Pleural fluid proteomics from patients with pleural infection shows signatures of diverse neutrophilic responses: The Oxford Pleural Infection Immunophenotyping Study (TORPIDS 2)

<bold>Background:</bold> Pleural infection is a severe and complicated disease with increasing incidence worldwide. Knowledge of the immune response to pleural infection remains incomplete. We designed a study to phenotype immune responses and assess correlation with clinical outcomes. <bold>Methods:</bold> We subjected 80 pleural fluid samples from the PILOT study (prospective study of pleural infection) to mass spectrometry. Proteins were retained if they were detected in at least 50% of the samples, with analysis resulting in 499 proteins. Data normalization and bias correction were performed using the Variance Stabilization Normalization method. The k-nearest neighbour algorithm was used to impute missing values. Simultaneous Decomposition Analysis was employed to integrate and dimensionally reduce proteomic and clinical data. <bold>Results:</bold> Pathway analysis identified pathways associated with fibrinolysis, neutrophil mediated immunity, neutrophil extracellular traps, and complement activation. Unsupervised hierarchical clustering based on neutrophil mediated immunity and neutrophil extracellular traps protein profiles separated samples into two groups, signalling the presence of immature neutrophils. A higher clinical RAPID score was negatively associated with expression of Paraoxonase 1 and 3 and positively associated with Cystatin C. Gene Set Enrichment Analysis identified 4 clusters of protein profiles. <bold>Conclusion:</bold> Pleural infection human samples exhibit proteomic signatures indicating diverse responses of neutrophil mediated immunity. Immature neutrophils may thus be a potential therapeutic target in pleural infection.