The Effect Of γδT Cells On Neutrophils And Liver Fibrosis In Mice Infected With Schistosoma Japonicum

Schistosoma japonicum have been existing in China for thousands of years,and mainly endemic in China,Japan,Philippines and other places.After the founding of the People’s Republic of China,it’s a great achievement that most of the country has been in transmission-blocking situation with decades of work on prevention and treatment of schistosomiasis.However,there are still ten thousands cases of advanced schistosomiasis in some areas,and several provinces have not yet reached the stage of elimination of schistosomiasis,so the schistosomiasis remains a significant threat to people’s health.Cercariae ofS.japonicuminfect people through skin and most of the pathogenic manifestations is mainly generated by a large number of eggs laid by the adults parasites pairing in the mesenteric vein.Eggs are deposited in the liver and induce a storm of immune response,causing chemotaxis of inflammatory cells and formation of granulomas.Along with the unique egg granulomas diffusing in the liver,the progressive hepatic fibrosis develops,terminally causing irreversible pathologic damage including hepaticcirrhosis,ascites and portal hypertension.Schistosomiasis is a serious hazard to human health.The diverse antigen expression derived from the individual multicellular structure and complex life history of S.japonicum,leading to complex immune pathogenesis,that is an important reason for the lack of knowledge on the complex immuno-pathology mechanism and the short of breakthrough on the prevention and vaccine research of this disease.Begining with the interaction between host and pathogen,this subject explored the change of immune environment in the mice infected with S.japonicum,especially focused on the Gammadelta(γδ)T cells and the change of their phenotypes and functions before and after infection.Different subsets of γδT cells were blocked to determine their effects on different cytokines,the proportion of neutrophils and granuloma fiberosis in host.Results of the research would deepen the recognition of mechanism of pathological changes in schistosomiasis and innate immune in host,and provide new clues for further work on design and optimization of new vaccines as well as immune therapy of protection against S.japonicum.PartⅠThe phenotype and function change ofγδT cells in mice model of Schistosoma japonicumThe depositionof S.japonicum eggs causing lots of granulomas in liver,that leads to strong immunopathology reaction,has been amain point of schistosomiasis research.The first part of this subject was discussing the phenotypes and functions of y8 T cells in the model of mice infected with S.japonicum,γδT cells are a special subset of T cells,which stand between innate immunity and adaptive immunity.Most of previous studies of Schistosoma focused on Th cells,and suggested that there was no significant effect of γδT cells in the formation of granulomas.However in recent years some reaserches supported that the IL-17 plays an important role in the process of hepatic schistosomiasis,andyγδT cells were the main sourse of IL-17.This study focused on elaboratingthe change of function and possible mechanism of γδT cells in the course of schistosomiasis,to offer some references for further studies on the function of IL-17 in schistosomiasis.Blood and spleen were collected from mice infected with S.japonicum at different stages of disease,for testing the phenotype and function of γδT cells by flow-cytometry.Liver tissueswere collectedsimultaneously,made into frozen specimens,stained by immuno-fluorescence for testing the location of γδT cells in granulomas.Meanwhile,theγδT cellsfrom spleens of multiple mice were isolated and analysed by transcriptome microarray,for the preliminary study on possible mechanism of γδT cells in the IL-17 pathway.Results showed that,although the percentage of γδT cells in lymphocytes didn’t vary significantly(P>0.05)after infection,the proportion of activated γδT cells increased4 weeks after infection(P<0.05);γδT cells existed in egg granulomas at different stage of schistosomiasis,and Vy2 subset also participated in the disease;Vyl subset produced only IFN-y,Vy2 subset produced IL17A and IFN-γ,and both subsets might be inhibited in late stage of infection.The possible functional ways of y8 T cells might be through CCL22,CCL1,IL-23a,IL-3,IL-4,MMP9,IL21,CXCL2,CCL2 and other factors.Part ⅡThe level and function location of neutrophils in mice model infected with Schistosoma japonicumPrevious studies confirmed that neutrophils,macrophages and other cells derived from myeloid increased substantially in mice infected with S.japonicum,and mightbe involvedin the formation of egg granuloma and liver fibrosis in the process of schistosomiasis.To determinethe possible recruitment neutrophils by γδTcells,we defined the basic expression level of neutrophils in different stages of schistosomiasis,and explore its location and function initially in the second part of the subject.Firstly,blood and spleen were collected from mice infected with S.japonicum,stained for dectecting the proportion of neutrophils in mature white blood cells by flow-cytometry.Secondly,neutrophils from spleen of infected mice were isolated,and co-cultured with T cell from spleen of unifected mice,to test the effect of neutrophils on the proliferation of T cells stained with CFSE in vitro;Thirdly,slicesof liver tissuefrom mice at the differentstage of schistosomiasis were observed under confocal laser scanning microscopy,to determine the location of neutrophils in egg granulomas.Fourthly,by blocking part of the neutrophils intravenously,the change of the serum