Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Abstract

SummaryChronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O2) due to production of superoxide (O2 − ). In this study, we show that the PMNs also consume O2 for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N G -monomethyl-L-arginine (L-NMMA) resulted in reduced O2 consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO3 − (P < 0·04, r = 0·66, n = 10) and NO2 − (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O2 for production of reactive oxygen species, the PMNs in CF sputum also consume O2 for production of NO.

Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum

“…The detection of N 2 O required supplementation with 10 mM of NO 3 − , which is higher than the reported concentrations in expectorated sputum (Kolpen et al, 2014a,b;Hassett, 1996). The actual spatiotemporal distribution of NO 3 − in the lungs is, however, not known, but the concentrations of involved electron acceptors may vary more than a 100-fold in sputum samples (Kolpen et al, 2014a). For P. aeruginosa, the ability to consume NO 2 − and increase the biomass during NO 2 − supplementation further confirms that this pathogen can perform denitrification.…”

“…This anaerobiosis is predominantly caused by host cells due to the intense depletion of O 2 by PMNs for production of superoxide (Kolpen et al, 2010) and to a lesser extent for production of nitric oxide (NO) (Kolpen et al, 2014a) and for respiration by the lung epithelium (Worlitzsch et al, 2002), while O 2 consumption by microbial aerobic respiration is diminutive (Kolpen et al, 2010). In spite of anaerobiosis, P. aeruginosa grows and persists in the endobronchial secretions (Yang et al, 2008;Kragh et al, 2014).…”

Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients

“…In the conductive zone, anaerobic conditions have been measured [64] mostly due to the oxygen consumption by PMNs [66]. P. aeruginosa grows by denitrification in the anaerobic environment [208] [209,210]. Low oxygen concentrations were also found in the paranasal sinuses of CF patients [211].…”

Inflammation: A Double-Edged Sword in the Response to <b><i>Pseudomonas aeruginosa</i></b> Infection

“…Recruited neutrophils consume and further deplete available oxygen [90], to which the “obligate aerobe” P. aeruginosa responds by upregulating pathways that allow it to respire using nitrate as a terminal electron acceptor, i.e. by denitrification [91–93]. This strategy may share features with that employed by pathogenic Enterobacteriaceae, which can use by-products of the host inflammatory response as terminal electron acceptors to outcompete obligate aerobes in the inflamed gut [85].…”