Abstract Rationale Acute lung injury (ALI) is a major cause of global mortality. ALI is defined by loss of alveolar barrier function, which leads to pulmonary edema formation and respiratory failure. Although recovery of barrier function is central to lung repair, there is little understanding of barrier recovery mechanisms. Since reports indicate cleavage of the Notch protein is barrier-strengthening in cultured cells, we tested the hypothesis that Notch cleavage drives alveolar barrier repair in a mouse model of ALI caused by S. aureus (SA) lung infection. Methods We intranasally-instilled mice with PBS, SA, siRNA, and plasmid DNA. We defined alveolar barrier function by two approaches: (1) indirectly, by quantifying pulmonary edema by lung wet weight to body weight (LW/BW) ratio; and (2) directly, by quantifying leak of fluorophore-tagged dextran (20 kD) from microvessels into airspaces in alveoli of live lungs that were inflated, perfused, and imaged by confocal microscopy. Results Confocal images show dextran leaked from microvessels into airspaces in alveoli of live lungs at 4h after SA instillation (p0.05 versus PBS), indicating SA caused loss of alveolar barrier function. Follow-up images in the same alveoli 3h later showed dextran did not leak into airspaces at 7h after SA instillation, indicating the barrier rapidly repaired. Adding the Notch cleavage inhibitor, DAPT, to the lung perfusate solution blocked barrier repair (p0.05 versus vehicle), indicating Notch cleavage was central to the barrier repair mechanism. Mouse models of SA lung infection support the imaging findings, in that SA instillation caused increase, then decrease of LW/BW ratio at 6h and 24h post-instillation, respectively, and the decrease was blocked by siRNA against the Notch-cleaving enzyme, PS1 (p0.05 versus non-targeting siRNA). Since data generated in cultured cells suggest expression of a Notch cleavage product, the Notch transmembrane domain (TMD), is barrier-strengthening, we considered Notch TMD expression in alveoli might promote barrier repair. Live lung imaging confirmed that intranasal instillation of plasmid DNA led to plasmid expression in the alveolar epithelium. Notch TMD plasmid pretreatment decreased LW/BW ratio at 24h after intranasal SA instillation (p0.05 versus vector), suggesting Notch TMD expression in the alveolar epithelium accelerated barrier repair. Conclusions Our findings provide the first evidence that alveoli regain barrier function after SA-induced barrier loss, and that barrier repair depends on Notch cleavage in the alveolar epithelium. Strategies that leverage the Notch cleavage pathway in the alveolar epithelium might promote barrier repair in lungs with SA-induced ALI. This abstract is funded by: Stony Wold-Herbert Fund Fellowship Grant (SM), American Lung Association COVID-19 and Emerging Respiratory Viruses Research Award 1031520 (JH), R01HL164821 (JH)
Moore et al. (Fri,) studied this question.