American Journal of Respiratory Cell and Molecular Biology

The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex responsible for pumping H+ into the cytosol, thereby maintaining intracellular pH; however, its role in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is unclear. In this study, the functional relevance of V-ATPase and hypoxia inducible factor (HIF) 1 were assessed using alveolar-specific ATP6V0C knockout mice (Atp6v0cAT2-KO) and HIF1A knockout mice (Hif1aAT2-KO), respectively. ATP6V0C expression levels were measured in serum and bronchoalveolar lavage fluid (BALF) of patients with ARDS. ATP6V0C expression was increased in lung tissues from ALI murine models and BALF from patients with severe ARDS. Genetic deficiency of ATP6V0C in alveoli attenuated the functional, histologic, and inflammatory hallmarks of lipopolysaccharide (LPS)–induced ALI but did not alter the host’s susceptibility to bacterial pathogens. Mechanistically, transcriptomic analyses revealed that ATP6V0C-regulated genes are highly enriched in the HIF-1 signaling pathway. HIF-1α was upregulated synchronously with ATP6V0C in injured lungs, while co-immunoprecipitation confirmed their interaction. Following LPS instillation, the signs of ALI were further exacerbated in Hif1afl/fl mice pretreated with lung epithelial tropic adeno-associated virus carrying ATP6V0C, but not in Hif1aAT2-KO mice. HIF-1α, as a transcriptional factor, in turn, regulated ATP6V0C expression, forming a positive feedback loop. ATP6V0C levels were increased in BALF but not serum in patients with ARDS. ATP6V0C levels in BALF correlate with ARDS severity. In summary, our study identified an ATP6V0C–HIF-1α detrimental feedback loop that exacerbates epithelial apoptosis and inflammation, thereby driving the progression of ALI. Targeting the ATP6V0C–HIF-1α loop may hence present a promising therapeutic strategy against ALI/ARDS.

Pneumonias remain a leading cause of death worldwide. Seeking novel strategies to protect susceptible patients, we have reported that inhaled delivery of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) protects animals against a broad range of infectious pneumonias by stimulating antimicrobial responses from the lung epithelium. Toll-like receptor 9 (TLR9) is well-established as the primary cellular receptor for CpG ODNs. However, we recently reported that ODNs also stimulate TLR9-independent generation of antimicrobial mitochondrial reactive oxygen species. By testing a variety of synthetic ODN molecules, we found that ODNs containing a phosphorothioate backbone, but not those with a phosphodiester backbone, induce TLR9-independent pathogen killing in lungs and improve mouse survival. Phosphorothioate-backboned ODN binds mitochondrial protein voltage-dependent anion channel 1 (VDAC1) at its N-terminus, initiating pneumonia-protective metabolic reprogramming in lung epithelial cells that yield the protective antimicrobial effect. Thus, the phosphorothioate backbone of ODN is a critical structural pattern that activates TLR9-independent, metabolically modulated innate immune protection that may be harnessed to protect vulnerable patients against pneumonia.

RationaleMechanical ventilation is integral to the medical management of the critically ill. However, prolonged exposure to pathologic airflow during mechanical ventilation places patients at risk for airway injury. Here, we developed an innovative microsurgical mouse model to examine how changes in airflow affect cellular regeneration and repair in the tracheobronchial airway. We examined wall shear stress (WSS), a known consequence of airflow constriction, and evaluated clinical significance using Heliox, a helium-rich environment, which reduces pathologic airflow in human patients.MethodsWe modulated airflow by altering airway geometry. Specifically, orthotopic airway transplants replaced a tracheal segment with either a bronchial scaffold or a tracheal scaffold (control) (n = 10/group). After surgery, mice were subjected to room air (1 or 2 weeks) or Heliox (1 week). Computational fluid dynamics models were used to quantify WSS. Epithelial and fibroblast cell number and phenotype, as well as collagen density, were analyzed using histology. Statistical analysis was performed using 1-way analysis of variance with a multiple comparison test.ResultsUsing our novel microsurgical model, we reduced airway cross-sectional area by 0.54-fold and increased WSS by 71.40-fold. This increase in WSS was associated with squamous epithelial differentiation, increased fibroblast activation, and significant fibrosis. These phenotypic changes were rescued when mice were recovered in Heliox.ConclusionsOur findings suggest a mechanical mechanism in which airflow regulates epithelial differentiation, fibroblast activation, and fibrosis in the tracheobronchial airway. These studies support the development of therapeutic interventions that modulate intraluminal WSS and have the potential to accelerate airway regeneration.

The alternative reading frame (ARF) protein, encoded by the CDKN2A locus, is well-recognized for its role in tumor suppression. Emerging evidence has highlighted ARF as a critical regulator of innate immunity and inflammation, with links to increased susceptibility to cardiometabolic diseases. This study investigates the role of ARF in lung homeostasis and reveals that its deficiency in mice affects lipid metabolism and leads to pulmonary abnormalities resembling pulmonary alveolar proteinosis (PAP). ARF-deficient mice exhibited abnormal surfactant clearance, characterized by lipid and protein accumulation in the alveoli, foamy alveolar macrophages (AMs) with enlarged and vacuolated morphology, and increased bronchoalveolar lavage fluid turbidity. These changes were linked to disrupted surfactant homeostasis resulting from an imbalance between increased lipid uptake (via upregulation of scavenger receptors such as SR-A1 and CD36) and impaired lipid efflux, evidenced by reduced expression of the cholesterol transporter SR-BI. These mice also display reduced AM numbers, increased eosinophil and neutrophil infiltration, consistent with secondary PAP. Additionally, a distinctive chemokine and cytokine profile (elevated Ccl12, Ccl2, Cxcl1, and IL-10) was observed, which may be associated with type 2 immune responses and alternative AM polarization. Interestingly, ARF deficiency also appears to compromise AM maintenance through effects on self-renewal and survival. Pulmonary function tests revealed increased tissue elastance and damping, suggesting early-stage lung stiffness. Collectively, these findings highlight the essential role of ARF in lung homeostasis and lipid regulation, providing insights into its potential involvement in PAP pathogenesis.