| Background: Fasciolosis,infection caused by the two trematode species Fasciola gigantica and Fasciola hepatica,results in threatening to public health security.Fasciola spp.actively release abundant soluble moleculethe and Extracellular Vesicles(EVs)to manipulate host immune microenvironment and benefit its own survival.Commensal microorganisms in the gastrointestinal tract of vertebrates play vital roles in maintaining the development and function of the vertebrate immune system,in addition to supporting the balance of nutrition and energy metabolism of the host.It is reasonable that both microbes and helminths interact with each other,and such interaction may affect host homeostasis.In this study,we aimed to obtain a much clearer picture of the functional links between host and microbiota during the time course of F.gigantica infection.It can realize the transformation expreimental study to clinical application.Methods : The model of infection with F.gigantica metacercarial Buffaloes,BALB/C mice or intraperitoneal injection of excretory-secretory product(ESP),15 k EVs,100 k EVs released by adult F.gigantica in BALB/C mice were establishmented successfully.The Th1-and Th2-type immune response-related cytokines,transcription factors,toll-like receptors and anti-F.gigantica antigen specific antibodies to refelct the immune response induced by F.gigantica infection.16 S r RNA gene amplicon sequencing on an Illumina Hi Seq2500 platform were applied to analyze the structure and function of the gut microbiota connect with F.gigantica infection.Immunological data were used to perform the statistical analysis of linear models.Redundancy analysis(RDA)and canonical correspondence analysis(CCA)were used to determine the interaction relationship between F.gigantica infection iduced immune response and microbiota community composition.Following ultracentrifugation-based protocols,we isolated two different forms of extracellular vesicles from F.gigantica culture media,15 k EVs(15,000 g pellet),100 k EVs(100,000 g pellet).Shotgun technology was used to find the protein components of 100 k EVs.Results: This study confirmed that host susceptible to F.gigantica infection that is closely related to the Th2-type immune response-related cytokine environment.Mixed Th1-and Th2-type immune response during the early infection stage,when the infection became chronic,the common Th2 immune response was induced.Meanwhile,the level of total IgG and IgG1 for the parasite-specific antibodies showed an increasing trend.This suggests that Th2-related cytokines and IgG1 induced by F.gigantica may mediate successful infection in the natural host swamp buffaloes.Meanwhile,the activated ERK pathway and the inhibition of the classical TLRs-IKB-NF-κB(P65)pathway in the mouse model were found to be involved in the Th2-type immune response induced by F.gigantica infection.We successfully isolated and identified two different forms of extracellular vesicles of 15 k EVs and 100 k EVs.The protein components of F.gigantica 100 k EVs were ditermed by shotgun analyses and identified these proteins related to immune regulation,diagnosis and vaccine candidate molecules.BALB/C mice received continuous peritoneal injection of ESP、15k EVs、100k EVs released by adult F.gigantica,induced Th2-and Treg-type immune responses in vivo,meanwhile by up regulation of CLAT-4and PD-1 to limite the development of Th1-type immune responses.We found that alpha diversities decreased during infection,and gut microbes differed considerably across different sections of the gut from Buffaloes at different stages.Peptostreptococcaceae and Family＿XIII families are closely linked to the upregulation of IgG1 and IL4,respectively.The F.gigantica infection significantly reduced the abundance of short-chain fatty acid(SCFAs)-producing microbes Lachnospiraceae＿NK3A20＿group and Alistipes,reduced the concentrations of SCFAs in Buffaloes colon.Meanwhile F.gigantica infected mice showed a similar decreased in the abundance of short-chain fatty acid(SCFAs)-producing microbes uncultured＿bacterium＿Lachnospiraceae in colon.Our data show that gut microbes may affect the host Th2-type immune response during F.gigantica infection via SCFAs metabolism.Alpha diversities of BALB/C mice colon contents were decreased after infection or intraperitoneal injection of ESP,15 k EVs and 100 k EVs isolated from F.gigantica.RDA/CCA results showed that Th2/Treg cytokines were correlated with Bacteroidetes and Proteobacteria,Bacteroidaceae and Cyanobacteria plays a key role in the development of liver fibrosis caused by F.gigantica infection.Conclusions: Host gut microbes may affect the host immune response during F.gigantica infection via gut microbes metabolism.A deeper understanding of the interaction between F.gigantica–microbiota-host,which could include the exploitation of such relationships to improve prevention and control Fasciolosis. |
