Schistosoma Japonicum SEA Alleviates Allograft Rejection By Inducing The Proliferation Of MDSCs

Objectives:At present,there are a total of six species of Schistosoma that are known to infect humans.Among them,Schistosoma japonicum is a widely prevalent parasitic worm in China,which once threatened the health of millions of people in the country.After strong prevention and control by the Chinese government,Schistosoma epidemic has been effectively controlled.Our center is located in an endemic area of schistosomiasis,and in daily clinical practice,we have encountered several cases of Schistosoma japonicum.Among them,our research team reported a case of Schistosoma japonicum donor liver transplantation,and the donor liver biopsy clearly confirmed the deposition of Schistosoma japonicum eggs in the portal area.Starting from 3 months after surgery,the recipient can maintain the stability of the graft by taking only low-dose immunosuppressants.Liver biopsy at 6 months after surgery further confirmed that there were no signs of rejection or fibrosis.Therefore,we speculate that in allogeneic liver transplantation,Schistosome eggs can exert immune regulatory functions and help induce immune tolerance in allogeneic grafts.The prerequisite for opportunistic disease caused by parasitic infections is a decrease in the body’s immune monitoring ability.Research has confirmed that the body’s immune status decreases after long-term chronic infection with parasites.However,as an important member of the immune regulatory network,myeloid-derived suppressor cells(MDSCs)have been shown to exhibit significant proliferation during parasitic infections and may participate in parasitic immune escape processes.Therefore,the proliferation of MDSCs may exist after infection with Schistosoma japonicum in humans,and it may play an important role in the immune regulation of Schistosoma japonicum.This study is the first time to investigate the changes in peripheral blood MDSCs and their impact on the progression of liver fibrosis in human with schistosomiasis.MDSCs are a group of myeloid-derived cells at different stages of development and differentiation.They are dendritic cells,monocytes,and granulocytes’progenitor cells,with strong heterogeneity.Therefore,the phenotype of human MDSCs is complex,and the phenotype of granulocytic-myeloid derived suppressor cells(G-MDSCs)is more controversial.This study attempts to explore new functional biomarkers of human MDSCs through single cell sequencing methods.MDSCs are important members of the immune regulatory network,which can effectively suppress T cell immunity and achieve negative regulation of immune function.T cell immune mediated rejection is the main cause of acute rejection after organ transplantation.We constructed a mouse skin transplantation model to investigate the role and mechanism of soluble egg antigen(SEA)of Schistosoma japonicum in allogeneic grafts,and then explored the effect and mechanism of SEA on MDSCs through in vitro experiments.Material and Methods:In this study,56 patients with schistosomiasis infection in Dongting Lake area,Hunan Province,China are included,of which 18 are diagnosed as advanced schistosomiasis with Schistosoma japonicum(ASJ),38 are diagnosed as chronic schistosomiasis with Schistosoma japonicum(CSJ),and 21 healthy people are recruited as the control group.Flow cytometry is used to detect the percentage and absolute cell count of MDSCs and lymphocyte subsets.Doppler ultrasound determines the degree of liver fibrosis.Four patients infected with Schistosoma japonicum and two healthy people are recruited.After collecting peripheral blood,peripheral blood mononuclear cells(PBMCs)are extracted with density gradient centrifugation solution for single cell sequencing.t SNE dimensionality reduction analysis groups and annotates all