CD4~+T Cell Subsets In The Role Of Schistosomiasis Egg Granuloma Formation

Objectives:The roles of CD4+T cell subsets and ratio of Th1/Th2and Th17/Treg during theformation process of schistosomiasis liver egg granuloma were researched by buildingthe schistosomiasis japonicum Balb/c mice model, in order to improve the pathogenicmechanism of schistosoma japonicum and provide new targets for treatingschistosomiasis.Methods：Infecting Balb/c mice with schistosoma japonicum cercaria, mice models ofschistosomiasis japonicum in different time points of0w,4w,6w，8w,10w,12w,16w,20w and24w were constructed. The percentages of Th1, Th2, Th17and Tregcells and the CD4+T subgroups change in liver and spleen were detected by FCM.The relevant cytokines level of IL-2, IFN-γ, IL-4, IL-10, IL-17, IL-6, IL-23, TGF-βand IL-1β were assayed by ELISA. Using Trizol method to extract the mRNA of livertissue, the mRNA expression levels of IL-2,IFN-γ,IL-12,IL-4,IL-10,IL-6,IL-23,IL-17,ROR-γt,Foxp3,TGF-β and IL-1β were identified by real-time PCR. The pathologicalchanges of liver tissue in mice models of schistosomiasis japonicum were observed byHE and Masson staining.Results：Through Mice liver biopsy HE staining and Masson staining, we observed that:the infected mice liver tissues emerged inflammation with different degrees; seriousegg granuloma and mild fibrosis appeared in8w; the degree of liver fibrosis becamesignificant in12w; the inflammatory reaction and pathological changes of liver in8w~12w were most serious. The inflammatory response was alleviated after16w,further alleviated in20w~24w, and large numbers of fibrous scar lesions weredeveloped, and the dead eggs were liquefied and hollowed.Inflammation FCM results showed that the percentage of Th1and Th2cells in infected mice liver and spleen was significantly higher than that of normal controlgroup.P<0.05, the proportion of Th1cells in the liver reached peak value at4w and16w after schistosomiasis infection,the values were4.29±0.74%and4.90±0.14%,respectively, which were significantly higher than that of other time points,Th2cells proportion reached maximum value6.47±0.18%at8w after schistosomiasisinfection, which was higher other time points; In the normal control group, mice liverTh1/Th2ratio is3.17%, percentage of Th1cells increased significantly at4w, ratio ofTh1/Th2is5.67%; percentage of Th1cells markblely decreased, however, theproportion of Th2cells increased significantly, Th1/Th2ratio inversed and became0.84%;, ratio of Th1/Th2inversed and became0.46%at8w; Th1/Th2ratio was0.47%at10w and0.53%at12w, proportion of Th1cells increased at16w, whileproportion of Th2cells decreased, the ration of Th1/Th2was1.55%, from16w to24w,the ratio of Th1/Th2was observably lower than control group, but comparing with16w, no significant difference was observed. proportion of Th1cells in the spleenreached peak value at4w and16w after schistosomiasis infecting, the values were6.86±0.99%and6.0±0.6%,respectively, proportion of Th2cells reached peak value at8w,the value was6.94±0.75%, In the normal control group mice spleen Th1/Th2ratiowas3.17%, schistosomiasis infection4w, Th1cells percentage significantly increased,Th1/Th2ratio was5.86%;, proportion of Th1cells significantly decreased at6w,while the Th2cells proportion increased markblely, Th1/Th2ratio appeared inversionand became0.8%; at8w after infection, inversion occurred in Th1/Th2ratio andbecame0.54%;ratio of Th1/Th2was0.58%at10w, Th1/Th2ratio is0.86%at12w,proportion of Th1cells significantly increased at16w after infection, proportion ofTh2cells significantly decreased, ratio of Th1/Th2was2.34%, Th1/Th2ratio aresignificantly lower than normal control group at16w and24w, but there was nosignificant difference compared with16w.The percentage of Th17and Treg cells in the liver and spleen in mice infectedgroup were significantly higher than normal control group, P<0.05, IL-17A+Th17liver in normal mice, IL-17F+Th17proportion of Treg cells