Abstract

BackgroundDifferent from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. However, there are different virulent isolates within China 1, such as virulent TgCtwh3 and avirulent TgCtwh6, and little is known about differences in macrophage activation between them. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates.MethodsAdherent peritoneal macrophages (termed Wh3-Mφ and Wh6-Mφ, respectively) harvested from infected mice were cultured for detection of Nitric Oxide and arginase activity, and activated markers on Wh3-Mφ/Wh6-Mφ were determined by flow cytometry. In in vitro experiments, the levels of IL-12p40 and TNF-α were measured using ELISA kits, and mRNA expressions of IL-12p40, TNF-α, iNOS, Arg-1 and Ym1 were assayed by real-time PCR. To confirm the activation state of NF-kB p65 in infected cells stained by IF, protein levels of iNOS, Arg-1, Ym1, nuclear NF-κB p65, and phosphorylation of STAT6/STAT3/IκBα were evaluated by Western Blotting. A one-way ANOVA test was used to compare differences among multiple groups.ResultsThe result revealed that contrary to the virulent TgCtwh3, the less virulent TgCtwh6 isolate induced a significant increase in IL-12p40 and TNF-α. Although both isolates down-regulated CD80, CD86 and MHCII molecule expression on macrophages, TgCtwh3 promoted up-regulation of PD-L2 and CD206. Wh6-Mφ generated a high level of NO whereas Wh3-Mφ up-regulated Ym1 and arginase expression at transcriptional and protein levels. In terms of signaling pathway, TgCtwh3 induced phospho-STAT6, conversely, TgCtWh6 led to NF-κB p65 activation.ConclusionsThe virulent TgCtwh3 isolate induced macrophages to polarize toward alternatively activated cells with STAT6 phosphorylation, whereas the less virulent TgCtwh6 elicited the development of classically activated macrophages with nuclear translocation of NF-κB p65. This discrepancy suggests that it is necessary to thoroughly analyze the genotype of TgCtwh3 and TgCtwh6, and to further study other effector molecules that contribute to the macrophage polarization in T. gondii.

Highlights

  • Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China

  • TgCtwh3 and TgCtwh6 strains display different virulence Our previous study showed that different from the three clonal lineages of T. gondii strains isolated in North America and Europe, the genotype China 1 (ToxoDB#9) is a major lineage prevalent in China with different virulent strains [12]

  • It was found that mice inoculated with 500 TgCtwh3 tachyzoites uniformly succumbed to infection within 9 days (Figure 1A), in contrast, no death was noted in animals given an equivalent dose of TgCtwh6 tachyzoites during the whole experiment

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Summary

Introduction

Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates. Tachyzoites, the rapidly replicative form of the parasite, elicits an extremely strong type Th1 immune response, characterized by proinflammatory cytokine production such as IL-6, IFN-γ and TNF-α. Macrophages can provide a niche permissive for parasite replication and are the most abundant cell type infected by Toxoplasma [3]. M1 exhibits antimicrobial functions against intracellular pathogens which is conducted by the production of reactive oxygen and nitrogen intermediates such as NO and promote strong Th1 responses, while M2 is accompanied by diminished proinflammatory cytokine secretion and shares high expression of scavenger, mannose (CD206) and galactose receptors [5]

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