| Objective:Echinococcus multilocularis(E.multilocularis)infection,also known as alveolar echinococcosis(AE),is a worldwide public health disease that needs to be solved urgently.An important reason that why E.multilocularis can parasitize in the host for a long time is that it can induce immune tolerance or immune escape.The function of CD8~+T cells is considered to be closely related to the progress of infectious diseases.Negative Tim-3/Galectin-9 pathway can induce T cells to produce immune tolerance.The purpose of this study is to investigate the effect of Tim-3/Galectin-9 pathway on CD8~+T cell function and its relationship with E.multilocularis infection.Methods:(1)Clinical study:the proportions of total CD8~+T cells,their subsets and the expression of Tim-3 on CD8~+T cells in peripheral blood of 30 healthy controls,62 AE patients[33 metabolically active AE(MAAE)patients and 29 metabolically inactive AE(MIAE)patients]were detected by flow cytometry;HE staining was used to observe the inflammatory cell infiltration around the liver lesions and the pathological manifestations of the liver in AE patients;Expressions of CD8,IL-10,TGF-β1,Tim-3 and Galectin-9 in infected liver tissue of AE patients were detected by immunohistochemistry.The expressions of CD107a,Foxp3,Tim-3 and Galectin-9 m RNA in peripheral blood mononuclear cells(PBMC)of AE patients were detected by real-time quantitative PCR(q RT-PCR).(2)In vitro experiment:Tim-3~+CD8~+T cell group and Tim-3~-CD8~+T cell group of AE patients were screened by flow cytometry.The culture was stimulated by CD3/CD28 antibody in vitro,and the levels of IFN-γ,perforin,granzyme B,IL-10 and TGF-β1 in the culture supernatant of each group was detected by enzyme-linked immunosorbent assay(ELISA);CD8~+T cells from healthy controls and AE patients were selected by immunomagnetic beads and co-cultured in vitro with PBS,E.multilocularis capsule fluid,E.multilocularis capsule fluid+Galectin-9 protein,E.multilocularis capsule fluid+anti-Tim-3m Ab and E.multilocularis capsule fluid+Galectin-9 protein+anti-Tim-3m Ab respectively.The proportions of CD8~+Tim-3~+T cells,CD8~+CD107a~+T cells and CD8~+Foxp3~+T cells in each group were detected by flow cytometry;The m RNA expressions of CD107a and Foxp3 were detected by q RT-PCR;The levels of IFN-γ,perforin,granzyme B,IL-10 and TGF-β1 in the culture supernatant of each group were detected by ELISA.(3)Animal experiment:BALB/c mouse models of normal control group,E.multilocularis infection group,(E.multilocularis+Galectin-9)stimulation group and(E.multilocularis+Tim-3-Fc)blocking group were prepared.The degree of intraperitoneal infection and pathological changes of liver in each group were observed at different stages of infection;The proportions of total CD8~+T cells,their subsets and the expression of Tim-3 on CD8~+T cells in mice peripheral blood in different infection stages were detected by flow cytometry;The expressions of CD8,IL-10,TGF-β1,Tim-3 and Galectin-9 in the liver of mice in different infection stages were dynamically observed by immunohistochemical method;The m RNA levels of CD107a,Foxp3,Tim-3 and Galectin-9 in the liver of mice in different stages of infection were detected by q RT-PCR.Results:(1)The proportions of peripheral blood total CD8~+T cells(43.15±3.25%,P<0.001)and CD8~+CD28~-T cells(30.07±3.31%,P<0.001)in AE patients are higher than that in control group(29.54±3.68%)(13.06±1.97%);The proportions of