| Objective This study aims to reveal the diversity between the ability of Toxoplasmagondii strains with different genotypes to induce biased/polarized activation of mousemacrophage. Simultaneously, strain virulence with genotype Chinese1and type I wasinvestigated.Methods(1)To revive and subculture the tachyzoite of RH and Wh3: Tachyzoites of RH andTgCtwh3are provided by our laboratory,106tachyzoites were intraperitoneallyinoculated to six to eight weeks of BALB/C mice. The mice were under closesurveilance after inoculation. The mice were sacrificed by cervical dislocation underanestheathia when the animals showed clinical manifestations such as weakness,roachback and piloerection. After being sterilized in75%alcohol for about5min, themice were fixed on the stencil plate, the peritoneal fluids were harvested with sterilenormal saline water and the abdominal cavity was washed with the same aline to collectas many parasites as possible, and the tachyzoites were counted. After3generations ofmouse passage The tachyzoites were collected for the next experiments.(2) Strainvirulence experiment: Fourty to six-week old BABL/c mice were divided into eight groups with5each, and were intraperitonealyl inoculated with101,102103, and104tachyzoites of Wh3and RH strain tachyzoites, respectively. Mice were monitored forsurvival.（3）Extract of the total RNA and qRT-PCR: RNA preparation and quantitativePCR for amplification of roptry protein16(ROP16) in both TgCtWh3and RH wereperformed following the manufacturer’s instructions. Amino acid sequences of ROP16of the two strains was predicted and subjected to Blast for comparision.(4) Thecultivation of RAW264.7: RAW264.7were infected with Wh3and RH tachyzoites,respectively in accordance with the ratiao of tachyzoite: cell of3:1, and continuouslycultured for24hours. Supernatant was collected and the total protein, nucleus protein,and total RNA of the cells were harvested according to the manual instructions.(5)Detection of M1/M2related cytokines: macrophage-generated cytokines and relatedtranscript factors, such as stat3, pstat3, stat6, pstat6, iNOS, Arg-1, NF-κB, IκBα，etc.were tested by Western blotting, and IL-10, IL-12p40, IL-12p70, iNOS and Arg-1inmRNA level were examined by fluorescence quantitative PCR. The nitric oxide (NO)was detected from the supernatant of the cell culture.ResμLts (1) T. gondii TgCtwh3strain with genotype Chinese1shares the samemutation of ROp16at amino acid residue leucine-503(L503) with the type I RH strain;(2) TgCtwh3strains showed the high virulence to mice but slightly lower than thevirulent RH strain;(3) TgCtwh3strain infected RAW264.7cells after24hours post infection gave rise tohigh expression of iNOS and Arg-1, whereas RH strains induced high expression ofArg-1. The NO secretion of TgCtwh3-infected macriphages was slightly higher thanthat of the RH strain and the normal control;(4)after24hours of TgCtwh3strain and RH strain infection, RAW264.7cells showed high expression of IL-12, but low expression of IL-10.Conclusions⑴TgCtwh3strain with genotype Chinese1, which is predominantlycirculationg in China, Differs in virulence and in ability to induce activation ofmacrophages. TgCtwh3strain-infected macrophages had a high expression of iNOS andArg-1, while RH-inoculated macrophages generated a high expression of Arg-1.⑵RAW264.7cells infected with TgCtwh3strain tended to the bias of classicallyactivated macrophages(M1) on24hours postinfection, producing a high level of IL-12and low level of IL-10. The cells infected with RH strain, however, biased toalternatively activated macrophages(M2). Additionally, at early stage of24hourspostinfection, the host cells seem to stay in a status of M1too. |
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