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Objective The formation of neutrophil extracellular traps (NETs) induced by influenza A virus (IAV) subtype H1N1 was investigated both qualitatively and quantitatively. Methods Mouse bone marrow neutrophils were isolated, purified, and characterized. NETs were induced in vitro using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). Additionally, IAV groups with three different titers: one hundred 50% tissue culture infective doses (100 TCID50), 50 TCID50, and 25 TCID50 as well as the normal control group were established, and the intracellular nucleoprotein (NP) mRNA expression levels of the IAV groups were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The effect of each factor on neutrophils was assessed by measuring the concentration of circulating cell-free DNA (cfDNA) in the supernatant of each group using the quantitative SYTOX Green staining method. The NETs structure in each group of cells was observed under a fluorescence microscope after Hoechst 33342 staining. An immunofluorescence assay was performed to detect the expression levels of NET characteristic markers citrullinated histone H3 (CitH3), peptidylarginine deiminase 4 (PAD4), myeloperoxidase (MPO), and neutrophil elastase (NE) proteins, as well as the nuclear co-localization and fluorescence intensity of PAD4 with CitH3, and MPO with NE in each group. The levels of reactive oxygen species (ROS) were determined by using a fluorescent probe assay, and the levels of intracellular CitH3 protein formation were determined by using Western blotting. Results The activity of neutrophils isolated from mouse bone marrow reached 98%, with purities of ≥87%. The expression levels of NP mRNA in the IAV groups were significantly higher than those in the control group. Compared with the control group, the cfDNA levels of the PMA, LPS, and IAV groups were significantly increased, with significant increases in the web-like structures of NETs. The immunofluorescence assay showed that the relative expression levels of MPO, NE, PAD4, and CitH3 proteins were elevated to varying degrees, with the co-localization of PAD4/CitH3 or MPO/NE increased after IAV infection. Moreover, the peak of MPO protein expression was observed before that of NE protein, whereas CitH3 expression paralleled that of PAD4 protein. Additionally, the ROS level was elevated, and the level of CitH3 protein formation was also significantly increased. Conclusion Stimulation of neutrophils by IAV (H1N1) induces NET formation, which may be related to the increased intracellular ROS and PAD4 levels.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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