Neutrophil Extracellular Traps of Cynoglossus semilaevis: Production Characteristics and Antibacterial Effect

Abstract

Neutrophil extracellular traps (NETs) are structures released by neutrophils as a cellular immune defense against microbial invasion. The process of NETs generation, netosis (NETosis), can take place via either a suicidal mechanism, during which the NETs-releasing cells became dead, or a "live" mechanism, during which the NETs-releasing cells remain vital. NETosis has been studied intensively in mammals in recent years, but very little is known about the NETosis in fish. In this study, we examined NETosis in tongue sole (Cynoglossus semilaevis), a species of teleost with important economic values. We found that following stimulation with phorbol 12-myristate 13-acetate (PMA) and three common fish bacterial pathogens, abundant NETs structures were released by neutrophils that were most likely in a live state. The released NETs captured, but did not kill, the bacterial pathogens; however, the replication of extracellular, but not intracellular, pathogens was inhibited by NETs to significant extents. Reactive oxygen species (ROS), nitric oxide (NO), and myeloperoxidase (MPO) production were observed to be enhanced in NETosing neutrophils, and blocking the production of these factors by inhibitors significantly decreased NETs production induced by PMA and all three bacteria. Taken together, these results indicate for the first time that in teleost there exists a non-cell death pathway of NETosis that produces NETs with antibacterial effects in a ROS-, NO-, and MPO-dependent manner.

Keywords: Cynoglossus semilaevis; NETosis; antibacterial; innate immune defense; neutrophil extracellular trap.

Figure 1

Production of neutrophil extracellular traps (NETs) by neutrophils in response to phorbol 12-myristate 13-acetate (PMA) treatment. Tongue sole neutrophils were treated with or without (boxed image) PMA and observed with a scanning electron microscope. Arrows indicate NETs.

Figure 2

Production of neutrophil extracellular traps (NETs) by live neutrophils. (A) Tongue sole neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and stained with Sytox Green and DAPI. The cells were then observed with a fluorescence microscope. (B) PMA-treated neutrophils were stained with Sytox Green and incubated with DNase I for different times. The cells were observed as above. Arrows indicate NETs. Bar, 20 μm.

Figure 3

Bacteria-induced production of neutrophil extracellular traps (NETs). Tongue sole neutrophils were treated with Pseudomonas fluorescens (A), Vibrio harveyi (B), and Edwardsiella tarda (C), and the cells were observed with a scanning electron microscope. Arrows indicate NETs-trapped bacteria.

Figure 4

Time-course production of neutrophil extracellular traps (NETs) by neutrophils in response to various treatments. Tongue sole neutrophils were treated with or without (control) phorbol 12-myristate 13-acetate (PMA), Pseudomonas fluorescens, Vibrio harveyi, and Edwardsiella tarda for various hours, and NETs production was determined. The experiment was performed three times, and the results are shown as means ± SEM. **P < 0.01; *P < 0.05.

Figure 5

Multiplication of neutrophil extracellular traps (NETs)-trapped bacteria. Neutrophils were stimulated with phorbol 12-myristate 13-acetate for NETs production (NETs-positive cells), and a portion of the cells were then treated with DNase I to degrade NETs (NETs-negative cells). NETs-positive and NETs-negative cells were incubated with Pseudomonas fluorescens (A), Vibrio harveyi (B), and Edwardsiella tarda (C) for different hours, and bacterial numbers were determined by plate count. The experiment was performed three times, and the results are shown as means ± SEM. **P < 0.01.

Figure 6

Production of reactive oxygen species (ROS), NO, and myeloperoxidase (MPO) in neutrophils after different treatments. Tongue sole neutrophils were treated with or without (control) phorbol 12-myristate 13-acetate (PMA), Pseudomonas fluorescens, Vibrio harveyi, and Edwardsiella tarda for different times, and ROS (A), NO (B), and MPO (C) productions were determined. The experiment was performed three times, and the results are shown as means ± SEM. **P < 0.01; *P < 0.05.

Figure 7

Production of neutrophil extracellular traps (NETs) in response to various inhibitors. Tongue sole neutrophils were treated with phorbol 12-myristate 13-acetate (PMA), Edwardsiella tarda, Pseudomonas fluorescens, or Vibrio harveyi in the presence or absence (control) of Trolox (A), L-NAME (B), and ABAH (C), and NETs production was determined. The experiment was performed three times, and the results are shown as means ± SEM. **P < 0.01.