Study On The Role And Its Mechanism Of γδ T Cells Promoting Early Liver Fibrosis In Mice Infected With Schistosoma Japonicum

ObjectiveSchistosomiasis is a severely harmed zoonosis parasitic disease caused by Schistosoma infection,and is also a tropical disease that has been seriously ignored.Schistosomiasis is endemic in more than 70 countries and regions in the world.Theinfected population in the world is conservatively estimated at 200 million.At the same time,about 800 million people are threatened by schistosomiasis.In addition to serious damage to health,animal schistosomiasis has also caused huge losses in the economy.Schistosoma japonicum is the most prevalent in China,and there are many unresolved problems in the prevention and treatment of schistosomiasis.S.japonicum has a complex life cycle,after cercariae infecting people or animals,all the subsequent developmental stages can cause disease.A large number of eggs which produced by the adult worm in the portal vein enter into the liver with the blood flow and deposit in the liver,inducing a serious inflammatory response.The innate immune system and the adaptive immune system in the host are activated successively,and the immune response changes from Th1 type to Th2 type,and Th2 type immune response gradually becoming the dominant immune response.At this time,pathological immunoreaction,including granuloma and liver fibrosis,begin to form in the liver of host.Adaptive immune cells such as CD4+cells,B cells and innate immune cells such as macrophages,NK cells,eosinophils,neutrophils,and γδ T cells participate in the formation of these pathology together.As an innate immune cell,γδ T cells recognize antigens without MHC restrictions and can quickly respond to S.japonicum antigens.It has been demonstrated that γδ T cells are closely related to liver fibrosis in S.japonicum infected mice,but the mechanism of γδ T cells promoting liver fibrosis has not been fully elucidated.This study focused on early stage of S.japonicum infection,employing TCRδ chain knockout(KO)mice to clarify the role of γδ T cells in fibrosis in S.japonicum-infected mice,and further explore the mechanism of early liver fibrosis formation.MethodsMice were divided into WT control group,WT infection group and TCR δ KO infection group,and the mouse model of S.japonicum infection was established by abdominal adherence method.In the WT infection group and the TCR δ KO infection group,16 mice were taken to monitor the survival rate,and the remaining mice were dissected at different stages of infection.Peripheral blood from different stages of infection was collected,part of which was used to detect liver function and compare the changes of liver function in two groups of mice after infection.Some serum was used to detect the expression level of cytokines by ELISA,and the expression level of IL-17A in two groups of mice was analyzed.Dissociate the complete liver,take an appropriate amount of liver for total RNA and protein extraction,and analyze the changes in fibrosis-related indicators of the two groups of mice;Then appropriate amount of liver tissue was fixed in 4%paraformaldehyde for pathological staining to show area of granuloma and the degree of fibrosis of the two groups of mice,which can further analyze the effect of TCR δ KO on granuloma and fibrosis.Meanwhile,another part of the liver were ground and lysated,obtained the supernatant after centrifugation.The expression level of cytokines in liver was detected by ELISA.The remaining liver and spleen were ground and filtered and centrifuged to isolate white blood cells and flow cytometry antibodies were used to stain the molecules in the cell membrane,intracellular and nucleus.The changes of γδ T cells,CD11b+Gr-1+ cells and their secreted cytokines in the process of infection were examined,and analyzed the role of γδ T cells on the relevant cells and their secreted cytokines.Hepatic stellate cell line were stimulated by cytokine IL-17A in vitro,and the direct activation effect of IL-17A on hepatic stellate cells has been analyzed.Result1.Flow cytometry analysis showed that the proportion of γδ T cells in the liver of schistosomiasis-infected mice did not change significantly during the infection process(0 weeks,4 weeks and 6 weeks post infection),but the absolute number of cells increased significantly,which were 3.2×104,7.3×104 and 9.8×105 at 0 weeks,4 weeks and 6 weeks,respectively,and showed statistical difference among groups,(P<0.0001);meanwhile,the proportion of Vγ 2 subtype γδ T cells increased,which were(24.72 ± 2.02)%,(30.36 ± 3.47)%and(44.46 ± 3.20)%at 0 weeks,4 weeks and 6 weeks,respectively,and showed statistical difference among groups(P<0.01),and the proportion of cells secreting IL-17A in this subset gradually increased,which were(17.06 ± 1.64)%,(25.32± 4.57)%and(48.06 ±1.03)%at 0 weeks,4 weeks and 6 weeks,respectively,and showed statistical difference among groups(P<0.0001).2.Survival rate of S.japonicum infected mice were monitored for 16 weeks,compared with mice in WT infection group,the survival rate of mice in TCR δ KO infection group was improved with statistical difference(P<0.05);compared with WT mice,the liver function(ALT and AST)of TCR δ KO mice improved at 4 weeks and 6 weeks post infection(P<0.05,P<0.01,P<0.01,P<0.05);compared with WT mice,the spleen index of TCR δ KO mice decreased at 4 weeks and 6 weeks post infection(P<0.05,P<0.001),the liver index of TCR δ KO mice decreased at 6 weeks post infection(P<0.01).3.HE staining showed that the granuloma area induced by single egg in liver,WT group and TCR δ KO group was(1.3 ± 0.095)X 105 μ m2 and(0.95±0.033)×105 μm2 at 6 weeks post infection,respectively,and the area in TCR δ KO group was significantly lower than that in WT group,and showed statistical difference(P<0.0001);and Masson staining showed the fibrotic area in liver,WT group and TCR δ KO group was(6.63±0.40)×104 μm2 and(3.76± 0.18)×104μm2 at 6 weeks post infection,respectively,similarly,the area in TCR δ KO group was significantly lower than that in WT group,and showed statistical difference(P<0.0001).4.The mRNA level of IL-1 7A in the liver of the WT group is(3.0±0.45)times that of the TCR δ KO group at 6 weeks post infection(P<0.05);in the supernatant of liver tissue lysate,IL-17A in the WT group and TCR δ KO group were(217.2±19.15)pg/ml and(101.6±18.95)pg/ml at 6 weeks post infection,respectively.The TCR δ KO group was lower than the WT group,and showed statistical difference(P<0.01).In peripheral blood,IL-17A in WT group and TCR δ KO group were(143.9± 33.82)pg/ml and(34.0±22.29)pg/ml at 6 weeks post infection,respectively.Similarly,the TCR δ KO group was lower than the WT group,and showed statistical difference(P<0.05).In addition,TCR δ KO liver tissue Col 1,TFG-β and other mRNA also decreased,while IL-13 mRNA levels did not change.5.The proportion of CD11b+Gr-1+cells in the livers and spleens from WT mice and TCR δ KO mice changed during the infection process.The proportion in livers from WT mice were(1.32± 0.15)%,(10.20 ± 1.47)%and(33.64±1.09)%,respectively;the proportion in spleen from WT mice were(1.00± 0.07)%,(3.20± 0.02)%and(15.05±0.99)%,respectively;the proportion in liver from TCR δKO mice were(1.46±0.10)%,(9.80 ± 0.81)and(22.22 ± 1.57)%,respectively;the proportion in spleens from TCR δ KO mice were(1.10 ± 0.13)%,(1.60 ± 0.07)%and(8.30± 0.64)%,respectively.At 0 weeks,there was no difference in liver and spleen CD11b+Gr-1+cells between the two groups of mice.At 4 weeks,CD11b+Gr-1+ cells in spleens from TCR δ KO mice were lower than that in WTmice,and showed statistical difference(P<0.001),but,there was no difference in the liver at this time.At 6 weeks,the CD11b+Gr-1+cells in the liver and spleen of the TCR δKO mice were lower than those of the WT mice,and showed statistical difference(P<0.001,P<0.001,respectively).In another group of infection experiments,IL-17A was injected into TCR δ KO mice at 3 weeks post infection,and the proportion of liver CD11b+Gr-1+cells returned to the level of the WT infection group.6.Vitro experiments showed that hepatic stellate cells were activated by IL-17A stimulation,and the expressions of α-SMA and Col 1 were up-regulated(3.7 ± 0.7)times(P<0.05)and(3.9±0.1)times(P<0.0001).7.Flow cytometry showed that the proportion of TFG-β secreted by CD11b+Gr-1+cells in livers from WT mice was(30.20±4.01)%at 6 weeks post infection,and the proportion of TFG-β secreted by CD11b+ Gr-1+cells in livers from TCR δ KO mice was(20.73±1.13)%at 6 weeks post infection,the latter proportion decreased,and showed statistical difference(P<0.01).In addition,the percentages of IL-17A receptor expressed on CD11b+Gr-1+cell in liver and spleen of WT mice were about 54.3%and 26.0%at 4 weeks post infection,similar,the percentages were 52.9%and 20.3%at 6 weeks post infection.8.Flow cytometry showed that the proportions of B cells in liver from WT mice and TCR δ KO mice were(15.56±0.75)%and(28.18±2.88)%,respectively.The B cells in liver from KO mice were higher than those in the WT mice,and showed statistical difference(P<0.01);the proportion of B cells in spleen from two groups of mice were(55.48±2.01)%and(62.83±1.46)%.Similarly,the B cells in the KO mice were higher than those in the WT mice,and showed statistical difference(P<0.05).Conclusion1.IL-17A-producing-γδ T cells are involved in the early liver pathological process of mice infected with Schistosoma japonicum and aggravate fibrosis.2.IL-17A recruits CD11 b+Gr-1+cells through receptor IL-17RA,and the secretion of TGF-βby CD11b+Gr-1+ cells promotes liver fibrosis.3.IL-17A can directly promote liver fibrosis via activating hepatic stellate cells.