| Cystic ehinococcosis (CE) is a zoonosis of global distribution caused by Echinococcus granulosus (E. granulosus), and does serious harm to the health of humanbeing, and causes significant economic losses, however the disease is difficult to control because appropriate diagnostic procedures are lacking and the available drugs are inefficient. During the life cycle of E. granulosus, protoscoleces (the larval or metacestode stage) can develop either into an adult worm, in the gastrointestinal tract of the definitive host (dog), or into a secondary hydatid cyst in the intermediate host (human). They also infect, and adapt to a variety of intermediate hosts. These characterizations make protoscoleces the important model to study parasite-host interactions. In this study we focused on (i) the overall effects of protoscoleces on the host immune cells, especially the regulational role in the myeloid-derived suppressor cells (MDSC); (ii) the characterization of secretory-excretory proteins in the protoscoleces transcriptome. Our study will deepen the knownlege of immune evasion strategies adopted by parasite and physiological adaptation, and will provide important molecules basis for the development of CE vaccines and drug targets.Part?Phenotype and function of immune cells in mice post Echinococcus granulosus protoscoleces infectionThe status of host immune cells is central to fight aginst parasite infection, while immune evasion adopted by the parasite is essential for parasite survival. Previous studies have showed that host immune resppnses to parasite antigens have been significantly downregulted (or inhibited) post Echinococcus granulosus protoscoleces (E. granulosus) infection, however, the mechanisms associated are not well declared. This part has focused on the overall status and function of the innate and adaptive immune cells in Balb/c mice post infection, to explore the potential escaping strategies of E. granulosus.The protoscoleces were collected from cysts in the livers (or lungs) of sheep infected by E. granulosus, and then the number of protoscoleces was counted. Each Balb/c mice was inoculated intraperitoneally with 200?L of a suspension containing 2000 live protoscoleces in PBS, whereas mice injected with 200?L PBS were used as controls. The single cell suspension was prepared from murine peripheral blood and spleens of mice that collected at different time point post infection (30,180,360 days). The state and phenotypic characterization of various immune cells before or after infection were evaluated using a multiplex flow cytometry assay, and combined with proliferation assay, to explore the overall effects of protoscoleces on host immune system.At 30 dpi, an increase in the number of CD11b+ and CD11c+ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1+ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b+/GR-1+. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4+ and CD8+ T cells following infection. Regulatory T cells expressing CD4+/CD25+/FoxP3+ increased significantly over the course of infection. The proliferation index was significantly inhibited post infection (P<0.05).Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest that these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection.Part IIStudy on the immunoregulatory molecules in M-MDSC of mice post Echinococcus granulosus protoscoleces infectionMDSC is a heterogeneous group of cells that derived from bone marrow progenitor cells and immature cells. They are the precursors of dendritic cells (DC), macrophages, granulocytes. Numerous studies have showed that MDSC play a strong suppressive effect on host immunity in various tumor models. In the first part, E. granulosus infection was found to induce the accumulation of MDSC in the peripheral blood and spleen of Balb/c mice, which might help the parasite to escape the host immune responses, however, the mechanisms of these actions remain unclear. Recent studies indicated that monocytic-type MDSC (M-MDSC) is the major cell group that play the inhibitory effects in MDSC. Therefore, this study concentred on the screening of immunoregulatory molecules in M-MDSC post infection using the DNA microassay.The murine spleens with different stages post E. granulosus protoscoleces infection and the corresponding normal spleens were firstly collected, then the single cell suspensions were prepared. The M-MDSC were isolated using the magnetic bead separation technology (Miltenyi Biotec, Germany), and total RNA was extracted for mouse genome spectral analysis. The differentiated expressed genes screened were annotated and analysed using bioinformatics, and were then validated using the Real-time PCR.The results showed that 1090 differentiated expressed genes were found in M-MDSC before or after infection. Among 27 selected genes for Real-time PCR,24 were consistent to the results of DNA microassay. Protein network analysis showed that the molecules associated with chemotaxis and transcriptome in M-MDSC were