level of IL-17A,the percentage of each subgroup of γδT cells and hepatic fibrosis were tested.Results showed that,the proportion of neutrophils/leukocytes in the blood and spleen increased significantly(P<0.05)in the mice model of S.japonicum infection;neutrophils could inhibit T cell proliferation in vitro;the location of neutrophils in liver egg granulomasvaried during the course ofschistosomiasis,recruited by eggs at least twice,and infiltratedinto granulomas eventually;after blocking part of neutrophils,the proportionof Vγ2/γ8 T cells increased(P<0.05).Part ⅢVy2T cells promote recruitment of neutrophils and promote fibrosis in liver in mice model infected with Schistosoma japonicumIt is well documented that γδT cells could recruit neutrophils through secreting IL-17 in some models of cancer and autoimmune disease.The second part of the subject confirmed that neutrophils increased a lot after infection with S.japonicum.It was assumedin the third part of the subject that γδT cells couldpromote recruitmentof neutrophils by secreting IL-17 inmice infected with S.japonicum and the experiments were designed to prove it.First,the serum from infected mice was measured by Cytometric Bead Array(CBA)kits repeatedly,in order to test the dynamic change of cytokines and determine the baseline level of IL-17A for time point of blocking experiments.Secondly,with blocking the γδT cells and subsets of them by injecting antibodies,the changes in the percentage of neutrophils in the blood and spleen,and the level of IL-17A in serum were tested.Meanwhile,to measure the degree of hepatic fibrosis,slides of liver tissue staining with Masson and HE were observed,and the hyaluronic acid(HA)and type III procollagen(PCⅢ)in serum were detected by ELISA kit.On the other hand,γδT cells and Th cells were adoptively transfered into the RAG-/-mice infected with S.japonicum,then the percentage of neutrophils in blood and spleen were observed.Results showed that,during the course of schistosomasias,IFN-γ significantly increased at 5th week,IL-1βincreased at 6th week,IL-10 increased since 7th week,IL-17A increased at 7th week,IL-4 increased at 7th week,G-CSF began increasing at 4th week and declined at 7th week,increased again at 9th week;when blocking total y8 T cells or solely Vyl T cells,neutrophils didn’t increase slower significantly;when blocking Vγ2 T cells independently,the IL-17A level in serum declined(P<0.05),the propotion of neutrophils in blood and spleen both reduced(P<0.05),the level of HA and PCIII in serum both declined(P<0.05),the collagen proportion in egg-granulomas declined(P<0.05).Vγ2 T cells couldsecrete IL-17A,help recruit neutrophils,might promote the formation of egg-granulomas,and accelerate fibrosis in the early stage of infection with S.japonicum.In addition,adoptive transfer experiments showed that neither y8 T cells nor Th cells could increase the proportion of neutrophils significantly,but Th cells might recruit eosinophils.Part ⅣThe effect of 4 common stimulants on the intercellular cytokines and CD62L in spleenic CD8+ T cells of C57BL/6 mice infected with Schistosoma japonicum.During the flow-cytometry assays in mice model infected with S.jaonicum,we designed a panel including IFN-γ and CD62L to exam the change of effective γδT cellsand CD8+T cells in vivo.However,with repeated experiments,the CD62L+ cells could never be found,but appeared in the un-stimulated group as control.We asume that the activated cells might be lack of CD62L.So the CD8+T cellswere chosen for detection because of the phenotype character of y8 T cells that we found in the first part of the subject.In order to verify the hypothesis,the fourth part of the subject focused on four kinds of common stimulants:Phorbol-12-myristate-13-acetate(P),Ionomycin(I),Brefeldin A(B)and Monensin(M).The splenic single cell suspension wasprepared at 8 and 12 weeks after infection,and were stimulated in vitro by P(50 ng/ml)+ I(1 μmol/L)+ M(2 μmol/L)for 4 h.Flow cytometry was performed to determine the proportion of CD8 + T cells secreting IFN-y and detect surface expression of CD62L.Meanwhile,the splenic single cell suspension at 4 weeks after infection was stimulated by I(1 μmol/L),P(50 ng/ml),B(1 μg/ml),M(2 μmol/L),P+ I,P + I + M,P + I + B,and P + I + M + B for 4 h,and supernatant levels of IL-4,IL-17A,IFN-y,IL-10,IL-1β and G-CSF were determined with cytometric bead array.CD62L expression on CD8 + T cells was examined by flow cytometry,and mean fluorescence intensity(MFI)was calculated.The results showed that in the late stage of schistosomiasis(8-12 weeks),there was a lower percentage of IFN-γ/CD8+ T cells among splenic cells.P +I stimulation decreased the expression of CD62L.ConclusionThis project focusd on the host-pathogen interactions,exploring function and mechanism of γδT cells in mouse model infected with S.japonicum,yielded following results:1.The γδT cells located in the egg-granulomas in the liver of mice infected with S.japonicum.Vγ1 T cells produced IFN-γ,Vγ2 T cells produced both IL17A and IFN-γ,and their function could be inhibited at late stage of schistosomiasis.2.The proporion of neutrophils increased with the progress of schistosomiasis.The neutrophils could inhibit T cell proliferation.It was observed that neutrophils were recruited twice during the formation of egg-granulomas.3.In mice infected with Sjaponicum,Vγ2 T cells could help recruit neutrophils and might promote fibrosis in the liver.When blocking Vγ2 T cells alone,the percentage of neutrophils declined,IL-17A in serum decreased,and the degree of liver fibrosis alleviated.4.During the flow-cytometry assay,we found the intracellular cytokines and CD62L should be avoided designing together.