In vitro investigation on extracellular traps formation of cat polymorphonuclear leucocytes against Toxoplasma gondii

Authors: NESLİHAN SÜRSAL ŞİMŞEK, AYŞE ÇAKMAK, KADER YILDIZ

Abstract: Neutrophil granulocytes are one of the most important defenders of the innate immune system in the host. Moreover, neutrophils are able to reach the inflammation area and kill the pathogens using various defense strategies including neutrophil extracellular traps (NETs). However, there is still not enough information available regarding the innate immunity against Toxoplasma gondii in cats that are both definitive and intermediate hosts of this parasite. Therefore, we investigated the in vitro NETs formation which is induced by cat polymorphonuclear leucocytes (PMNs) against T. gondii tachyzoites. Firstly, PMNs were isolated from cat venous blood samples by using discontinues Percoll dilutions (72%, 63%, 54%, and 45%). Afterward, MN-tachyzoites cocultures were stained against histone (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) by using monoclonal antibodies and were examined under a fluorescence microscope. The effect of different parasite doses (1:1, 1:3, and 1:5) and incubation times (30, 60, 90, and 120 min) on NETs formation was also evaluated. The presence of the extracellular DNA content was measured using a fluorometer. Confluent Vero cell cultures were used to assess the effect of NETs on the tachyzoites viability. The classical structures of NETs, such as extracellular DNA, NE, H3, and MPO were microscopically observed in the NETs formation released from cat PMNs. The amount of extracellular DNA increased in parallel with the incubation time (p ? 0.001). The influence of the tachyzoites dose on the NETs formation was not statistically significant (p > 0.05). Zymosan was used as a positive control in the experiments and it was shown to be an important inducer for the NETs formation. In conclusion, as mentioned previous studies and considering our results, the NETs may be a conserved strategy to control T. gondii infection in hosts because of the immobilization and lethal effects.

Keywords: Cat, in vitro, neutrophil extracellular traps, Toxoplasma gondii

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