How do tissues sense proteolytic stress?

Introduction/Rationale: Excess proteolytic activity – from endogenous proteases, microbial toxins, venoms, and industrial enzymes – occurs during inflammation, infection, tissue injury, and genetic disorders (α1-antitrypsin deficiency). These activities contribute to major diseases including COPD, ARDS, rheumatoid arthritis, aneurysm, chronic wounds, and cancer, yet how tissues detect and resist proteolytic stress is poorly understood.

Methods: To address this, we developed an in vivo lung model using the bacterial protease LasB delivered either once (acute exposure) or three times at 24-hour intervals (repeated exposure). Acute exposure caused marked vascular injury, erythrocyte extravasation with heme release, neutrophil influx, and loss of lung function. Strikingly, repeated exposures produced a resilient phenotype: reduced protein leakage, fewer erythrocytes and neutrophils, and preserved lung function.

Results: Mechanistic studies using genetic mouse models, transcriptomics, and pharmacology showed this adaptation depended on sensing of oxidative molecules – erythrocytic heme – and activating tissue oxidative stress response. Alveolar macrophages emerged as the principal sensors activating NRF2 transcription factor which induces an antioxidant gene program including heme oxygenase 1 (Hmox1). Hmox1 catabolized heme to bilirubin, a metabolite that can relay stress information to neighboring fibroblasts. In response, fibroblasts produced several protease inhibitors in NRF2-dependent manner that directly bound and inhibited LasB activity, suggesting active resistance to proteolytic stress.

Conclusion: These results identify tissue-resident macrophages and an NRF2-dependent program as central mediators of lung adaptation to proteolytic stress, revealing a cellular circuit by which tissues detect protease-driven damage and actively restore homeostasis.