CD8~+CD28~+T cells(CTL)(12.01±2.32%,P<0.001)in AE patients is lower than that in control group(19.44±1.12%).There is no significant difference about the proportions of CD8~+CD28~+perforin~+T cells(16.38±5.45%)and CD8~+CD28~+granzyme B~+T cells(2.62±0.53%)between AE patients and control group(15.60±4.39%)(2.42±0.42%).The proportions of CD8~+CD28~+IFN-γ~+T cells(3.32±0.57%,P<0.001),CD8~+CD28~-IL-10~+T cells(5.37±1.34%,P<0.001)and CD8~+CD28~-TGF-β~+T cells(18.12±2.48%,P<0.001)in AE patients are higher than that in control group(2.16±0.27%)(1.68±0.31%)(7.48±1.34%).The proportions of CD8~+Tim-3~+T cells(15.28±3.46%,P<0.001),Tim-3~+CTL(6.31±2.13%,P<0.001)and Tim-3~+CD8~+Treg cells(13.48±3.97%,P<0.001)in peripheral blood of MAAE patients are significantly higher than those in control group(2.58±0.43%)(1.46±0.33%)(5.13±1.87%)and MIAE patients(3.45±0.46%)(1.62±0.36%)(5.32±2.07%).Compared with the control group,the expression of CD107a m RNA in AE patients’PBMC is lower(P<0.001)and the m RNA levels of Foxp3(P<0.001),Tim-3(P<0.001)and Galectin-9(P<0.001)are significantly higher.Furthermore,the m RNA level of CD107a is negatively correlated with Tim-3(r~2=0.411,P<0.001)and Galectin-9(r~2=0.180,P=0.019),and the m RNA level of Foxp3 is positively correlated with Tim-3(r~2=0.489,P<0.001)and Galectin-9(r~2=0.366,P=0.019).The expressions of CD8(P<0.001),IL-10(P<0.001),TGF-β1(P<0.001),Tim-3(P<0.001)and Galectin-9(P<0.001)in the proximal end of infection liver are higher than those at the distal end of infection of AE patients.(2)After Tim-3~+CD8~+T cells and Tim-3~-CD8~+T cells from AE patients were stimulated in vitro,it is found that IFN-γ(P<0.001),perforin(P<0.001)and granzyme B(P<0.001)in the supernatant of Tim-3~+CD8~+T cells are significantly lower than the secretion level of Tim-3~-CD8~+T cells,the levels of IL-10(P<0.001)and TGF-β1(P<0.001)are significantly higher than that of Tim-3~-CD8~+T cells;After co-culture with PBS,E.multilocularis capsule fluid,E.multilocularis capsule fluid+Galectin-9 protein,E.multilocularis capsule fluid+anti-Tim-3m Ab and E.multilocularis capsule fluid+Galectin-9+anti-Tim-3m Ab in vitro about AE patients’CD8~+T cells,it is found that the proportions of CD8~+CD107a~+T cells(P<0.001),the m RNA level of CD107a(P<0.001)and the levels of IFN-γ(P<0.001),perforin(P<0.001)and granzyme B(P<0.001)in culture supernatant of E.multilocularis capsule fluid+Galectin-9 group are lower than those in other groups.The proportions of CD8~+Foxp3~+T cells(P<0.001),the level of Foxp3 m RNA(P<0.001)and the levels of IL-10(P<0.001)and TGF-β1(P<0.001)in culture supernatant are higher than that of other groups;After blocking by E.multilocularis capsule fluid+anti-Tim-3m Ab,the proportions of CD8~+CD107a~+T cells(P<0.001),the m RNA level of CD107a(P<0.001)and the levels of IFN-γ(P<0.001),perforin(P<0.001)and granzyme B(P<0.001)in the culture supernatant are higher than those in other groups.However,the proportions of CD8~+Foxp3~+T cells(P<0.001),the level of Foxp3 m RNA(P<0.001)and the levels of IL-10(P<0.001)and TGF-β1(P<0.001)in culture supernatant of E.multilocularis capsule fluid+anti-Tim-3m Ab group are no significant difference between E.multilocularis capsule fluid group and PBS group,but they are significantly lower than those in E.multilocularis capsule fluid group(P<0.001,P<0.001,P<0.001),E.multilocularis capsule fluid+Galectin-9 group(P<0.001,P<0.001,P<0.001)and E.multilocularis capsule fluid+Galectin-9+anti-Tim-3m Ab group(P<0.001,P<0.001,P<0.001).