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.

Key words: Giardia lamblia, Neutrophil, Extracellular traps, Immunofluorescence