significantly down-regulated, which may affect the normal differentiation of M-MDSC. Moreover, functional molecules were found to influence DC, M0 function and the interaction with T cells. Overall, this study will play a molecular basis for studying the immune regulation mechanisms of M-MDSC.Part IIITranscriptomic characterizations of potential excretory/secretory proteins expressed in Echinococcus granulosus protoscolecesDuring the life cycle of E. granulosus, protoscoleces (the larval or metacestode stage) can develop either into an adult worm, in the gastrointestinal tract of the definitive host (dog), or into a secondary hydatid cyst in the intermediate host (human). And they can also infect, and adapt to, a variety of intermediate hosts. These features make protoscoleces an excellent model to study parasite-host interactions. The excretory/secretory proteins (ESPs) released by parasite playing a central role in parasite-host interface, are important targets to vaccine, diagnosis and drug development. The identification of ESPs composition is still incomplete using modern proteomics, due to their complex elements, diffentiated expressed abundance, and the mixed host proteins. In this study, we used Roche 454 sequencing technology to explore the transcriptome profiles of the larval stage of E. granulosus protoscoleces, especially elucidate the characterization of ESPs using in silico secretome analysis, for better understanding its physiological adaptation.Total RNA was extracted in TRIzol reagent (Invitrogen, USA), and RNA quality was performed by gel electrophoresis and a 2100 BioAnalyzer (Agilent Technology, Santa Clara, CA). cDNA was synthesized using 2 ?g of total RNA with the SMART cDNA synthesis kit (Clontech Laboratories, USA). The cDNA library was constructed using GS-FLX Titanium General Library Preparation Kit (Roche, USA), and sequenced using a half run on the Roche 454 GS-FLX Titanium platform. After removal of the low quality reads and the sequencing adapters, the clean reads were then assembled using Newbler 2.5.3 (Roche, USA) as the protoscolex transcriptome. Using a series of bioinformatics softwares, the potential ESPs were predicted from the transcriptome, and were then analysed to explore their functions in parasitism.A large number of unigenes were generated (26,514), of which 22,910 (86.4%) were mapped in the newly published E. granulosus genome and 17,705 (66.8%) were distributed within the coding sequence (CDS) region. Of the 2,280 ESPs predicted from the transcriptome,138 ESPs were inferred to participate in carbohydrate metabolism, while 124 ESPs were inferred to participate in protein metabolism. Eleven ESPs were identified as intracellular enzymes that regulate glycolysis/gluconeogenesis (GL/GN) pathways, while a further 44 antigenic proteins, 25 molecular chaperones and four proteases were highly represented. We found that many proteins were significantly enriched in development-related signaling pathways, such as the TGF-P receptor pathways and insulin pathways.Our study suggested that ESPs released by parasite are not only involved in mediating host immune responses, more importantly, in carbohydrate metabolism, fighting against hostile host environments, and key development-related signal pathways. Our data will deepen the knowledge of parasite metabolic adaptation, and some of these molecules will be novel targets for CHD diagnosis and intervention.Conclusions1. Using multiplex flow cytometry, we described the key events in the cellular immune infiltration and activation following E. granulosus protoscoleces infection in Balb/c mice:(i) The expression of MHC ? molecules in APCs was significantly downregulated, thereby affecting parasite antigens presentation; (ii) these accumulated immunosuppressive cells such as Treg and MDSC, might inhibit T cell immune responses although that T cells were persistently activated post infection.2. The results showed that 1090 differentiated expressed genes were screened in M-MDSC before or post infection using Agilent mouse microassay. Bioinformatics analysis revealed that molecules associated with chemotaxis and transcription were significantly downregulated following infection, affecting normal differentiation and maturation of M-MDSC. Furthermore, some functional molecules might influence the interaction of M-MDSC with T cells, providing an important molecular basis for studying the exact mechanisms of the immune suppression of M-MDSC in pathogenic conditions.3. The transcriptome of potential ESPs in E. granulosus was firstly characterized. ESPs released by the parasite are not only involved in mediating host immune responses, more importantly, in carbohydrate metabolism, fighting against hostile host environments, and key development-related signal pathways. Our data will deepen the knowledge of parasite metabolic adaptation, and some of these molecules will be novel targets for CHD diagnosis and intervention. |
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