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Abstract

Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by a severe inflammation and infiltration of neutrophiles which secrete neutrophil elastase (NE), which leads to lung parenchyma destruction and disease progression. In CF, disease severity has been linked with airway dysbiosis. As the relationship of dysbiosis, respiratory tract inflammation and impaired lung function is not fully understood this study aimed to assess this interplay. In an initial cross-sectional study, the inflammatory profile of spontaneous sputum samples of CF patients was analyzed. Cytokines were measured by Cytometric Bead Array, anti-proteases levels by enzyme-linked immunosorbent assay and free NE activity via Förster resonance energy transfer (FRET) probe NEmo-1 in the supernatant fraction. The interplay of those parameters together with lung function were evaluated in a multiparameter analysis. The increase in the inflammatory markers NE, IL-1β, IL-8 and TNF-α correlated negatively with the lung function parameter forced expiratory volume in one second percent (FEV1 %) predicted and α-diversity of the airway microbiome. Based on the microbiome composition, patients were grouped in 7 clusters. One cluster had a diverse microbiome, which was rich in species of the oropharynx-like flora and had a high evenness. These patients had the highest FEV1 % predicted and the lowest inflammatory load. In contrast, the dominance of a particular pathogen in other clusters was associated with higher levels of inflammation and lower lung function; this was most pronounced in the cluster dominated by Pseudomonas aeruginosa. The time-resolved interdependencies of the parameters was evaluated in a longitudinal follow-up study. Repeated sampling over a year revealed a strong decrease of the microbiome diversity while inflammatory markers were rather volatile. However, during a 3-year study, inflammatory markers continuously increased, and FEV1 % predicted declined. Moreover, the patients’ initial condition and disease stage had an important effect on the disease progression. Thus, the combined investigation of microbial clusters, inflammation parameters and lung function may provide information about disease severity and allow a more directed therapeutic treatment. Previous studies considered only free NE activity, though recent evidence suggests importance in pathogenesis of its membrane associated fraction. As a rapid and sensitive quantification method to measure membrane bound NE is missing, a flow cytometric approach based on the FRET probe NEmo-2E was established. With the new method it was shown that, a saturation of the membrane NE was detected in CF and COPD, whereas free NE activity was substantially elevated in CF sputum samples. This indicates that NE first associates to the membrane before excess NE is detectable in the supernatant fraction. Thus, membrane associated NE activity could be a potential early inflammation marker for both, COPD and CF.