| Cystic echinococcosis,also known as hydatid disease,is a serious zoonotic disease infected by the larvae stage of Echinococcus granulosus parasitizing in human and animal liver and lung organs.It is a major disease that our country focuses on prevention and control,endangering human health and animal husbandry production.Cystic echinococcosis is widespread in China,and the number of threatened people and patients rank first in the world.Dendritic cells(DC)are the most powerful antigen-presenting cells that can effectively initiate the primary immune response and determine the direction of immune response during parasite infection.Myeloid-derived suppressor cells(MDSC)and regulatory T cells(Treg)are two major groups of cells with immunosuppressive effects.Both were significantly enriched in the spleen,abdominal cavity and peripheral blood of mice infected with the protoscoleces of Echinococcus granulosus.DC and MDSC can induce the production of Treg,while MDSC can differentiate into DC.Parasites can release exosomes that carry a variety of biologically active substances(proteins,lipids,nucleic acids,etc.),participating in parasite-parasite information exchange,parasite-host interactions,and playing a key role in the pathogenesis of parasite infection.Parasite-derived exosomes contain a large number of miRNAs,which can be taken up by host cells to regulate the host's immune response.However,the dynamic changes of DC and suppressor cells(MDSC and Treg)in the livers of mice infected with E.granulosus protoscoleces(Eg-psc)is unclear,and the function and mechanism of protoscoleces-derived exosomes and their miRNAs in the pathogenesis of E.granulosus infection need to be elucidated.In this study,a mouse model of Eg-psc infection was constructed,and dynamic changes of DC,MDSC and Treg in hepatic leukocyte of mice at early,middle,and late stages(3,6 and 12 months)post-infection were detected by flow cytometry.Eg-psc derived exosome like vesicles(PSC-ELVs)and hydatid fluid exosome like vesicles(HF-ELVs)were obtained by ultracentrifugation,and high-throughput sequencing and bioinformatics analysis were performed to obtain ncRNAs(including miRNAs,lncRNAs,and circRNAs)expression profiles of PSC-ELVs and HF-ELVs.And we screened and identified top 20 miRNAs in PSC-ELVs,predicted their potential target genes and constructed lncRNA-mRNA-miRNA regulatory networks.The results demonstrated that the most abundance miRNAs in the PSC-ELVs might be might be involved in host immunity and pathogenesis and provided valuable resources for further analyses of regulatory potential of miRNAs in both ELVs at the parasite-host interface.E.granulosus excretion-secretion antigens(ES)regulate the maturation and function of DC,affecting the release of inflammatory factors,and regulating the Th1/Th2 immune response.Exosomes are an important part of ES,and are widely involved in nucleic acid transport,cell-to-cell material exchange,and antigen presentation.In this study,co-culture of PSC-ELVs and bone marrow-derived dendritic cells(BMDC)was performed to analyze their effects on the expression of BMDC surface MHC II and costimulatory molecules and cytokine secretion,to study the function and of mechanism of PSC-ELVs and their miRNAs on DC.These results provide a theoretical basis for the role of PSC-ELVs and their miRNAs in the host immune response,as well as a new idea for the clinical diagnosis and prevention and treatment of hydatidosisPart 1 Dynamic changes of dendritic cells and suppressor cells in livers of mice infected with Echinococcus granulosusWe established Eg-psc infected mouse model,the infected group mice were injected with 2 000 protoscoleces via peritoneal injection,while the control group mice were injected with equalvolume of normal saline.Mouse hepatic leukocyte were harvested 3(early stage),6(medium stage)and 12 months(late stage)post-infection,and the proportions of DC and suppressor cells(MDSC and Treg)were assessed by flow cytometry.The results showed that the proportions of DC were(5.27 ± 0.55)%,(6.87 ±0.32)%and(18.5 ± 0.64)%in mouse liver white blood cells in the infection group 3,6 and 12 months post-infection,and 5.92±0.43)%,(5.99 ± 0.1 2)%and(13.73±0.22)%in the control group,and there were significant differences in the proportion of DC in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection(P<0.05).The proportions of MDSC were(1.61±0.36)%,(5.68 ± 0.69)%and(16.18 ± 0.69)%in mouse liver white blood cells in the infection group 3,6 and 12 months post-infection with E.granulosus,and(2.19±0.42)%,(0.99± 0.07)%and(4.18 ± 0.84)%in the control group,and there were significant differences in the proportion of the MDSC in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection(P<0.01).The proportions of M-MDSC were(0.69 ± 0.27)%,(5.30 ± 0.72)%and(10.75± 0.29)%in mouse liver white blood cells in the infection group 3,6 and 12 months post-infection,and(0.42 ± 0.24)%,(0.69 ± 0.02)%and(2.12 ± 0.13)%in the control group,and there were significant differences in the proportion of the M-MDSC in the mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection(P<0.01).The proportions of PMN-MDSC were(0.93 ± 0.23)%,(0.32 ± 0.02)%and(5.14±1.03)%in mouse liver white blood cells in the infection group 3,6 and 12 months post-infection,and(1.77 ± 0.26)%,(0.28±0.05)%and(1.99 ± 0.90)%in the control group,and there were significant differences in the proportion of