were1.367±0.071,1.267±0.55and3.64±0.255,respectively; IL-17A+Th17and IL-17F+Th17cells proportion reach maximum value at8w and16w after schistosomiasis infection,thevalues were29.58±1.195%,27.16±1.94%and30.057±1.54%and30.057±2.7%,respectively, which were significantly higher than other time points; at8w Treg cellsproportion reached peak value31.8±0.4%,which was significantly higher than that ofother time points; In the normal control group,mice liver of Th17/Treg ratio was0.72%, the ratio of Th17/Treg in mice infection group were significantly higher thannormal control group, the degree of increased Th17cells were significantly higherthan Treg cells; peak value of Th17/Treg ratio was4.35%at16w. in the spleen ofnormal mice, the percentage of IL-17A+Th17and IL-17F+Th17and Treg cells were1.79±0.07,1.34±0.112and3.613±0.14; the proportions of IL-17A+Th17and IL-17F+Th17cell reached peak value at16w and8w after schistosomiasis infection, thevalues were21.07±1.12%,17.22±1.12%,28.2±1.94%and28.2±1.62%, respectively,significantly higher than other time points; Treg cells proportion reach peakvalue23.04±1.21%at8w after schistosomiasis infection, which was significantlyhigher than other time points; In normal control group, ratio of Th17/Treg in micespleen was0.86%; The ratio of Th17/Treg in mice infection group were significantlyhigher than normal control group, the increase degree of Th17was higher than that ofTreg cells, the peak value of Th17/Treg ratio was5.99%at16w after infection.Real-time PCR results showed that the expression amount of mRNA of livertissue molecular IL-2, IFN-γ, IL-12, IL-4, IL-10, IL-6, IL-23, IL-17, ROR-γt, Foxp3,TGF-β, and IL-1β was much higher than normal control group, P<0.05, IFN-γ andIL-12mRNA expression level reached peak value at4w after infection, were52.52±3.13and52.52±2.48, IL-2mRNA expression level reach maximum value at4w and16w after infection, were123.9±2.48and123.9±2.95, which wereconsistented with the time point of the Th1cells increased; IL-4and IL-10mRNAexpression amount reached peak at8w,the value were140.6±5.36and140.6±1.94,respectively, which were consistentd with the time point of the Th2cellsincreased; IL-6, IL-23, IL-17, ROR-γt mRNA expression reached peak values, were84.05±5.6,57.01±4.68,757.6±2.45and157.14±2.4,which were consistented withtime point of Th17, the increased amount of Foxp3and TGF-β mRNA expression reached peak at8w after infection, the values were87.88±8.25and87.88±7.4,consistenting with the time point of the Treg cells increased; Amount of IL-1β mRNAexpression at8w and16w reached peak value, were38.05±4.82and4.82±4.82respectively.ELISA test results showed that the mice serum IL-2, IFN-γ, IL-4, IL-10, IL-6,IL-23, IL-17, TGF-β, and the expression of IL-1β volume were significantly higherthan normal control group, P<0.05, content of IL-2and IFN-γ reached maximumvalue at4w and16w after infection, the values were87.77±1.58,35.97±0.8and35.97±1.45,30.32±0.86; IL-4, content of IL-10reached peak at8w, the values were58.8±0.41and58.8±3.46; content of IL-6, IL-23reached peak at8w, the values were976.7±1.53and976.7±6.84; IL-17content reached peak at8w and16w afterinfection, the values were174.47±0.2and174.47±0.43; TGF-β content achieved peakvalue at8w, the value was1212.8±0.5; IL-1β content achieved peak value at10w and16w after infection, the values were43.33±0.96and43.33±0.86; ELISA test resultsand real-time PCR results were identical with each other.Conclusions：After mice infected with schistosoma japonicum, along with the increase of thedegree of liver pathological damage, the percentage and relatecytokines of Th2, Th17and Treg cells significantly rised, which caused the ratios of Th1/Th2and Th17/Tregcells unbalanced, the degree of increasing of Th2and Th17were consistented with theliver egg granuloma lesion severity，the combined action of Th2and Th17cells andTh1/Th2and the imbalance of Th17/Treg cells schistosomiasis liver egg granulomaformation in Japan played an important role in immune pathological injury of liver.