| Objective:Cystic echinococcosis is a zoonotic disease caused by infection with the larvae of Echinococcus granulosus(E.granulosus),causing serious threat to human health and hindering the development of animal husbandry.The parasite’s ability to establish persistent infection for 40 years or more is partly due to its evolving immune evasion strategies.It is one self-protection function for E.granulosus when coexisting with hosts,and the key of prevention and treatment for cystic echinococcosis is understanding the evasion mechanisms.Previous research has found the protective effect of arginase,which impedes the control of pathogens or tumors,whereas it remains largely unknown during E.granulosus infection.Here,we analyzed whether arginase was produced and assessed its role in immunosuppression in mice infected by protoscolex of E.granulosus.Methods:Balb/c mice were injected with protoscolex of E.granulosus through intraperitoneal inoculation.After infection for 270 d,the blood,peritoneal lavage fluids and livers were collected from mice in infection group and control group respectively,and then the serum,peritoneal cells and liver leukocytes were isolated.Firstly,Western Blotting and immunofluorescent assay were used to evaluate the expression of arginase 1(ARG-1),arginase 2(ARG-2)and inducible nitric oxide synthase(iNOS)in peritoneal cells and liver leukocytes in protein level.The arginase activity in peritoneal cells and liver leukocytes was tested by a commercial Arginase Activity Colorimetric Assay Kit.Furthermore,the profiles of dynamic ARG-1 expression in peritoneal cells were assessed at different time points(90 d,180 d,270 d and 360 d post-infection)by flow cytometry.Secondly,the expression levels of enzymes involved in arginine metabolism were analyzed by RT-qPCR.Meanwhile,the presences of amino acids and urea in serum were determined using a High Speed Amino-acid Auto-Analyzer;the nitric oxide(NO)and total polyamine content were measured by the NO and Total Polyamine Detection Kits,in order to understand the global metabolism of arginine.And the urea and NO content in livers were analyzed by commercial kits.Besides,the profiles of dynamic CD3ζ expression in CD4+ and CD8+T cells from spleens were assessed at different time points(90 d,180 d,270 d and 360 d post-infection)by flow cytometry.Likewise,the levels of CD3ζexpression in CD4+and CD8+ T cells from liver were determined after infection for 270 d.In addition,peritoneal cells(270 d post-infection)were co-cultured with purified T cells in a transwell system for 48 h in vitro,and the levels of CD3ζ re-expression in CD4+and CD8+T cells were compared by flow cytometry.The aim was to elucidate the relation between CD3ζ and ARG-1 for E.granulosus-infected mice in vivo and in vitro.Furthermore,the favorable effect of arginine on CD3ζexpression was verified in E.granulosus-infected mice and in vitro.Finally,purified T cells were respectively cultured in RPMI 1640 medium with or without arginine,then microRNA(miRNA)microarray was used to identify the differentially expressed miRNAs related to arginine regulation.And the difference was respectively verified in vitro and in E.granulosus-infected mice by RT-qPCR.The targeted genes of differentially expressed miRNAs were predicted by the miRanda software,and functional annotation was completed according to the KEGG and GO databases.Furthermore,the targeted genes involved in amino acid deficiency-related pathways were screened and analyzed,in order to evaluate the roles of miRNAs in CD3C expression and T cell function regulated by arginine in E.granulosus-infected mice.Results:The results of Western Blotting and immunofluorescent assay showed increased level of ARG-1 protein(P<0.001),unchanged ARG-2 and iNOS(P>0.05)in the peritoneal cells and liver leukocytes from infected mice compared to those from the control group.Also,elevated arginase activity in the peritoneal cells and liver leukocytes was present in infected mice(P<0.05).The proportions of ARG-1 expression in SSChhighCDllb+F4/80+,SSClowCD1 