ORIGINAL ARTICLES

Giardia lamblia trophozoites induce the formation of neutrophil extracellular traps in vitro

  • Shu-ning LI ,
  • Wen-lin LI ,
  • Hai-e SHEN ,
  • Yang WANG ,
  • Xi-feng TIAN
Expand
  • 1 College of Clinical Medicine, North China University of Science and Technology, Tangshan 063000, China
    2 College of Life Sciences, North China University of Science and Technology, Tangshan 063210, China
    3 College of Nursing, North China University of Science and Technology, Tangshan 063210, China

Received date: 2021-03-15

  Revised date: 2021-06-19

  Online published: 2021-08-05

Supported by

Natural Science Foundation of Hebei Province(H2017209143);Innovation and Entrepreneurship Project of North China University of Science and Technology(X20191810)

Abstract

Objective To study the induction of neutrophil extracellular traps (NETs) by Giardia lamblia using qualitative and quantitative methods. Methods Neutrophils were collected from blood samples of a healthy participant by using isolation reagent kit, and cultured with G. lamblia (4 × 105/ml) in M199 medium for 30, 60, 120, 180 or 240 min. The concentration of double-stranded DNA (dsDNA) in the supernatant at the defined time points was determined by PicoGreen staining method. The concentration of myeloperoxidase (MPO) in the supernatant was assayed by ELISA. In the test group designed, 200 μl of neutrophils was added with 200 μl of G. lamblia (both 4 × 105/ml) into 24-well plates and cultured at 37 ℃ for 4 h. Neutrophils producing extracellular trap nets were double-stained with anti-H3Cit and anti-MPO fluorescence. For visualization, DNA was labeled with blue fluorescence, citrullinated histone H3 protein was labeled with green fluorescence, and MPO was labeled with red fluorescence. Laser confocal microscopy was used to observe the formation of NETs. The positive group was set by using the neutrophils treated with 100 nmol/L phorbol ester, while the negative control group was the untreated neutrophils. SPSS software was used for statistical analysis, and the experimental data were analyzed with independent sample t-test. Results The purity of neutrophils isolated from the blood was over 75%. The neutrophils with rod-shaped or lobulated nuclei could be observed under a microscope. The fluorescence intensities after mixing the neutrophils with Giardia (4 × 105/ml) for 30, 60, 120, 180, and 240 min were 7.30 ± 1.12, 11.15 ± 1.10, 21.69 ± 2.71, 26.35 ± 2.26, and 29.29 ± 3.27, respectively, while those in the negative control group and the positive control group were 3.79 ± 0.48 and 35.78 ± 2.83, respectively. The level of dsDNA in the supernatant of the test group was higher than that of the negative control group, and the content of dsDNA increased with prolonged culture time(P < 0.05). ELISA results showed that the absorbance related to the MPO content in the supernatant of the negative control group, test group and positive control group were 0.209 4 ± 0.018 2, 0.611 5 ± 0.060 7, and 0.721 7 ± 0.087 9, respectively. The MPO content of the test group was higher than that of the negative control group (P < 0.05). Laser confocal microscopy revealed presence of a reticular substance between neutrophils and Giardia, which was enriched with histone and MPO. Conclusion Giardia trophozoites have the potential to activate neutrophils releasing extracellular traps in vitro.

Cite this article

Shu-ning LI , Wen-lin LI , Hai-e SHEN , Yang WANG , Xi-feng TIAN . Giardia lamblia trophozoites induce the formation of neutrophil extracellular traps in vitro[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2021 , 39(4) : 455 -460 . DOI: 10.12140/j.issn.1000-7423.2021.04.006