PMN-MDSC in mouse liver white blood cells between the infection and control groups 3 and 12 months post-infection(P<0.05).The proportions of Treg cells were(3.35±0.14)%,(6.24±0.38)%and(3.41 ± 0.07)%in mouse liver white blood cells in the infection group 3,6 and 12 months post-infection,and(3.48±0.46)%,(3.65±0.45)%and(3.12±0.12)%in the control group,and there were significant differences in the proportion of Treg cells in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection(P<0.01).The percentages of DC,MDSC and Treg cells increase in mouse liver white blood cells 6 and 12 months post-infection with E.granulosus,and a more remarkable increase is seen in the percentage of MDSC,whichis mainly found in M-MDSC.Meanwhile,DC differentiated by M-MDSC also increased significantly in the same stages.These findings suggest that M-MDSC may play a major immunosuppressive role in the medium and late stages of E.granulosus infection in mice.Part 2 Non-coding RNA profiles of exosome-like vesicles from the protoscoleces and hydatid cyst fluid of Echinococcus granulosusThe ultracentrifugation was performed to isolate the PSC-ELVs and HF-ELVs from the culture medium for E.granulosus PSCs in vitro and the hydatid fluid(HF)of fertile sheep cysts,respectively.Transmission electron microscopy,nanoparticle tracking analysis,and Western blotting were used to confirm that vesicles produced by the PSCs and hydatid cysts were ELVs,based on their size,morphology,and specific ELVs markers.The miRNAs,lncRNAs,and circRNAs profiles of the two types of ELVs were analyzed using high throughput sequencing and their functions were predicted via Gene Ontology enrichment(GO)and the Kyoto Encyclopedia of Genes and Genomes pathway analysis(KEGG).In PSC-ELVs and HF-ELVs,118 and 58 miRNAs were identified,respectively,among which 53 miRNAs coexisted in both ELVs,while 65 and 5 miRNAs were unique to PSC-ELVs and HF-ELVs,respectively;2 361 and 1 254 lncRNAs were identified in PSC-ELVs and HF-ELVs,respectively,among which 1 004 IncRNAs coexisted in both ELVs,while 1 357 and 250 lncRNAs were unique to PSC-ELVs and HF-ELVs,respectively.Intriguingly the spilled PSCs from cysts excrete ELVs with more number of and highly expression levels of miRNAs and circRNAs than HF-ELVs.The miRNA sequencing data was validated by quantitative RT-PCR.Furthermore,the target lncRNAs and mRNAs regulated by the 20 most abundant miRNAs were screened and a ceRNA regulatory network containing 5 miRNAs,41 lncRNAs,and 23 mRNAs was constructed.The 20 most abundant miRNAs in the PSC-ELVs were predicted to mediate inflammatory response,MAPK cascade and collagen catabolic process during parasite infections.Moreover,egr-miR-4989-3p was the most abundant miRNA in PSC-ELVs and HF-ELVs,sharing identical seed sites with egr-miR-277a-3p and belonging to same miRNA family.In conclusion,for the first time,we identified the ncRNAs profiles in PSC-ELVs and HF-ELVs that might be involved in host immunity and pathogenesis.The most abundant miRNAs may play an immunoregulatory role in both ELVs participating in parasite-host interactions through related signaling pathways or by interacting with target genesPart 3 The function and mechanism of PSC-ELVs acting on dendritic cellsConfocal microscopy was performed to observed that PSC-ELVs could be internalize by BMDC after co-culture with PSC-ELVs.The miRNAs in PSC-ELVs could be transported to BMDC,which is confirmed by qRT-PCR.qRT-PCR and ELISA were used to detect the mRNA and protein expression levels of BMDC cytokine after treatment.Flow cytometry was performed to detect the expression levels of MHCII and co-stimulatory molecules on the surface of BMDC.The results showed that PSC-ELVs effectively induced BMDC to produce pro-inflammatory cytokines IL-6,IL-12,TNF-?,IL-?,IFN-y and anti-inflammatory cytokine IL-10,and most of them have a synergistic effect with LPS,and upregulated the cell surface marker MHC II and costimulatory molecules CD80,CD86,CD40,PDL1 and PDL2.These results suggest that PSC-ELVs may carry certain virulence molecules to induce the maturation of BMDC and polarize towards stimulating DC,showing obvious pro-inflammatory properties,and may generate Thl-type cell-mediated immune responses,which may help kill and clear the invading pathogens to protect the host.By transfecting two miRNAs(miR-277a-3p and miR-4989-3p)mimic and inhibitor into BMDC,qRT-PCR detection found that miR-277a-3p and miR-4989-3p induced the mRNA levels of pro-inflammatory cytokines IL-6,IL-12 and TNF-? significantly increased,and the mRNA level of IL-10 was significantly reduced in BMDC,suggesting that miR-277a-3p and miR-4989-3p can induce BMDC to produce inflammatory cytokines and polarize towards stimulating DC.MiR-277a-3p and miR-4989-3p mimic induced the mRNA levels IGF1 and NF-?B1 were significantly reduced in BMDC,while the mRNA and protein levels of NF-?B P65 were significantly increased,and its phosphorylation levels were increased,suggesting that IGF1 and NF-?B1 are the potential target genes of miR-277a-3p and miR-4989-3p,may induce the production of pro-inflammatory cytokines by activating NF-?B P65 and its phosphorylation.The results will provide new research strategiesfor the further study of the mechanism of PSC-ELVs and their miRNA regulating the host immunity response,and the prevention and treatment of echinococcosis. |
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