The Immunomodulatory Effect About Myeloid-derived Suppressor Cells In A Mouse Model Of Schistosomiasis

Schistosomiasis is a worldwide distribution parasitic diseases of zoonoses,Schistosoma japonicum(Sj)is the main pathogens harmful to livestock and human health in China.Schistosomiasis is an immune pathological disease too.There are many effects of immune cells and immune molecules in the formation of schistosomiasis pathological changes,such as the imbalance of Th1/Th2?Th17/Treg cell subsets,the significant increase of inflammatory cytokines as IL-1?IL-6?TNF-?,and the imbalance between IL-4 and IFN-?.The recent studies have found that with the parasite infects the host,the myeloid-derived suppressor cells(MDSC)in vivo have mass proliferation and aggregation in diseased tissues,significantly inhibits the host immune response,thus facilitating the worms to escape the host immune attack.To explore the distribution and immuno-modulatory effects of MDSC in Schistosoma japonicum infection host,this study used flow cytometry to identify,isolate and analyze the MDSC proliferation status,characteristics and dynamic levels in bone marrow,blood,liver and spleen of Schistosoma japonica model mice;to study the effects of MDSC subsets on CD4+T and CD8+T cell proliferation in Schistosomiasis model mice;and to analyze the effects of MDSC on Treg cell levels.thus provide experimental basis for MDSC immuno-modulatory effect in the mouse model of Schistosomiasis,further to supply and perfect the immuno-modulatory network in the host of Schistosoma japonicum infection,for providing a new research target and laboratory basis for the control of Schistosomiasis.Part ? The dynamic level analysis and subsets identification of MDSC in Schistosomiasis japonica model miceObjective:To construct a mouse model of Schistosomiasis japanica,study the distribution of MDSC in different tissues in the model mice and the dynamic changes in the natural course of disease,isolate and identify the subpopulations and cell morphology,explore the relationship between dynamic changes of MDSC and immunopathologic changes of Schistosomiasis japanica.Methods :1.The mice were infected with 30±cercariae of Schistosoma japonicum by abdominal application,and 40 BALB/c mice were infected as the experimental group.At the same time,10 normal mice were set as the control group,The mice were sacrificed after anesthesia at the planned time point,and the liver pathological changes of mice with schistosomiasis japonica model were observed by HE staining and Masson staining.Note: the actual number of infected mice is 120% of the planned number to prevent death due to disease or other causes during the long course of feeding and experiments.The number of experimental mice involved in the construction of the model in this paper is calculated using this method.All animal experiments in this paper are carried out in strict accordance with the regulations of the ethics committee of the university of South China.Experimental animals are killed under anesthesia and every effort is made to minimize its pain,suffering and accidental death.2.HE and Masson staining were used to observe the pathological changes of the liver in the mouse model of Schistosomiasis japonica.3.Flow Cytometry was used to detect the dynamic changes of MDSC in blood,liver,spleen and bone marrow of the model mice of Schistosomiasis japonica at the 0 week,2nd week,4th week,6th week,8th week,12 th week,16 th week,20 th week.5 mice were taken at each time point for the experiment.Meanwhile,the control group mice were detected for MDSC level changes of four tissues in mice at 4th week?8th week synchronized with the natural course of the infected mice.4.At the 8th week time point of the natural course of the model mice,Using CD11b\GR1\Ly6G as the identification molecule,the mononuclear subsets(M-MDSC)with CD11b+Gr1+Ly6G-phenotype and the granulocytic subsets(G-MDSC)with CD11b+Gr1+Ly6G+ phenotype were sorted by flow cytometry.The morphological identification and analysis to these sorted cells were performed by Write-Giemsa staining.Results :1.The overall appearance of the liver and spleen samples of the model mice were consistent with those of Schistosomiasis japanica: the liver was browned and hardened after 6 weeks of Schistosoma japonicum(Sj)infection,obvious granular nodules of worm eggs were seen on the surface.The spleen volume began to increase after the 4th week of Schistosoma japonicum infection.2.HE staining and Masson staining of liver tissues