References

[1] Adam RD. Biology of Giardia lamblia[J]. Clin Microbiol Rev, 2001, 14(3):447-475.
[2] Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria[J]. Science, 2004, 303(5663):1532-1535.
[3] Rada B. Neutrophil extracellular traps[J]. Methods Mol Biol Clifton N J, 2019, 1982:517-528.
[4] Mori YK, Yamaguchi M, Terao Y, et al. α-Enolase of Streptococcus pneumonia induces formation of neutrophil extracellular traps[J]. J Biol Chem, 2012, 287(13):10472-10481.
[5] Abi Abdallah DS, Lin CY, Ball CJ, et al. Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps[J]. Infect Immun, 2012, 80(2):768-777.
[6] Simon D, Simon HU, Yousefi S. Extracellular DNA traps in allergic, infectious, and autoimmune diseases[J]. Allergy, 2013, 68(4):409-416.
[7] Goldmann O, Medina E. The expanding world of extracellular traps: not only neutrophils but much more[J]. Front Immunol, 2013, 3:420.
[8] Abi Abdallah DS, Denkers EY. Neutrophils cast extracellular traps in response to protozoan parasites[J]. Front Immunol, 2012, 3:382.
[9] Wei FR, Yang YT, Wang JY, et al. Leishmania infantum infection stimulates neutrophils to generate extracellular traps in mouse[J]. Chin J Parasitol Parasit Dis, 2019, 37(1):18-22. (in Chinese)
[9] (危芙蓉, 杨玥涛, 汪俊云, 等. 婴儿利什曼原虫感染小鼠体内中性粒细胞形成胞外诱捕网能力的研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1):18-22.)
[10] Ventura-Juarez J, Campos-Esparza M, Pacheco-Yepez J, et al. Entamoeba histolytica induces human neutrophils to form NETs[J]. Parasite Immunol, 2016, 38(8):503-509.
[11] Sousa-Rocha D, Thomaz-Tobias M, Diniz LF, et al. Trypanosoma cruzi and its soluble antigens induce NET release by stimulating Toll-like receptors[J]. PLoS One, 2015, 10(10):e013956.
[12] Zhu H, Lu SQ, Guo ZZ, et al. The establishment of axenic culture of a Sichuan strain of Giardia lamblia[J]. Chin J Parasit Dis Con, 1992, 5(1):42-44. (in Chinese)
[12] (祝虹, 卢思奇, 郭增柱, 等. 蓝氏贾第鞭毛虫四川株纯培养的建立[J]. 中国寄生虫病防治杂志, 1992, 5(1):42-44.)
[13] Rodríguez-Espinosa O, Rojas-Espinosa O, Moreno-Altamirano MM, et al. Metabolic requirements for neutrophil extracellular traps formation[J]. Immunology, 2015, 145(2):213-224.
[14] Park SY, Shrestha S, Youn YJ, et al. Autophagy primes neutrophils for neutrophil extracellular trap formation during Sepsis[J]. Am J Respir Crit Care Med, 2017, 196(5):577-589.
[15] Armstrong CL, Klaes CK, Vashishta A, et al. Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA[J]. Innate Immun, 2018, 24(4):210-220.
[16] Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs)-formation and implications[J]. Acta Biochim Pol, 2013, 60(3):277-284.
[17] Azzouz L, Cherry A, Riedl M, et al. Relative antibacterial functions of complement and NETs: NETs trap and complement effectively kills bacteria[J]. Mol Immunol, 2018, 97:71-81.
[18] Villagra-Blanco R, Silva LMR, Görtner U, et al. Molecular analyses on Neospora caninum-triggered NETosis in the caprine system[J]. Dev Comp Immunol, 2017, 72:119-127.
[19] Wei ZK, Wang ZD, Liu X, et al. Toxoplasma gondii triggers neutrophil extracellular traps release in dogs[J]. Front Cell Infect Microbiol, 2020, 10:429.
[20] de Bont CM, Boelens WC, Pruijn GJM. NETosis, complement, and coagulation: a triangular relationship[J]. Cell Mol Immunol, 2019, 16(1):19-27.
[21] Guimarães-Costa AB, DeSouza-Vieira TS, Paletta-Silva R, et al. 3′-nucleotidase/nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps[J]. Infect Immun, 2014, 82(4):1732-1740.
[22] Freitas-Mesquita AL, Dick CF, Dos-Santos ALA, et al. Cloning, expression and purification of 3′-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps[J]. Mol Biochem Parasitol, 2019, 229:6-14.
[23] Mendez J, Sun DL, Tuo WB, et al. Bovine neutrophils form extracellular traps in response to the gastrointestinal parasite Ostertagia ostertagi[J]. Sci Rep, 2018, 8(1):17598.
[24] Sousa-Rocha D, Thomaz-Tobias M, Diniz LF, et al. Trypanosoma cruzi and its soluble antigens induce NET release by stimulating toll-like receptors[J]. PLoS One, 2015, 10(10):e0139569.
[25] Aley SB, Zimmerman M, Hetsko M, et al. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides[J]. Infect Immun, 1994, 62(12):5397-5403.
[26] Cotton JA, Bhargava A, Ferraz JG, et al. Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis[J]. Infect Immun, 2014, 82(7):2772-2787.
[27] Araújo NS, Mundim MJ, Gomes MA, et al. Giardia duodenalis: pathological alterations in gerbils, Meriones unguiculatus, infected with different dosages of trophozoites[J]. Exp Parasitol, 2008, 118(4):449-457.
[28] Aratani Y. Myeloperoxidase: its role for host defense, inflammation, and neutrophil function[J]. Arch Biochem Biophys, 2018, 640:47-52.
[29] Saha P, Yeoh BS, Xiao X, et al. PAD4-dependent NETs generation are indispensable for intestinal clearance of Citrobacter rodentium[J]. Mucosal Immunol, 2019, 12(3):761-771.
[30] Eghbalzadeh K, Georgi L, Louis T, et al. Compromised anti-inflammatory action of neutrophil extracellular traps in PAD4-deficient mice contributes to aggravated acute inflammation after myocardial infarction[J]. Front Immunol, 2019, 10:2313.
[31] Thålin C, Daleskog M, Göransson SP, et al. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma[J]. Immunol Res, 2017, 65(3):706-712.
Outlines

/

〈 〉