cells,and we annotate low density neutrophils in peripheral blood PBMCs after sample de batch effect.Further UMAP analysis,cluster analysis,quasi time series analysis,and KEGG analysis are performed.Search for a group of cells most likely to be MDSCs at the single-cell sequencing level using classic MDSCs markers,and name them“myeloid-derived suppressor cell like cells”(MDSCLCs).Thus further searching for new functional biomarkers that may be used to define MDSCs.In vivo experiments of Schistosoma japonicum SEA and MDSCs:5～6w male C57 mice are selected as recipients,and Balb/c mouse tail skin is used as donors(Balb/c mouse’s tail skin is transplanted to the right chest and abdomen of C57 mice)to construct a mouse allogeneic skin transplantation model.The experiment is divided into four groups:Control group,SEA group,Control+MDSCs elimination group(Control+Anti-Gr1 group),SEA+MDSCs elimination group(SEA+Anti-Gr1 group).The MDSCs elimination group is treated with Anti-Gr1 neutralizer to eliminate MDSCs.On the Day 0 of the experiment(Day0)and Day7,100μg of Anti-Gr1 neutralizer is intraperitoneally injected into each animal.SEA is administered subcutaneously to the back of mice with a body weight of 3.125μg/g,and four groups are administered subcutaneously starting from Day-21(Control group or Control+Anti-Gr1 group:physiological saline+Freund’s incomplete adjuvant;SEA group or SEA+Anti-Gr1 group:SEA+Freund’s incomplete adjuvant),with a medication interval of 14 days.All four groups of mice undergo allogeneic skin transplantation on Day0,and skin graft scores are recorded daily starting from Day 7.Blood,skin grafts(excluding Day0),and spleen are taken from mice during experiments on Day0,Day7,Day10,and the day of complete rejection with skin grafts.Flow cytometry is used to detect MDSCs and Treg in the blood and spleen of mice,and HE staining is performed on skin grafts.The complete rejection of skin grafts is used as the endpoint event of the entire experiment,and the K-M(Kaplan Meier)survival curve is used to reflect the survival rate of skin grafts on Day7 to Day15In vitro experiment of Schistosoma japonicum SEA and MDSCs:bone marrow cells of femur and tibia of male C57 mice aged 5～6w are taken for primary culture.The experiment is divided into three groups:(1)Control group:40ng/ml granulocyte-macrophage colony stimulating factor(GM-CSF);(2)IL-6 group:40ng/ml GM-CSF+40ng/ml IL-6;(3)SEA group:40ng/ml GM-CSF+SEA 10μg/ml.After 7 days of in vitro culture,flow cytometry is used to detect CD11b~+Ly6C~+monocytic-myeloid derived suppressor cells(M-MDSCs)and CD11b~+Ly6G~+G-MDSCs.Real time quantitative PCR(RT-q PCR)method is used to detect the relative m RNA expression of MDSCs and their immune regulatory related genes.Results:The percentage of MDSCs in peripheral blood PBMCs[MDSCs/PBMCs(%)]of patients infected with Schistosoma japonicum is much higher than that of healthy controls(HCs)group(34.85±18.61%vs 5.03±2.07%,P<0.001).Regarding the MDSCs subgroup,G-MDSCs/PBMCs(%)in patients infected with Schistosoma japonicum significantly increases compared to the HCs group(32.25±18.76%vs 0.30±0.36%,P<0.001),while M-MDSCs/PBMCs(%)slightly decreases compared to the HCs group(2.60±2.24%vs 4.73±2.01%,P<0.001).There is no statistically significant difference in MDSCs and their subgroups between the CSJ and ASJ group.Pearson correlation analysis shows that the absolute count of CD3~+T cells is negatively correlated with MDSCs/PBMCs(%)(r=-0.305,P=0.022).G-MDSCs/PBMCs(%)in patients with Schistosoma japonicum infection diagnosed as liver fibrosis(grade>0 group)by Doppler ultrasound is significantly higher than that in patients without liver fibrosis(grade=0 group)(35.61±17.34%vs.18.86±11.39%,P=0.003).Pearson correlation analysis shows that there is a positive correlation between G-MDSCs/PBMCs(%)and the degree of liver