| Though great efforts have been made in the past 50 years, schistosomiasis japonica remains a serious public health problem in China. The comprehensive measures for controlling schistosomiasis were based on the population chemotherapy with praziquantel, combined with public health promotion and killing snails in endemic areas, which have made great achievements. However, the control task is being challenged by the change of natural circumstance and decreased financial resources, and the most important challenge is high rate of reinfection after chemotherapy due to large numbers of reservoir hosts and difficulties in controlling the areas with Oncomelania hupensis infestation. In 1980s, the worldwide scientists reached consensus that is the priority to develop an effective anti-schistosomiasis vaccine in order to prevent reinfection after chemotherapy.During the past decades, vaccine development and related basic immunological researches were mainly concerned on schistosomiasis mansoni. Consequently, the development of vaccines against schistosomiasis japonica is far behind that of schistosomiasis mansoni, especially the understanding of basic immunology, which greatly limits our choice of rational strategies for vaccine development for schistosomiasis japonica. Thus, fundamental immunological research should be highlighted to develop effective and specific vaccines against schistosomiasis japonica. Many vaccine studies for schistosomiasis mansoni have estabilished that antigen-specific CD4+ T cell-mediated immunity and humoral response are fundamental to the acquired resistance against schistosome, which concentrate on the acquired immunity. On the other hand, the understanding of innate immunity involved is just in the very initial stage.So far, many studies of the innate immune response during the infection of Schistosoma mansoni (S. mansoni) focused on egg antigen to illuminate why host developed a predominant Th2 response after egg deposition. All these data suggested that antigens derived from parasite could activate the cascade pathway induced by pattern recognition receptors (PRRs) to initiate and direct the acquired immune response. Thus, we could speculate that during the early stage of infection, parasite molecules might also cause a similar effect through specific PRRs pathway. Nevertheless, up to now few studies reported characteristics of immune response and related molecules during TLRs recognition of Schistosoma japonicum (S. japonicum). Those results from studies of S. mansoni also suggested that, Toll-like receptor 2 (TLR2) and TLR4 were two important immune receptors involved in recognition of components of SEA; and TLR4 also played an important role in the immunorecognition of cercaria molecules. Therefore, the purpose of this study is to investigate the effects of TLR2 and TLR4 on the outcomes of S. japonicum infection.TLR2 knock out (TLR2-/-), TLR4 null mutated (TLR4-/-) mice and their counterpart wild-type (WT) C57BL/6J and C57BL/10J mice were infected with S. japonicum. Mice were sacrificed to measure the parasite burdens at 6 weeks after infection.To evaluate the immune responses after infection, the levels of schistosoma antigen- specific IgG were determined for each mouse by indirect ELISA; and the production of Th1/Th2 cytokines by spleen mononuclear cells was determined by Bio-plex system. Then we further compared the gene expression profiles from gene deficient and their WT control 6 weeks post infection using microarrays (Mouse Genome Expression 430 2.0, Affymetrix Co.). Those genes with more than 2-fold change were further analyzed with both GO and pathway anaylsis, and the mRNA levels of cytotoxicity associated genes were validated by real-time quantitative PCR (RTQ-PCR). Finally, the cytotoxicity of natural killer (NK) cells and CD8+T cells from each groups of mice primed with SEA were compared.The main results we got are as follows:1. Both worm and egg burdens were reduced in TLR2-/- mice, but increased in TLR4-/- mice. Six weeks after challenge with 40 S. japonicum cercariae, parasite burdens including total worms, total eggs in livers and eggs produced by each pair of worms (EPP) were evaluated. The parasite burdens were decreased in TLR2-/- group, but increased in TLR4-/- group when compared to the WT mice, respectively. Significant differences were observed in total eggs and EPP but not in total worms.2. The level of cellullar immune response in TLR2-/- mice was higher than that in B6 mice, while the expression of cytokines in TLR4-/- mice was lower than that in B10 mice during the acute phase of S. japonicum infection. In the supernatant of spleen mononuclear cells stimulated with SEA or ConA, the concentrations of IL-12p70, IFN-?, IL-2, GM-CSF and IL-4 in TLR2-/- mice were markedly higher than those of B6 mice. The production of TNF-?and IL-10 in TLR2-/- mice were more than that in WT mice when stimulated with ConA, but the expression of IL-10 stimulated with SEA was less than that in B6 mice. On the contrary, the production of IL-12p70, IFN-?, IL-2, IL-5 and IL-4 in TLR4-/- mice were significantly less than that in B10 group; the levels of IL-10 stimulated with ConA was much less. After stimulated with SEA, the expression of TNF-?was significantly less than that in the B10 mice.3. After infection with S. japonicum, the level of antigen-specific IgG in TLR4-/- mice was significantly higher than that in B10 mice, but there was no significant difference between TLR2-/- and B6 mice. Following S. japonicum infection, schistosome antigen-specific IgG antibody in the sera of all three groups of mice kept on rising. In acute phase of infection, the level of SWAP- and SEA-specific IgG antibody in TLR4-/- mice were significantly higher than those in B10 mice; but there were no significant difference in both SWAP and SEA-specific antibody between TLR2-/- and B6 mice.4. Using the microarrays to compare the gene expression profiles in splenocytes 6 weeks post-exposure, we found that two categories of genes involved in T cell responses and cytotoxicity were significantly upregulated in TLR2-/-mice; and these genes were downregulated in TLR4-/- mice. In TLR2-/- mice, there were 251 probe sets found to have significantly higher signal values and 444 probe sets were downregulated when compared to B6 mice; and in TLR4-/- mice, only 91 probe sets were found to be enhanced more than 2-fold, and 121 genes have lower signal values. Interestingly, those genes enhanced in TLR2-/- mice were downregulated in TLR4-/- mice correspondingly. Further analysis of GO and pathway indicated that these genes and pathway mainly belong to two categories: T cell response and cytotoxicity pathway.5. After being primed with SEA, the cytotoxicity of NK cells in both TLR2-/- and TLR4-/-mice was slightly higher than that in WT mice without significant difference. And the mRNA levels of cytotoxicity-associated genes from CD8+T cell were increased in TLR2-/- mice, but decreased in TLR4-/- mice. The cytotoxicity of CD49b+ NK cells from spleens of both TLR2-/-and TLR4-/-mice to YAC-1 cells was similar to that in their WT controls. On the other hand, the mRNA levels of cytotoxicity-associated genes (gzma, gzmb, gzmk, pfr1 and fasl) from CD8+ T cells of TLR2-/- mice were higher than that of B6 mice, and only the level of pfr1 was significantly different; and the expression of gzma, gzmb and fasl from CD8+ T cells of TLR4-/- mice were significantly lower than those of B10 mice.In conclusion, after infection with S. japonicum, parasite burdens, T cell-mediated immune response as well as the cytotoxicity pathway were significantly different in TLR2-/- mice and TLR4-/- mice, when compared with the wild-type mice, respectively. These results indicate that TLR2 and TLR4 might influence the outcomes of S. japonicum infection through both T cell immune response and cytotoxicity pathway. |
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