(3)Compared with the mice in the control group and blocking group,with the extension of infection time,the levels of Tim-3 and Galectin-9 in the infection group and stimulation group are continued to increase,the vesicles grow faster,the volume is larger,the number is more,and the degree of liver damage is heavier.Blocking Tim-3/Galectin-9 pathway in vivo can control the growth rate of vesicles and reduce the degree of mice liver damage to a certain extent;Compared with 90 and 180 days in the infection group,the proportions of CTL(P=0.046,P<0.001),IFN-γ~+CTL cells(P<0.001,P=0.001),perforin~+CTL cells(P=0.008,P=0.007)and granzyme B~+CTL cells(P<0.001,P<0.001)are decreased significantly in the stimulation group,and the proportions of CD8~+Treg cells(P=0.011,P<0.001),CD8~+IL-10~+T cells(P=0.040,P<0.001)and CD8~+TGF-β~+T cells(P=0.040,P<0.001)are increased;At the same time in Tim-3 blocking group,the proportion of peripheral blood CTL(P=0.020,P=0.037),IFN-γ~+CTL cells(P<0.001,P<0.001),perforin~+CTL cells(P=0.026,P<0.001)and granzyme B~+CTL cells(P<0.001,P<0.001)are increased,the proportions of CD8~+Treg cells(P=0.443,P=0.015),CD8~+IL-10~+T cells(P<0.001,P<0.001)and CD8~+TGF-β~+T cells(P<0.001,P<0.001)are decreased;With the extension of infection time,the positive expression rate of CD8,IL-10,TGF-β1,Tim-3,Galectin-9 and the m RNA levels of Foxp3,Tim-3 and Galectin-9 are gradually increased in varying degrees in the liver of mice in infection group,stimulation group and blocking group,while the m RNA level of CD107a is gradually decreased.However,compared with the 90and 180 days of the infection group,the positive expression rate of IL-10(P=0.003,P=0.002),TGF-β1(P=0.032,P=0.001),Tim-3(P=0.017,P=0.030),Galectin-9(P=0.002,P=0.031)and the m RNA levels of Foxp3(P<0.001),Tim-3(P<0.001)and Galectin-9(P<0.001)are increased significantly in the liver of mice in the stimulation group,and CD107a m RNA level(P<0.001)is decreased significantly;At the same time,the positive expression rate of IL-10(P=0.021,P=0.033),TGF-β1(P<0.001,P<0.001),Tim-3(P<0.001,P<0.001),Galectin-9(P=0.007,P<0.001)and the m RNA levels of Foxp3(P<0.001),Tim-3(P<0.001)and Galectin-9(P<0.001)in the liver of mice in Tim-3blocking group are decreased significantly,and CD107a m RNA level(P<0.001)is increased significantly.Conclusion:(1)The function of CD8~+T cells has changed in E.multilocularis infection:CTL ratio and the expression of CD107a,IFN-γ,perforin and granzyme B are decreased,the proportions of CD8~+Treg cells and the expression of Foxp3,IL-10 and TGF-βare increased.It indicates that CTL function is exhausted and the inhibition of CD8~+Treg cells is enhanced in E.multilocularis infection.(2)The functional changes of CD8~+T cells in E.multilocularis infection are related to Tim-3/Galectin-9 pathway.The high expression of Tim-3/Galectin-9 pathway can weaken the function of CTL about killing pathogens or target cells,and can promote the inhibitory function of CD8~+Treg cells.Therefore,it is not conducive to the clearance of E.multilocularis by host.Blocking this pathway can restore the effector function of CTL and reduce the inhibition of CD8~+Treg cells to a certain extent.(3)Tim-3/Galectin-9 pathway is related to the E.multilocular infection process.Blocking this pathway can control the development of the infection to a certain extent. |
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