Characteristic Of Exosomes From Cysticercus Pisiformis And Their Mechanism On Regulating Macrophage Polarization

Cysticercosis pisiformis is one of the most common and important parasitic disease,causing serious economic losses to the rabbit breeding industry.Exosomes,as a new communication vehicle between parasite and host,transport the bioactive molecules from the parasites to host cells,thus regulating the function of receipient cells.Let-7c can induce the macrophages(M?)polarization to the M2 phenotype through regulation of transcription factor C/EBP ? in vertebrates.In addition,the infection of metacestodes could induce the host Th2-type immune response.Therefore,whether the immunosuppression is related to the exosomes secreted by the metacestodes or the exosomal let-7,and how does the let-7 regulate the M? polarization? Because of this,the following research was carried out in this thesis:1.The exosomes derived from Cysticercus pisiformis(C.pisiformis)were isolated and purified by differential centrifugation.Electron microscopy and nanoparticle tracking analysis were employed to characterize the exosomes,which indicated that the obtained vesicles were C.pisiformis exosomes.To evaluate the effect of exosomes on macrophages,the RAW264.7 M? cells were treated with C.pisiformis exosomes,the production of NO was significantly inhibited,and M? proliferation were significantly promoted by exosome incubation.The expression level of Th2 immune related molecules IL-4,IL-6,IL-10,IL-13 and Arg-1 were significantly up-regulated in exosomes-stimulated M?(P < 0.05),whereas the Th1 type immune-related molecules,IL-12,IFN-? and iNOS were obviously decreased(P < 0.05).The above data suggested that exosomes from C.pisiformis could promote the M? to secret Th2 type immune related molecules.Apart from the exosomal effect in vitro,the immune response of exosomes in vivo was confirmed subsequently.Rabbits were immunized with purified exosomes and ESP from C.pisiforms,and the level of serum antibodies and cytokines were detected by ELISA.Moreover,the immunoprotective effects were assessed after artificial challenge infection with T.pisiformis.The results showed that the levels of specific antibodies,IL-4 and IL-10 cytokines were significantly increased in rabbits immunized with exosomes compared to ESP group(P < 0.05),while the level of IFN-? was significantly decreased(P < 0.05)and TNF-? showed no difference.The rabbits necropsy results revealed that the worm reduction rate in exosomes group(24.88%)was lower than that in ESP group(65.17%).These results enable us to estimate that the exosomes of C.pisiformis induce a Th2 type cellular immune response in host.2.To identify the proteins and miRNAs in exosomes,we performed protein profiling and small RNA sequence analysis,and verified the molecules by western blot and qPCR.87 proteins,including 14-3-3 and enolase,41 known miRNAs and 18 novel miRNAs were identified in the exosomes of C.pisiformis,and 5 miRNAs were significantly enriched in exosomes.Target gene prediction of miRNAs displayed that the target genes were mostly involved in signal transduction and immune system.3.Since let-7-5p was enriched in exosomes,and mammalian let-7c related to regulation of M? polarization.Let-7-5p mimics were synthesized and transfected into M?,the expression level of M1/M2 phenotype marker was detected by qPCR,ELISA and western blot.The results demonstrated that overexpression of let-7-5p significantly decreased the iNOS,IL-12 and NO expression(P < 0.05),and up-regulated the expression of Arg-1 and IL-10(P < 0.05).On the contrary,knockdown of let-7-5p showed an opposite result.As described above,let-7-5p could induce the M? polarization to the M2 phenotype.Furthermore,C/EBP ? was considered as the target gene of let-7-5p through TargetScan,miRNA database,Pictar and seed sequence analysis,which was further validated by qPCR and western blot analysis,the results showed that the transcription and expression levels of C/EBP ? were significantly decreased with the treatment of let-7-5p mimics,while knockdown of let-7-5p significantly up-regulated the expression level of C/EBP ?.Double luciferase reporter assay proved that let-7-5p bonded to 3' UTR of wild type C/EBP ?.Furthermore,knockdown of C/EBP ? strongly reduced the IL-12 and iNOS expression(P < 0.05),and induced the expression of IL-10 and Arg-1(P < 0.05).These results proved that C/EBP ? is the target gene of let-7-5p,contributing to M? polarization through regulation of the C/EBP ? expression.Conclusively,in this study exosomes were isolated and identified from C.pisiformis.The exosomes could induce the Th2-type immune response in vitro and in vivo.The let-7-5p enriched in exosomes can induce the M? polarization to M2 phenotype through regulation of the target gene C/EBP ?,and subsequent immunosuppressive response in host cells was produced.This study demonstrated that exosomes and the packaged miRNA let-7-5p played an important role in the immunosuppressive mechanism of C.pisiformis infection,establishing a solid foundation for clarifying molecular mechanism of immunosuppression and immune evasion of metacestodes infection.