial cells, neutrophil elastase increased MUC4 mRNA levels in both a concentration- and time-dependent manner. RNA stability assays revealed that neutrophil elastase increased MUC4 mRNA levels by prolonging the mRNA half-life from 5 to 21 h. Neutrophil elastase also increased MUC4 glycoprotein levels as determined by Western analysis, using a monoclonal antibody specific for a nontandem repeat MUC4 sequence. Therefore, airway epithelial cells respond to neutrophil elastase exposure by increasing expression of MUC4, a potential activator of epithelial repair mechanisms.

This editorial summarizes the challenges of exhaled breath biomarker research particularly for nasal NO. We also introduce a new focus for Pediatric Pulmonology, a section on Translational Medicine.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air–liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α–converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

The body first encounters deleterious inhaled substances, such as allergens, industrial particles, pollutants, and infectious agents, at the airway epithelium. When this occurs, the epithelium and its resident inflammatory cells respond defensively by increasing production of cytokines, mucus, and reactive oxygen and nitrogen species (ROS/RNS). As inflammation in the airway increases, additional infiltrating cells increase the level of these products. Recent interest has focused on ROS/RNS as potential modulators of the expression of inflammation-associated genes important to the pathogenesis of various respiratory diseases. ROS/RNS appear to play a variety of roles that lead to changes in expression of genes such as interleukin-6 and intercellular adhesion molecule 1. By controlling this regulation, the reactive species can serve as exogenous stimuli, as intercellular signaling molecules, and as modulators of the redox state in epithelial cells. Unraveling the molecular mechanisms affected by ROS/RNS acting in these capacities should aid in the understanding of how stimulated defense mechanisms within the airway can lead to disease. Images Figure 1