Unraveling the immunosuppressive role of Elastase B produced by cystic fibrosis isolates of <i>Pseudomonas aeruginosa</i> in an organotypic 3D lung epithelial cell model

<i>Pseudomonas aeruginosa</i> chronically infects the lungs of people with cystic fibrosis (pwCF), where it produces a broad range of proteases that impact host immunity and inflammation. While some studies suggest these enzymes induce inflammation and may contribute to the excessive inflammatory response in pwCF, others indicate that they degrade cytokines and suppress the immune response. Here, we investigate the role of <i>P. aeruginosa</i> proteases in CF lung inflammation by culturing clinical isolates under physiologically relevant conditions and studying the inflammatory response in an organotypic 3D lung cell culture model. Exposure to supernatants from isolates with proteolytic activity led to cytokine degradation and reduced cytokine release in the 3D model. To identify the specific protease(s) responsible, we performed a proteomics analysis on the supernatants of the isolates with and without proteolytic activity. In total, 79 proteases were identified in the supernatants, with extracellular proteases among the most differentially expressed in the proteolytically active isolate group. Metalloprotease Elastase B (LasB) exhibited the highest level of differential expression. Therefore, we determined the contribution of LasB to the observed reduction in cytokine levels by utilizing <i>P. aeruginosa lasB</i> knockout mutants. Compared to the <i>lasB</i> mutant, the wild-type strain significantly decreased inflammation by degrading MCP-1, IL-1β, GM-CSF, and IL-8. While this study brings to light a key role for LasB, our findings demonstrate that immunomodulation by <i>P. aeruginosa</i> most likely involves the combined action of multiple proteases. This study provides a foundation for future research into protease-mediated immune evasion during chronic infection.