BackgroundNeutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.MethodsThe effect of two natural surfactants (Alveofact® and Curosurf®) on spontaneous and phorbol-12-myristate-13-acetate–induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.ResultsBoth surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.ConclusionsThe described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.

BackgroundNeutrophil extracellular traps (NETs)—as double-edged swords of innate immunity—are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.MethodsWe analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).ResultsUntreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.ConclusionsG-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.

Besides performing phagocytosis and degranulation, neutrophils are capable of eliminating microorganisms by releasing neutrophil extracellular traps (nEts). nEt formation was found to be associated with increased mortality in sepsis. during sepsis levels of interleukin 1β (iL-1β), a cytokine, increases significantly and also was associated with increased mortality. Blocking of the interleukin 1 (iL-1) receptor by anakinra leads to less nEt formation in gout patients. However, nEt formation is crucial during infection by trapping pathogens and thereby slowing the process. total or early blocking of cascades leading to nEts may lead to aggravation of infection in otherwise mild cases. the dose-and time-dependent effect of the iL-1 receptor antagonist anakinra was tested on spontaneous, lipopolysaccharide (LPs)-induced and phorbol-12-myristate 13-acetate (Pma)-induced formation of nEts in vitro. Quantitative detection of nEts was performed for nEtspecific proteins and cell-free dna. immunostained microscopy imaging was used for visualization.our study shows a dose-and time-dependent inhibitory effect of anakinra that involves the change of intracellular calcium mobilization on the formation of nEts in vitro for Pma-stimulated neutrophils but not for LPs-stimulated neutrophils. it may be useful for treatment of sepsis as part of a multimodal treatment concept, but it seems that timing and dose need to be carefully chosen.