Neutrophils are crucial mediators of host defense that are recruited to the central nervous system (CNS) in large numbers during acute bacterial meningitis caused by Streptococcus pneumoniae . Neutrophils release neutrophil extracellular traps (NETs) during infections to trap and kill bacteria. Intact NETs are fibrous structures composed of decondensed DNA and neutrophil-derived antimicrobial proteins. Here we show NETs in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis, and their absence in other forms of meningitis with neutrophil influx into the CSF caused by viruses, Borrelia and subarachnoid hemorrhage. In a rat model of meningitis, a clinical strain of pneumococci induced NET formation in the CSF. Disrupting NETs using DNase I significantly reduces bacterial load, demonstrating that NETs contribute to pneumococcal meningitis pathogenesis in vivo. We conclude that NETs in the CNS reduce bacterial clearance and degrading NETs using DNase I may have significant therapeutic implications.

Key Points Saliva induces bactericidal and DNase resistant NETs in the oral cavity via sialyl LewisX- L-selectin signaling. Disordered homeostasis in the oral cavity may lead to deficient saliva-mediated NETosis.

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Severe COVID-19 can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hall mark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe COVID-19 patients reveal high levels of neutrophil extracellular trap(s) (NETs) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and can lead to acute respiratory distress syndrome (ARDS). Recombinant human DNase (rhDNase/Pulmozyme) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated 5 COVID-19 patients presenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill COVID-19 patients was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of haemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high flow oxygen therapy in patients. Targeting NETs using rhDNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

Neutrophil extracellular traps (NETs) that are extensive webs of DNA covered with antimicrobial proteins into the extracellular environment during infection or inflammation as a part of their defense arsenal. Image acquisition of fluorescently labeled NETs and subsequent image-based quantification is frequently used to analyze NET formation (NETosis) in response to various stimuli. However, there are important limitations in the present methods for quantification. Manual methods tend to be error-prone, tedious, and often quite subjective, whereas the software-rooted options are either semi-automatic or difficult to operate. Here, we present an automated and uncomplicated approach for quantifying NETs from fluorescence images, built as a freely available app for MATLAB®. It is based on detection of a set of clearly defined parameters, all related to the biological manifestation of NETs and allowing for single-cell resolution quantification and analysis.