Kader YILDIZ, Ayşe ŞİMŞEK, Neslihan SÜRSAL ŞİMŞEK

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

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.

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1. Dubey JP. History of the discovery of the life cycle of Toxoplasma gondii. International Journal for Parasitology 2009; 39 (8): 877- 882. doi: 10.1016/j.ijpara.2009.01.005.
2. Dubey J, Lindsay D, Speer C. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews 1998; 11 (2): 267-299. doi:10.1128/CMR.11.2.267
3. Dubey JP. Toxoplasmosis of Animals and Humans.2nd ed.Boca Raton, Florida:CRC Press; 2010.
4. Sheffield HG, and Melton ML (1968): The fine structure and reproduction of Toxoplasma gondii. Journal of Parasitology 1968; 54: 209-226. doi:10.2307/3276925.
5. Tizard IR. Veterinary Immunology. 10 th ed. St. Louis, MO, USA: Elsevier; 2018
6. Dmitry G. Neutrophils, The: New Outlook for Old Cells: World Scientific; 2013.
7. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303 (5663): 1532-1535. doi: 10.1126/science.1092385
8. Guimaraes-Costa AB, Nascimento MT, Froment GS, Soares RP, Morgado FN et al. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proceedings of the National Academy of Sciences of the United States of America 2009; 106 (16): 6748-6753. doi: 10.1073/ pnas.0900226106
9. Wang Y, Chen Y, Xin L, Beverley SM, Carlsen ED et al. Differential microbicidal effects of human histone proteins H2a and H2b on Leishmania promastigotes and amastigotes. Infection and Immunology 2011; 79 (3): 1124-1133. doi: 10.1128/iai.00658-10
10. Gabriel C, McMaster WR, Girard D, Descoteaux A. Leishmania donovani promastigotes evade the antimicrobial activity of neutrophil extracellular traps. The Journal of Immunology 2010; 185 (7): 4319-4327. doi: 10.4049/jimmunol.1000893
11. Ventura-Juarez J, Campos-Esparza M, Pacheco-Yepez J, LópezBlanco J, Adabache-Ortíz A et al. Entamoeba histolytica induces human neutrophils to form Net s. Parasite Immunology 2016; 38 (8): 503-509. doi: 10.1111/pim.12332
12. Behrendt JH, Hermosilla C, Hardt M, Failing K, Zahner H et al. Pmn-mediated immune reactions against Eimeria bovis. Veterinary Parasitology 2008; 151 (2-4): 97-109. doi: 10.1016/j. vetpar.2007.11.013
13. Silva LM, Caro TM, Gerstberger R, Vila-Vicosa MJ, Cortes HC et al. The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitology Research 2014; 113 (8): 2797-2807. doi: 10.1016/j.apjtb.2016.01.001
14. Muñoz-Caro T, Hermosilla C, Silva LM, Cortes H, Taubert A. Neutrophil extracellular traps as innate immune reaction against the emerging apicomplexan parasite Besnoitia besnoiti. PloS One 2014; 9 (3). doi: 10.1371/journal.pone.0091415
15. Munoz-Caro T, Lendner M, Daugschies A, Hermosilla C, Taubert A. Nadph Oxidase, Mpo, Ne, Erk1/2, P38 Mapk and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation. Developmental & Comparative Immunology 2015; 52 (2): 245-254. doi: 10.1016/j.dci.2015.05.007
16. Villagra-Blanco R, Silva LMR, Gartner U, Wagner H, Failing K et al. Molecular analyses on Neospora caninum-Triggered NETosis in the caprine system. Developmental & Comparative Immunology 2017; 72: 119-127. doi: 10.1016/j.dci.2017.02.020
17. Villagra-Blanco R, Silva LMR, Munoz-Caro T, Yang Z, Li J et al. Bovine polymorphonuclear neutrophils cast neutrophil extracellular traps against the abortive parasite Neospora caninum. Frontiers in Immunology 2017; 8: 606. doi: 10.3389/ fimmu.2017.00606
18. Abi Abdallah DS, Lin C, Ball CJ, King MR, Duhamel GE et al. Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infection and Immunity 2012; 80 (2): 768-777. doi: 10.1128/IAI.05730-11
19. Wanderley CW, Silva CM, Wong DV, Ximenes RM, Morelo DF et al. Bothrops jararacussu snake venom-induces a local inflammatory response in a prostanoid- and neutrophildependent manner. Toxicon 2014; 90: 134-147. doi: 10.1016/j. toxicon.2014.08.001
20. Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs) - formation and implications. Acta Biochimica Polonica 2013; 60 (3): 277-284. PMID: 23819131
21. Alghamdi AS, Foster DN. Seminal Dnase frees spermatozoa entangled in neutrophil extracellular traps. Biology of Reproduction 2005; 73 (6): 1174-1181. doi: 10.1095/ biolreprod.105.045666
22. Aulik NA, Hellenbrand KM, Klos H, Czuprynski CJ. Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils. Infection and Immunology 2010; 78 (11): 4454-4466. doi: 10.1128/ IAI.00840-10
23. Chuammitri P, Ostojic J, Andreasen CB, Redmond SB, Lamont SJ et al. Chicken Heterophil Extracellular Traps (HETs): Novel defense mechanism of chicken heterophils. Veterinary Immunology and Immunopathology 2009; 129 (1-2): 126-131. doi: 10.1016/j.vetimm.2008.12.013
24. Ermert D, Urban CF, Laube B, Goosmann C, Zychlinsky A et al. Mouse neutrophil extracellular traps in microbial infections. Journal of Innate Immunity 2009; 1 (3): 181-193. doi: 10.1159/000205281
25. Palic D, Ostojic J, Andreasen CB, Roth JA. Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils. Developmental & Comparative Immunology 2007; 31 (8): 805- 816. doi: 10.1016/j.dci.2006.11.010.
26. Wardini AB, Guimaraes-Costa AB, Nascimento MT, Nadaes NR, Danelli MG, M et al. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. Journal of General Virology 2010; 91 (1): 259- 264. doi: 10.1099/vir.0.014613-0
27. Yildiz K, Gokpinar S, Gazyagci AN, Babur C, Sursal N et al. Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle. Veterinary Immunology and Immunopathology 2017; 189: 1-10. doi: 10.1016/j.vetimm.2017.05.005
28. Sursal N, Cakmak A, Yildiz K. In vitro investigation on extracellular traps formation of cat polymorph nuclear leucocytes against to Toxoplasma gondii. In: 2nd International Conference on Innate Immunity & Immune System Diseases. Berlin, Germany: Journal of Clinical and Cellular Immunology; 2016; pp. 82.
29. Muñoz-Caro T, da Silva LMR, Rentería-Solis Z, Taubert A, Hermosilla C. Neutrophil extracellular traps in the intestinal mucosa of Eimeria-infected animals. Asian Pacific Journal of Tropical Biomedicine 2016; 6 (4): 301-307. doi: 10.1016/j. apjtb.2016.01.001.
30. Wei Z, Hermosilla C, Taubert A, He X, Wang X et al. Canine neutrophil extracellular traps release induced by the apicomplexan parasite Neospora caninum in vitro. Frontiers in Immunology 2016; 7: 436. doi: 10.3389/fimmu.2016.00436
31. Reichel M, Munoz-Caro T, Sanchez Contreras G, Rubio Garcia A, Magdowski G et al. Harbour seal (Phoca Vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii. Developmental & Comparative Immunology 2015; 50 (2): 106-115. doi: 10.1016/j.dci.2015.02.002
32. Wartha F, Beiter K, Normark S, Henriques-Normark B. Neutrophil extracellular traps: casting the NET over pathogenesis. Current Opinion in Microbiology 2007; 10 (1): 52-56. doi: 10.1016/j.mib.2006.12.005
33. Guimaraes-Costa AB, DeSouza-Vieira TS, PalettaSilva R, Freitas-Mesquita AL, Meyer-Fernandes JR et al. 3’-Nucleotidase/Nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps. Infection and Immunity 2014; 82 (4): 1732-1740. doi: 10.1128/IAI.01232-13
34. Macedo IS, Lima MVA, Souza JS, Rochael NC, Caldas PN et al. Extracellular traps released by neutrophils from cats are detrimental to Toxoplasma gondii Infectivity, Microorganisms 2020; 8 (11): 1628. doi: 10.3390/microorganisms8111628
35. Makni-Maalej K, Chiandotto M, Hurtado-Nedelec M, Bedouhene S, Gougerot-Pocidalo MA et al. Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of P47phox and the activation of Rac2: involvement of protein tyrosine kinases, Pi3Kinase, PKC, ERK1/2 and P38MAPkinase. Biochemical Pharmacology 2013; 85 (1): 92-100. doi: 10.1016/j.bcp.2012.10.010