Aims. Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia‐induced lung injury at very high oxygen levels (>95%). Results. Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild‐type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase‐3 and poly (ADP‐ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling‐ (TUNEL‐) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis in vivo. EGFR inhibition decreased hyperoxia (95%)‐induced apoptosis and ERK in murine alveolar epithelial cells in vitro, and CRISPR‐mediated EGFR deletion reduced hyperoxia‐induced apoptosis and ERK in human alveolar epithelial cells in vitro. Innovation. This work defines a protective role of EGFR inhibition to decrease apoptosis in lung injury induced by 100% oxygen. This further characterizes the complex role of EGFR in acute lung injury and outlines a novel hyperoxia‐induced cell death pathway that warrants further study. Conclusion. In conditions of severe hyperoxia (>95% for >24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury.

Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly develops antibiotic resistance; thus, novel therapeutics are urgently needed to effectively combat this pathogen. Previously, we have shown that a novel cationic Zinc (II) porphyrin (ZnPor) has potent bactericidal activity against planktonic and biofilm-associated PsA cells, and disassembles the biofilm matrix via interactions with eDNA In the present study, we report that ZnPor caused a significant decrease in PsA populations in mouse lungs within an in vivo model of PsA pulmonary infection. Additionally, when combined with an obligately lytic phage PEV2, ZnPor at its minimum inhibitory concentration (MIC) displayed synergy against PsA in an established in vitro lung model resulting in greater protection of H441 lung cells versus either treatment alone. Concentrations above the minimum bactericidal concentration (MBC) of ZnPor were not toxic to H441 cells; however, no synergy was observed. This dose-dependent response is likely due to ZnPor’s antiviral activity, reported herein. Together, these findings show the utility of ZnPor alone, and its synergy with PEV2, which could be a tunable combination used in the treatment of antibiotic-resistant infections.

While delivery of supplemental oxygen is a life-saving therapy, exposure to high levels of oxygen, called hyperoxia, is associated with increased mortality in the intensive care unit (ICU). Hyperoxia leads to oxidant-mediated acute lung injury (ALI) and pulmonary cell death, called hyperoxic acute lung injury (HALI). Elucidation of molecular mechanisms in HALI could identify therapeutic targets in ALI. In the current study, we examined in vivo effects of HALI on Beclin-1 (BCN1), a molecule that regulates autophagy and cell death. Effects of HALI on BCN1 and autophagy markers were examined in wildtype mice. Analysis of BCN1 and autophagy was completed via Western blot, RT-qPCR, and immunohistochemistry. In wildtype mice, HALI led to increased BCN1 in the lung and in the alveolar epithelium. HALI resulted in significant alterations in markers of autophagy in the lung, including reduced microtubule-associated protein 1B-light chain (LC3B)-II/-I ratios, suggesting reduced autophagic flux. HALI caused increased LDH release in human alveolar type-II cells derived from induced pluripotent stem cells (AT2s ^iPSC ), as well as reduced LC3B-II/-I ratios. We previously showed that inhibition of the tyrosine kinase receptor epidermal growth factor receptor (EGFR) is protective in HALI. EGFR ^Wa5/+ mice, which have genetically reduced EGFR activity and improved survival in HALI, showed increased total BCN1, reduced phosphorylated-(p-)/total BCN1 ratios, and decreased LC3B-II/-I ratios in the lung in HALI compared with wildtype. Administration of wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor which decreases BCN1-mediated autophagy, led to increased mortality in HALI in wildtype mice. These data support that regulation of BCN1 and autophagy by EGFR is a protective mechanism in HALI, a pathway which warrants further study for its therapeutic potential. KEY MESSAGES: HALI is associated with increased mortality in the ICU and causes alveolar epithelial cell death, but the role of BCN1 in HALI is not well defined. This study shows that BCN1, a molecule involved in autophagy and cell death, is regulated by EGFR in HALI in vivo . These results are significant because regulation by BCN1 and autophagy by EGFR is a novel pathway in HALI with therapeutic potential that warrants further study.