lb+F4/80+,CD11b+CD11c+,CD11b+Gr-1+Ly-6C+Ly-6G-,CD11b+Gr-1+Ly-6C-Ly-6G+,CD11b+Gr-1+and CD11b+Ly-6G+cells from enterocoelia exhibited a rising trend along with the extension of infection time(P<0.05).Compared to the control group,the peritoneal cells from E.granulosus-infected mice(270 d post-infection)showed higher level of ARG-1 mRNA(over 60 times,P<0.001),slightly enhanced ARG-2 and AGAT(P<0.01),unchanged ADC(P>0.05),lower iNOS、ODC、OTC and OAT(P<0.01).Meanwhile,compared to the control group,the concentrations of arginine,citrulline and NO decreased(P<0.05),and those of ornithine and urea increased(P<0.01)in serum post-infection,indicating that the activity of arginase increased and the activity of nitric oxide synthase decreased in E.granulosus-infected mice.Also,compared to the control group,increased urea and decreased NO in livers were present in infected mice(P<0.05).Kinetic studies showed that the relative expression of CD3ζ remained unchanged in CD4+and CD8+T cells from spleens during the first 180 d after E.granulosus infection(P>0.05),and the decline occurred at 270 d and 360 d in the infection group compared to the control group(P<0.001).This was negatively correlated with dynamic ARG-1 expression(P<0.05).And decreased level of CD3ζ expression was found in CD4+and CD8+T cells from livers post-infection(P<0.01).In addition,purified T cells showed declined re-expression of CD3ζ when co-cultured with peritoneal cells from infected mice(P<0.001),and CD3ζ was regenerated by supplement of arginine or arginase inhibitor BEC(P<0.05),rather than nitric oxide synthase inhibitor L-NMMA or catalase(P>0.05).It demonstrated that the CD3ζ expression in T cells was inhibited by the arginase expressed in peritoneal cells from E.granulosus-infected mice.Furthermore,extra arginine could promote CD3ζ expression in E.granulosus-infected mice and in vitro(P<0.05),but not the urea(P>0.05).These results indicated that the arginine deficiency may be the dominant reason for immunosuppression of T cells mediated by arginase.46 differentially expressed miRNAs related to arginine regulation in cultured T cells were identified by miRNA microarray in present study,and the accuracy of difference was up to 90%verified by RT-qPCR.Likewise,70%differentially expressed miRNAs related to arginine regulation were found in E.granulosus-infected mice compared to the control group.Total 2 907 targeted genes were predicted,and the functions of targeted genes were mainly involved in 44 pathways,such as Signal transduction and Sensory system.Among these,15 miRNAs and 40 targeted genes were related to mTOR pathway;5 miRNAs and 6 targeted genes were related to eukaryotic initiation factor,indicating the important roles of miRNAs in CD3ζ expression and T cell function regulated by arginine deficiency.Conclusion:1.Elevated ARG-1 expression,but not ARG-2,was present in peritoneal cells and liver leukocytes from E.granulosus-infected mice compared to those from the control group,together with increased activity.Along with the extension of infection time,the expression of ARG-1 showed a rising trend in multiple myeloid cells from enterocoelia.2.ARG-1 was the principal enzyme for arginine metabolism in E.granulosus-infected mice.Arginine was consumed largely and ARG-1 antagonized nitric oxide synthase in E.granulosus-infected mice,reducing NO,which facilitates the parasite’s growth and development.3.Along with the extension of infection time,the expression of the CD3ζ chain showed a decreasing trend in E.granulosus-infected mice.And the arginine deficiency may be the dominant reason for immunosuppression of T cells mediated by arginase.4.MiRNAs may play an important role in CD3ζ expression and T cell function regulated by arginine deficiency in E.granulosus-infected mice,which provided a molecular basis for studies on the mechanism of immunosuppression mediated by arginase in E.granulosus-infected mice.Understanding the underlying mechanism of arginase in immune evasion of E.granulosus infection will pave the way for targeted treatment. |
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