fibrosis in patients with Schistosoma japonicum infection(r=0.356,P=0.017).The results of single-cell sequencing analysis indicate that low-density neutrophils extracted from PBMCs are mainly divided into 5groups in the Schistosoma japonicum infection group(SJ group)and healthy individuals.Among them,low-density neutrophil group 1 is a cell subgroup with MDSCs markers,mainly characterized by specific high expression of Integration suite beta 2(ITGB2),Fos proto-oncogene(FOS),Fos B proto-oncogene(FOSB),S100 calcium binding protein A6(S100A6),Arachidonate 5-lipoxygenase activating protein(Al OX5AP),Proline rich nuclear receptor coactivator 1(PNRC1),Eukaryotic translation initiation factor 1A Y-linked(EIF1AY),S100A8,S100A9 genes.Low density neutrophil group 0 serves as a resting subgroup,and quasi temporal analysis revealed a bidirectional differentiation relationship between low density neutrophil group 0 and group 1.In the mouse skin transplantation model,the average survival time of skin grafts in the SEA group is significantly longer than that in the Control group(14.67±2.739 vs 10.5±1.932 days,P=0.002)and MDSCs clearance group(14.67±2.739 vs11.2±2.728 days,P=0.008).Compared with the Control group,peripheral blood MDSCs(21.73±5.752%vs.15.31±2.98%,P=0.036),G-MDSCs(18.61±5.598%vs.12.65±3.127%,P=0.046),and Treg(33.23±4.933%vs.21.63±4.736%,P=0.015)in the SEA group show significant proliferation at time point of Day0.Compared with the Control group,peripheral blood MDSCs(47.82±8.336%vs 35.26±6.388%,P=0.015)and G-MDSCs(44.4±8.329%vs 30.67±5.737%,P=0.008)in the SEA group show significant proliferation at the time points of Day10.After using Anti-Gr1 in the SEA group,the percentage of MDSCs(29.74±4.784%vs 47.82±8.336%,P=0.001)or G-MDSCs(26.28±3.787%vs 44.4±8.329%,P<0.001)significantly decreases compared to Anti-Gr1use before.The percentage of M-MDSCs shows almost no change(3.453±1.184%vs 3.419±1.653%,P=0.969).When MDSCs are eliminated,the SEA+Anti Gr1 group experienced increased skin graft rejection on the10th day after transplantation compared to the SEA group.In vitro induction of MDSCs,compared with the Control group,the percentage of MDSCs in CD45~+cells[MDSCs/CD45+cells(%)]in the SEA group shows an upward trend(50.430±10.700%vs.36.300±1.462%,P=0.086).Compared to the Control group,the absolute count of MDSCs in the SEA group significantly increased(1267.000±414.400*10~3/well vs.587.800±56.240*103/well,P=0.048).Compared with the Control group,SEA significantly induced differentiation of G-MDSCs,with an increasing percentage(18.390±4.282%vs.11.830±0.361%,P=0.057)and a significant increase in absolute counts(464.000±167.300*10~3/well vs.192.000±23.130 x 10~3/well,P=0.049).Compared with the Control group,the functional gene Inducible nitric oxide synthase(i NOS)of MDSCs induced by SEA in vitro is significantly upregulated,but arginase1(Arg1)is significantly downregulated,while Il-10 and Il-1βare significantly upregulated;Toll-like receptor 1(Tlr1),Tlr2,and Tlr4 are significantly downregulated,while Myeloid differentiation factor 88(Myd88)shows no significant changes.NF-κB signaling pathway related genes are significantly downregulated.Conclusion:This study reveals for the first time that infection with Schistosoma japonicum leads to the proliferation of G-MDSCs in human peripheral blood,which thereby promotes immune escape of Schistosoma and exacerbates the degree of Schistosoma liver fibrosis.ITGB2 can serve as a marker for human MDSCs,and the localization or role of FOS,FOSB,and Al OX5AP in human MDSCs deserve further exploration.SEA significantly inhibits allograft skin graft rejection by inducing the proliferation of G-MDSCs,and its mechanism may be related to TLR/NF-κB pathway’s inhibition.In summary,SEA of Schistosoma japonicum is expected to become a new method for treating rejection.