Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 μg/mL—azithromycin and 10 and 50 μg/mL—chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.

Neutrophil extracellular traps (NETs) are threads of nuclear DNA complexed with antimicrobial proteins released by neutrophils to extracellular matrix to bind, immobilise, and kill different pathogens. NET formation is triggered by different physiological and non-physiological stimulants. It is also suggested that antibiotics could be non-physiological compounds that influence NET release.The aim of the study was to investigate the effect of clindamycin and amoxicillin on NET release and the phagocyte function of neutrophils.Neutrophils isolated from healthy donors by density centrifugation method were incubated with amoxicillin or clindamycin for two hours, and then NET release was stimulated with phorbol 12-myristate 13-acetate (PMA). After three hours of incubation with PMA NETs were quantified as amount of extracellular DNA by fluorometry and visualised by immunofluorescent microscopy. The percent of phagocyting cells was measured by flow cytometry.We showed that amoxicillin induces NET formation (increase of extracellular DNA fluorescence, p = 0.03), while clindamycin had no influence on NET release (p > 0.05), as confirmed by quantitative measurement and fluorescent microscopy. Regarding phagocyte function, both antibiotics increased bacterial uptake (43.3% and 61.6% median increase for amoxicillin and clindamycin, respectively).We concluded that the ability of antibiotics to modulate NET release depends on the antibiotic used and is not associated with their ability to influence phagocytosis.

Macrophages are essential components of the immune system and play a role in the normal functioning of the cardiovascular system. Depending on their origin and phenotype, cardiac macrophages perform various functions. In a steady-state, these cells play a beneficial role in maintaining cardiac homeostasis by defending the body from pathogens and eliminating apoptotic cells, participating in electrical conduction, vessel patrolling, and arterial tone regulation. However, macrophages also take part in adverse cardiac remodeling that could lead to the development and progression of heart failure (HF) in such HF comorbidities as hypertension, obesity, diabetes, and myocardial infarction. Nevertheless, studies on detailed mechanisms of cardiac macrophage function are still in progress, and could enable potential therapeutic applications of these cells. This review aims to present the latest reports on the origin, heterogeneity, and functions of cardiac macrophages in the healthy heart and in cardiovascular diseases leading to HF. The potential therapeutic use of macrophages is also briefly discussed.

Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach To the Editor: Neutrophil Extracellular Traps (NETs) are structures composed of DNA, histones, and intracellular enzymes, which are released from granulocytes to immobilize and kill pathogens in blood and tissues [1]. Several approaches are used to quantify and assess the effectiveness of NETs' release, based on fluorescent microscopy, immunoenzymatic measurement of neutrophil elastase (NE) release, and fluorometric measurement of DNA release. Unfortunately, these widely used methods have several drawbacks. Quantification of NETs using fluorescent microscopy is laborious and susceptible to observer bias. On the contrary, the release of NE and DNA is not strictly specific for NETosis as other forms of cell death, such as necrosis, could also result in the elevated levels of extracellular NE and DNA. Thus, it would be very beneficial to develop new and reliable methods to assess NETs' formation. Recently, Gavillet et al. published an exciting study that describes the flow cytometric method of NETs' quantification [2]. Because we have also been working on the application of flow cytometry for NETs' studies, we love to share our observations with scientific community.Our major concern regarding the aforementioned work is that Gavillet et al. validated their assay using mice models. But, it is disputable to use murine neutrophils as a reference for human cells-mice release NETs less efficiently than humans, and the formation lasts longer. Thus, it is easier to observe murine NETs using flow cytometry technique because NETs structures are compact and do not show typical morphology of DNA threads [3]. It has been reported that only 30% of murine neutrophils release NETs after 16 h of stimulation with 100 nM phorbol-12-myristate-13-acetate (PMA), while approximately 60-80% of human eutrophils release NETs 3 h after stimulation with PMA [3,4]. Gavillet et al. analyzed human samples after 30-min incubation, and CORRESPONDENCE the average amount of neutrophils which expressed citH3 after 2 min was 7.54 6 4.46% and did not differ between unstimulated, PMA-or CI-treated cells (n56). There were no changes in the level of citH3 in unstimulated samples over time. The amount of citH3 increased in samples stimulated with PMA (7.25 6 4.8%, 8.55 6 5.0%, 10.2 6 5.8% in 2nd, 5th, and 10th min, respectively) with significant difference between 2nd and 10th min (p50.03). In CI-stimulated samples, the level of citH3 also increased to 7.7 6 5.0, 9.03 6 5.8, 9.03 6 3.8% over time, but there was no statistical significance (p50.06 and 0.09 for 2nd vs 5th and 2nd vs 10th min) (Fig. 1B). The level of citrullination after 10th min did not change significantly up to the 3rd h of incubation with NETs' stimulators.The flow cytometry method for NETs' quantification is a real challenge as it allows to analyze cells only in suspension. Undoubtedly, Gavillet et al. opened a new field in NETs' studies. However, the methodology of flow-assisted ...