showed that inflammatory reactions and increased collagen expression started at the4 th week after Schistosoma japonicum infection,worm egg granuloma and fibrosis appeared after the 6th week,and increased over time.It suggested the mouse model of Schistosomiasis japanica was constructed successfully.3.Flow cytometry showed that at the 4thw and 8thw,there was no significant difference in the MDSC levels of liver,spleen,bone marrow and blood tissues in the control group,P>0.05;Compared with the experimental group(schistosomiasis model group)at the same time,the MDSC of all the four tissues was significantly lower than that of the experimental group,P?0.05.4.The dynamic MDSC level changes of liver,spleen,bone marrow and blood tissues were detected by Flow cytometry at the 0 week?2nd week?4th week?6th week?8th week?12th week?16th week?20th week in the natural course of Schistosomiasis japonica model mice.The results showed that the level of MDSC in bone marrow,spleen,liver and peripheral blood of Schistosomiasis japonica model mice was significantly higher than that in normal mice,P?0.05.It showed the first peak in bone marrow and blood tissue at the 2nd week of infected mice.At the 8th week of Schistosoma japonicum infection,the MDSC reached a high level and maintained for weeks in the four tissues,it was consistent with the severity of pathological changes in liver tissues of model mice.So the result showed a MDSC enrichment state in these tissues.5.The granulocyte subpopulations of MDSC(G-MDSC,CD11b+Gr1+Ly6G+)and mononuclear subpopulations of MDSC(M-MDSC,CD11b+Gr1+Ly6G-)were isolated and purified from the bone marrow tissue of Schistosomiasis model mice at the 8th week of the natural course by Flow cytometry,of which the number of granulocyte subsets(G-MDSC)account for about 85% and mononuclear subsets(M-MDSC)account for about 15% in all myeloid cells(CD11b+Gr1+).Wright-Giemsa staining revealed that granulocyte MDSC(G-MDSC)subset cells were mainly composed of immature granulocytes dominated by mid-late juvenile granulocytes,and mononuclear MDSC(M-MDSC)subset cells mainly composed of naive monocytes.Conclusion:In the mouse model of Schistosomiasis japonica,companied with the formation of egg granuloma and the aggravation of inflammatory reaction,the MDSC level increased significantly and was enriched in the bone marrow,spleen,peripheral blood and liver.The proliferated MDSC were heterogeneity,mainly composed of granulocyte MDSC subgroup(G-MDSC,CD11b+Gr1+ Ly6G+).Part ? The study of immune effects of MDSC on CD4+ T and CD8+ T cells in Schistosomiasis japonica model miceObjective:The BM-MDSC in Schistosomiasis japonica model mice were isolated and purified to study the immuno-suppressive effect on CD4+ T and CD8+ T cells,and explore the mechanism of MDSC action.Methods:1.To construct 45 BALB/c mice model of Schistosomiasis japonica infected by abdominal application of cercariae,then put into SPF animal house,At the 8th week of the natural course,the 12 mice were sacrificed without pain after anesthesia,and extracted the bone marrow tissue from the bilateral femur and tibia bone.The mononuclear subsets MDSC(M-MDSC)and granulocytic subsets MDSC(G-MDSC)were sorted out from the bone marrow MDSC(BM-MDSC)by Flow cytometry,and the CD4+ T cells and CD8+ T cells were sorted out from the normal BALB/c mice spleen by immune magnetic bead separation method.Two type of T cells were tagged by CFSE(carboxyfluorescein diacetate,succinimidyl ester).The granulocyte MDSC and mononuclear MDSC subpopulations co-cultured with two type of T cells were divided into three experimental groups respectively,in the ratio of 1:2,1:4,1:8,and cultured for 72 hours.At the same time,T cells only cultured were set up as blank control group and T cells plus dynabeads cd3/28-T-activator group as stimulation group,the proliferation inhibition peaks of CFSE staining in each group were detected by Flow cytometry.2.To take 6 mice model of Schistosomiasis japonica,at the 8th week of the natural course,the mice were sacrificed without pain after anesthesia,and extracted the bone marrow tissue of the bilateral femur and tibia bone.The BM-MDSC were sorted out by flow cytometry to detect the m RNA level of related cytokines and enzymes,such as TGF-??IL-10?IL-4?IFN-? and Arginase1(Arg1),induced nitric oxide synthase2(NOS2)by Real-time fluorescence quantitative PCR.3.To take 27 BALB/c mice model of Schistosomiasis japonica,At the 8th week after infected Sj,the mice were sacrificed without pain after anesthesia,and