A33-51 The ARDS Proteome During Pulmonary Infection Reveals Distinct Matrix Scaffold Signatures in the Airspace

Rationale

Pneumonia can elicit severe neutrophil-driven inflammation and acute respiratory distress syndrome (ARDS), a condition characterized by disruption of airspace barrier integrity. Whether ARDS exhibits a distinct neutrophilic and matrix injury signature during pulmonary infection remains unclear. We hypothesize that infection-related injury alters the airspace composition of neutrophil-derived tissue degradative enzymes and structural matrix proteins in a manner conserved across species.

Methods

Label-free quantitative mass spectrometry was performed on bronchoalveolar lavage (BAL) fluid collected in mouse models of infection-mediated lung injury and prospectively in 35 critically ill patients with ARDS with or without pulmonary infection as determined by clinical microbiological testing who also underwent clinical bronchoscopy. In mice, lung injury was induced by intratracheal inoculation with live Pseudomonas aeruginosa. BAL was collected at baseline and one day post-injury from wild-type mice and from susceptible mice with cell-specific deletion of the matricellular protein thrombospondin-1 involved in cell-matrix interactions. Mass spectrometry data were analyzed using Mascot/Scaffold and Spectronaut to generate protein identifications and quantifications. Protein accessions were mapped to gene identifiers using STRING. Reactome pathway enrichment analysis was performed using over-representation testing with hypergeometric testing. Statistical group comparisons were performed using t-testing after log transformation of spectral counts. All mouse and human studies were conducted under approved IACUC and IRB protocols.

Results

The airspace proteome during injury is defined by prominent neutrophil and matrix signatures (Neutrophilic: 280 proteins, p = 3.12x10-51 in mice, 339 proteins, p = 1.16x10-96 in humans; Matrix: 80 proteins, p = 8.93x10-7 in mice and 136 proteins, p = 6.99x10-23 in humans). When comparing infectious injury to controls in mice, there is heightened expression of neutrophil enzymes ELANE, MMP9, MMP8, and CTSG, with reduced expression of alveolar basement membrane matrix protein COL4A2. Mice susceptible to lung injury demonstrated heightened MMP2 and MMP9 expression alongside significant reductions in basement membrane proteins COL4A2 and COL6A1. During pulmonary infection in humans, ARDS was characterized by increased expression of MMP8, MMP9, ELANE, and CTSG, and reduced expression of basement membrane proteins COL5A1, COL6A3, COL6A2, COL15A1, and ELN, compared to ARDS without pulmonary infection.

Conclusion

Pneumonia-associated ARDS exhibits extensive extracellular matrix proteolysis in the airspaces, reflecting an imbalance between neutrophil-derived degradative enzymes and structural matrix scaffold proteins. In both mice and humans, heightened MMP9 coupled with reduced COL6 reveals neutrophil-mediated basement membrane degradation. These data suggest the existence of distinct ARDS proteotypes, which may provide a framework for personalized diagnostics and treatment.

This abstract is funded by: R01 HL136143, HL177904, T32 HL007317