Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Exposure to high FiO2 from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans-blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG [400μM] exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG [400μM] or amiloride [1μM] showed a reduction in alveologenesis and increased lung injury compared to age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10μM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high FiO2 exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.

COVID‐19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS‐CoV‐2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID‐19 testing for symptoms at drive‐through COVID‐19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real‐time polymerase chain reaction assays and quantitative proteomics to 20 virus‐positive and 20 virus‐negative samples. ACE‐2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78–63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10–1.23). Transcripts for two interferons (IFN) were elevated, IFN‐λ1 (OR =71, CI =7.07–713) and IFN‐λ2 (OR =40.2, CI =3.86–419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23–1.49 and OR =1.33 CI =1.20–1.47, respectively). Only transcripts for IP‐10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01–2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN‐γ OR =0.90 CI =0.33–0.79, IFN‐λ2,3 OR =0.60 CI =0.48–0.74) suggesting viral‐induced shut‐off of host antiviral protein responses. However, proteins for IP‐10 (OR =3.74 CI =2.07–6.77) and several interferon‐stimulated genes (ISG) increased with viral load (BST‐1 OR =25.1, CI =3.33–188; IFIT1 OR =19.5, CI =4.25–89.2; IFIT3 OR =245, CI =15–4020; MX‐1 OR =3.33, CI =1.44–7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS‐CoV‐2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral‐induced host shut‐off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

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In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p<0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p<0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p<0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung. Support or Funding Information This work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.