Abstract:

Objective To investigate the effect of hepatocyte local complement on the development of the liver-stage of Plasmodium parasites. Methods Hepa1-6 cells and HepG2-CD81 cells were collected, lysed, and RNA was extracted, respectively. Reverse transcription PCR amplified C3, C3aR1, C5, C5aR1 gene. Hepa1-6 cells and HepG2-CD81 cells suspension were lysed with protease and phosphatase, respectively. The protein concentration was measured using the bicinchoninic acid (BCA) assay and the expression of complement and receptor in hepatocytes was measured using Western blotting. Kunming mice fed with the BY265-RFP strain of P. yoelii expressing red fluorescence and the ANKA strain of P. berghei were fed with Anopheles stephensi for blood sucking. After 17-19 days, anatomically separated salivary glands from An. stephensi mosquitoes and added 50 000 sporozites into 24-well plate containing 10⁵ HepG2-CD81 cells per well for co-incubation, set an additional group that the cells were incubated with C5aR antagonist (40 nnmol/L, 500 μl/well）3 h after co-incubation. Cells were fixed with 4% paraformaldehyde and blocked, the non permeable group was incubated overnight at 4 ℃ with primary C3a rabbit anti human IgG antibody (1∶500) or rC5a rabbit anti human IgG antibody (1∶500), followed by 1 hour of incubation with green Dylight 488 fluorescent labeled goat anti rabbit IgG secondary antibody (1∶400), and 5 minutes of incubation with DAPI staining solution; added a primary antibody UIS4 goat polyclonal antibody (1∶500) to the permeable group and incubated overnight at 4 ℃, then added green IFKine^TM fluorescent labeled donkey anti rabbit IgG secondary antibody (1∶400) and incubated for 1 h, added CD88 rabbit polyclonal antibody (1∶400) to incubate overnight at 4 ℃, added the red Dylight 649 fluorescent labeled goat anti rabbit IgG secondary antibody (1∶400) to incubate for 1 h, added DAPI staining solution to incubate for 5 min, and observed the enrichment of complement around parasitophorous vacuoles under laser confocal microscopy. Take cobra venom factor (CVF) and inject it intraperitoneally into C57BL/6 mice as the CVF group; and set up a C3^-/- group (C3 whole gene knockout C3^-/- mice) and a control group (C57BL/6 mice). The mice in each group were challenged with 10 000 P. yoelii sporozoites. Total RNA was extracted from the liver and reverse transcribed to synthesize cDNA. The content of Plasmodium 18S rRNA was determined by fluorescence quantitative PCR, and the liver parasite burden were indicated as the relative content of 18S rRNA. Ten mice in control group (C57BL/6 mice) and C3aR^-/- group were challenged intravenously with 200 P. yoelii sporozoites, respectively, via tail vein. Five mice in control group (C57BL/6 mice), 6 C5aR whole gene knockout C5aR^-/- mice and 5 liver C5aR conditional knockout Alb-Cre^+/+C5aR^flox/flox hybrid mice were challenged with 1 000 P. yoelii sporozoites. Six mice in control group (C57BL/6 mice) and C5aR^-/- group were challenged with 1 000 P. berghei sporozoites. Tail vein blood was daily collected 3 d after challenge for making a thin blood smear and observed parasitemia after Giemsa staining, till all mice were founded malaria blood stages. Statistical analysis was conducted using Graphpad Prism 9.0 software. Normal distribution data is compared to continuous variables using t-tests, and pairwise differences are compared using one-way analysis of variance (ANOVA) for multiple comparisons; non normal distribution data are compared using Mann-Whitney U test. Results The PCR results showed that C3 (358 bp), C5 (267 bp) and their receptors C3aR (222 bp) and C5aR (388 bp) genes were amplified in Hepa1-6 cell line; C3 (202 bp), C5 (220 bp), C3aR (299 bp), and C5aR (374 bp) genes were amplified in HepG2-CD81 cell line. Western blotting results showed that both types expressed C3, C5, C3aR, and C5aR proteins. Laser confocal microscopy imaging showed that the cell nucleus showed blue fluorescence after DAPI staining, while the P. yoelii liver stages showed red spontaneous fluorescence. In the non permeable group, the highly expressed C3a and C5a in HepG2-CD81 cells show green fluorescence, overlapping with the distribution of parasitophorous vacuoles. The C5aR (pink) of the transmembrane group overlaps with the membrane of parasitophorous vacuoles (green); C5aR expression is still present on the membrane of parasitophorous vacuoles treated with C5aR antagonists. After infection with P. yoelii sporozoites, the relative content of 18S rRNA in the liver of the control group was 0.954 ± 0.523, the CVF group was 0.958 ± 0.231, and the C3^-/- group was 0.638 ± 0.437. There was no statistically significant difference among the three groups (P > 0.05). After infection with P. yoelii sporozoites, the control group and C3aR^-/- mice showed the blood stages after (5.30 ± 0.78) d and (5.30 ± 0.78) d, respectively. After infection with P. berghei sporozoites, the control group and C5aR^-/- mice showed the blood stages after (3.67 ± 0.47) d and (3.83 ± 0.69) d, respectively. There was no statistically significant difference in the appearance time of the the blood stages between the two groups of mice and the control group mice (P > 0.05). After infection with P. yoelii sporozoites, the appearance time of the blood stages in the control group, Alb-cre^+/+C5aR^flox/flox mice, and C5aR^-/- mice were (4.00 ± 0.00), (4.00 ± 0.00), and (4.17 ± 0.37) d, respectively, with no statistically significant difference (P > 0.05). Conclusion The activation of hepatocyte local complement had no significant effect on the development of Plasmodium liver-stage.

Key words: Plasmodium, Liver stage, hepatocyte local complement