Influence of Different Bacteria Strains Isolated from Septic Children on Release and Degradation of Extracellular Traps by Neutrophils from Healthy Adults.

Neutrophils are the first line of immune defense against pathogens. They use three major antimicrobial mechanisms: phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). NETs are structures which consist of nuclear DNA conjugated with antibacterial proteins. They are formed to entrap and kill pathogens. The aim of the study was to evaluate the influence of Escherichia coli (E. coli), Streptococcus pneumoniae (S. pneumoniae), Stenotrophomonas maltophilia (S. maltophilia), and Pseudomonas aeruginosa (P. aeruginosa), isolated from the peripheral blood of children with sepsis, on the release and degradation of NETs by neutrophils isolated from blood healthy adult subjects. Neutrophils were stimulated with the bacterial strains outlined above. The quantitative and qualitative analyses of NETs release were performed by fluorometric measurement and immunofluorescence, respectively. The ability of bacteria to degrade NETs was studied qualitatively. Oxidative burst was assessed by flow cytometry. Histone H3 citrullination was evaluated by Western blot. We found that NETs were formed only when neutrophils were incubated with S. pneumoniae. E. coli, P. aeruginosa, and S. maltophilia did not induce the release of the NETs. P. aeruginosa, S. pneumoniae, and E. coli induced the production of reactive oxygen species (ROS) by neutrophils. Two studied bacterial strains (S. pneumoniae and E. coli) were able to degrade NETs. However, none of the strains induced the citrullination of histone H3. We conclude that the ability of bacteria to induce and degrade NETs depends on the specific bacterial strain.

The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release.

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.

A Comparison of Mindray BC-6800, Sysmex XN-2000, and Beckman Coulter LH750 Automated Hematology Analyzers: A Pediatric Study.

BACKGROUND: Modern automated laboratory hematology analyzers allow the measurement of over 30 different hematological parameters useful in the diagnostic and clinical interpretation of patient symptoms. They use different methods to measure the same parameters. Thus, a comparison of complete blood count made by Mindray BC-6800, Sysmex XN-2000 and Beckman Coulter LH750 was performed. MATERIALS AND METHODS: A comparison of results obtained by automated analysis of 807 anticoagulated blood samples from children and 125 manual microscopic differentiations were performed. This comparative study included white blood cell count, red blood cell count, and erythrocyte indices, as well as platelet count. RESULTS: The present study showed a poor level of agreement between white blood cell enumeration and differentiation of the three automated hematology analyzers under comparison. A very good agreement was found when comparing manual blood smear and automated granulocytes, monocytes, and lymphocytes differentiation. Red blood cell evaluation showed better agreement than white blood cells between the studied analyzers. CONCLUSION: To conclude, studied instruments did not ensure satisfactory interchangeability and did not facilitate a substitution of one analyzer by another.

The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release.

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.