Background: While delivery of supplemental oxygen is a life-saving therapy, exposure to high oxygen, called hyperoxia, leads to increased intensive care unit mortality. Hyperoxia induces oxidant-mediated acute lung injury (ALI) and pulmonary cell death, called hyperoxic ALI (HALI). Elucidating molecular mechanisms in HALI could identify therapeutic targets in ALI. Methods: In the current study, we examined in vivo effects of HALI on Beclin-1 (BCN1), which regulates autophagy, and modulation of BCN1 by epidermal growth factor receptor (EGFR). Effects of HALI on BCN1 and autophagy were examined in mice with genetically decreased EGFR (EGFR ^Wa5/+ ). Wildtype (WT) and EGFR ^Wa5/+ mice were exposed to 100% oxygen for 24–72 hours along with normoxia controls (eight groups; n=4–6/group), and analysis of pulmonary BCN1 and autophagy was completed. Results: In WT, HALI led to increased BCN1 (59% increased total BCN1/β-Actin; p<0.01) in lung and alveolar epithelium (484% increased H-score; p<0.001). HALI led to decreased microtubule-associated protein 1B-light chain (LC3B)-II/-I ratios (43% decrease; p<0.05) and increased p62 (93% increase; p<0.05), suggesting reduced autophagic flux. In human alveolar type-II cells derived from induced pluripotent stem cells (AT2s ^iPSC ), HALI caused increased LDH release (130% increase; p<0.0001) and decreased LC3B-II/-I ratios (32% decrease; p<0.05). We previously showed that EGFR ^Wa5/+ mice are protected in HALI. In the current study, EGFR ^Wa5/+ mice showed increased pulmonary BCN1 (122% increase; p<0.05), reduced phosphorylated-(p-)/total BCN1 (47% decrease; p<0.05) and decreased LC3B-II/-I ratios (70% decrease; p<0.01) in HALI compared with WT. In addition, wortmannin (1 mg/kg), which decreases BCN1-mediated autophagy, increased mortality in HALI compared with vehicle control (n=10/group; 18% decreased hours survived; p<0.001). Conclusions: These data delineate a novel cell death pathway in HALI involving BCN1 and EGFR with therapeutic potential.

High oxygen damages human lung, called hyperoxic acute lung injury (HALI). HALI pathogenesis involves formation of reactive oxygen species (ROS) which overwhelm antioxidant mechanisms and lead to alveolar epithelial and endothelial cell death and interstitial inflammation. Resultant alveolar flooding causes ventilation-perfusion (V/Q) mismatch, hypoxic respiratory failure, and death. 12–16 h of severe hyperoxic exposure potentiates symptoms that include substernal chest discomfort, cough, and sore throat. Alterations in pulmonary function that ensue are decreased vital capacity (VC), decreased arterial oxygen tension (PaO ₂ ), increased intrapulmonary shunt, and increased dead space-to-tidal volume ratio (V _D /V _T ). A multitude of observational studies demonstrate harm of hyperoxic exposure in critically ill patients. While one randomized clinical trial (RCT) found targeting a lower oxygenation target in critically ill patients was beneficial compared with a higher target, another RCT demonstrated harm of such a strategy in patients with acute respiratory distress syndrome (ARDS). Multiple subsequent RCTs examining effects of lower- and higher-oxygenation strategies in critically ill patients identify no clear benefit of either strategy in the general ICU population. However, heterogeneous effects of lower- and higher-oxygenation approaches in ICU patient subgroups warrant further investigation. It is paramount that ICU physicians recognize that lethal and sublethal amounts of hyperoxia result in lung damage and that patients who require hyperoxia to avoid deleterious hypoxia can be at risk for HALI. For each patient in the ICU, it is likely that ideal oxygen targets depend on the specific medical conditions affecting the patient and is a promising area of future study.