Pleural infection is a severe disease with increasing incidence worldwide. The subphenotypes of pleural infection remain unknown.We designed a study to endotype the disease and assess the association between patient phenotype, microbiology and clinical outcome.

We subjected 80 pleural fluid samples to unlabelled mass spectrometry.

Pathway analysis of the differentially expressed proteins identified the neutrophil degranulation, glycolysis, pentose phosphate pathway, and the liver and retinoid X receptors (LXR-RXR) activation. Higher neutrophil degranulation was associated with increased glycolysis and pentose phosphate activation.

Pleural infection patients exhibit proteomic signatures indicating diverse responses of neutrophil mediated immunity, glycolysis, and pentose phosphate activation.

Pleural infection is a common and severe disease with increasing incidence worldwide. The endotypes of pleural infection remain unknown. A better understanding of patient variation in underlying biological response to infection may lead to improved treatments and clinical outcomes. We designed a study with the aim to endotype the disease and assess the association between patient phenotype, microbiology and clinical outcome.

We subjected 80 pleural fluid samples from the PILOT study, a prospective study on pleural infection, to unlabelled mass spectrometry. Proteins were retained if they were detected in at least 50% of the samples resulting in a total of 449 proteins. Unsupervised hierarchical clustering and UMAP analyses were used to cluster samples, Spearman and exact Fischer's methods were used for correlation assessment and protein expression was correlated with clinical outcomes.

UMAP plotting separated the samples in to two different and distinct cohorts. Pathway analysis of the differentially expressed proteins identified neutrophil degranulation, glycolysis, the pentose phosphate pathway, and the liver and retinoid X receptors (LXR-RXR) activation. Higher neutrophil degranulation was associated with increased glycolysis and pentose phosphate activation. Specimens dominated by Streptococcus Pneumoniae exhibited high neutrophil degranulation. Increased activity of the LXR-RXR pathway was associated with better survival.

Pleural infection patients exhibit proteomic signatures indicating diverse responses of neutrophil mediated immunity, glycolysis, and pentose phosphate activation which were associated with microbiology. Therapeutic targeting the LXR-XRX pathway with agonists may improve survival.

Samples collected for the PILOT clinical trial (DOI: 10.1183/13993003.00130-2020) used for the TORPIDS-2 study.

Patients were included if they had a clinical presentation consistent with pleural infection and any of the following criteria: 1. pleural fluid that was macroscopically purulent; or 2. pleural fluid that was positive on culture for bacterial infection; or 3. pleural fluid that demonstrated bacteria on Gram staining; or 4. pleural fluid with a pH ≤7.2 (measured by blood gas analyser) or low glucose level (≤3 mmol·L-1 or ≤55 mg·dL-1) in a patient with clinical evidence of infection; or 5. contrast-enhanced computed tomography (CT) evidence of pleural infection (consolidation of underlying lung with enhancing pleural collection) in a patient with clinical evidence of infection, alongside exclusion of other sources of infection. Evidence of infection was assessed by the recruiting physician on the basis of fever, an elevated peripheral blood white-cell count, or elevated serum inflammatory markers such as C-reactive protein (CRP). Study exclusion criteria were as follows: 1. age <18 years; 2. no pleural fluid available for analysis; 3. previous pneumonectomy on the side of pleural infection; and 4. expected survival of <3 months due to co-morbid disease, as judged by the recruiting physician.

Patients with evidence of pleural infection

Anonymised and non-identifiable data could be shared with other researchers. The mass spectrometry data are deposited in PRIDE to share with other researchers.