sacrificed 18 normal BALB/c mice at the same time,extracted the bone marrow tissue from their bilateral femur and tibia bone and sorted the BM-MDSC.Added 10 ?g/ml of Sj SEA(soluble egg protein),10 ?g/ml of Con A(Concanavalin A)and PBS to Sj model mice BM-MDSC respectively as experimental groups and PBS to nomal mice MDSC as control group.These pre-cocultured cells were cultured for 72 hours after stimulation in vitro.The expression levels of related cytokines such as TGF-?? IL-10? IL-4 and IFN-? in the supernatant of culture medium were detected by enzyme-linked immunosorbent assay(ELISA).Results:1.Both mononuclear subsets MDSC(M-MDSC)and granular subsets MDSC(G-MDSC)can reduce the proliferation activity of CD4+T and CD8+T cells,and show a concentration/quantity dependence.Which is mean the greater number of MDSC in the co-culture system,the stronger inhibitory effect on T cell proliferation.The inhibition effect of G-MDSC was more significant than M-MDSC.2.Real-time quantitative PCR(real-time PCR)showed that the expression levels of cytokines including TGF-??IL-10?IL-4?IFN-? and Arg1 and NOS2 of MDSC in Sj model mice were significantly higher than those in control group.3.ELISA showed that the levels of cytokines such as TGF-? ?IL-10?IL-4?IFN-? in the Sj model mice were significantly higher than those in the control group?Conclusion:The two MDSC subsets can significantly inhibit the proliferation of CD4+T?CD8+T cells,in which the inhibition effect of G-MDSC subgroup is stronger than M-MDSC;the mechanism of action may be related to the secretion of cytokines such as TGF-??IL-10?IL-4 and IFN-?,and the up-regulated expression of Arg1?NOS2 enzyme.Part ? The influence of MDSC to Treg cells in schistosomiasis japonica model miceObjective:To study the dynamic changes of Treg cells in liver and spleen tissues of schistosomiasis japonica model mice,analyze the correlation between MDSC and Treg in the immunopathological process of schistosomiasis,and discuss the interaction between MDSC and Treg cells.Methods:1.30 BALB/c model mice infected with schistosoma japonicum were constructed.The number and proportion of Treg cells in liver and spleen tissues of the model mice were detected by flow cytometry at the 0week?4th week?8th week?12th week?16th week?20th week in the natural course of Schistosomiasis japonica model mice.And the dynamic changes trend of Treg cells in the mouse model were analyzed.2.The correlation between Treg cells and MDSC at the same time point were analyzed by pearson analyse method.Results:1.The FCM results showed that the percentage of Treg cells in the liver and spleen of the infected group was significantly higher at the 8th week,12 th week and 16 th week of the disease,statistically significant than the normal mice,p<0.05.The proportion of Treg cells reached a peak value in the 16 th week of the course of schistosomiasis,it was37.2±3.04% in the liver tissue,and was 30.2±1.2% in the spleen tissue,both were significantly higher than other time points,P<0.01.2.Pearson correlation analysis was used to analyze the relationship between MDSC and Treg cells on the dynamic trend,and it was found that r=0.429,P<0.05,in the liver tissue,and r=0.498,P<0.01,in the spleen tissue.These results indicated that there was a moderate positive correlation between them in the liver and spleen tissues,and the correlation was more stronger in the spleen tissue.Conclusion:The MDSC and Treg cells were significantly increased in liver and spleen tissues with the increasing severity of hepatic granuloma lesions in the model mice of Schistosomiasis japanica.There was a moderate positive correlation between them,which may play a synergistic inhibitory role in the process of schistosomiasis lesions.Summary The MDSC in schistosomiasis japonica model mice were enriched in bone marrow,peripheral blood,liver and spleen to varying degrees;Both of granuclear MDSC(CD11b+Gr1+Ly6G+,G-MDSC)and mononuclear MDSC(CD11b+Gr1+Ly6G-,M-MDSC)showed the inhibition effect to proliferation of CD4+T and CD8+T cells in a quantity-dependent manner,and the G-MDSC were more effective than M-MDSC.The mechanism of action may be related to the secretion of cytokines such as TGF-??IL-10?IL-4 and IFN-?,and the up-regulated expression of Arg1?NOS2 enzyme.Moreover,there is a moderate positive correlation between MDSC and Treg cells in the liver and spleen tissues of Sj model mice at the same period,maybe both of them cooperatively play an immunosuppressive role in the process of schistosomiasis japonica.