The Negative Immune Regulation Of PD-1/PD-L1 Together With Their Related Molecules In Echinococcus Multilocularis Infection

Objective:To study the relation between the dynamic changes of PD-1/PD-L1 and focis during Echinococcus multilocularis infection and to reaserch the imnune function of PD-1/PD-L1 and the immune escape mechniasm of Echinococcus multilocularis,we observed expressions of PD-1/PD-L1 and its related molecules in Echinococcus multilocularis infection.Methods:112 female BALB/c mice were randomly divided into infection group and control group.The infection group was injected protoscoles of Echinococcus multilocularis by intraperitoneal injection.The control group was received corresponding volume of physiology saline.And then mice were sacrificed on 2 days,8 days,30 days,90 days,120 days,180 days and 330 days post inoculation,at dayl20 we collected spleen for stimulation in vitro.Flow cytometry was used to analyze the percentage of CD4+CD25+PD-1+T cells and CDllc?MHC ?+PD-L1+DC in spleen.The level of liver pathological alteration was observed by HE;the protein expression and location of PD-1 and PD-L1 were analyzed by immuhistochemistry respectively.The mRNA of PD-1,PD-L1,IL-10,IFN-Y and IL-4 were analyzed by qRT-PCR;Cytometry Beads Assay was used to messure the levels of IL-4?IL-10 and TGF-p(including levels after PD-1 blockade)in mice,and the levels of IL-2?IL-4?IL-6?IL-10?IL-17 and IFN-Y before treatment(stimulation of sPD-Ll was involved)in AE patients;We measured the serum levels of sPD-1 and sPD-Ll in each group by ELISA.Results:(1)Animal experiments:1)Flow cytometry results showed that the ratio of CD4+CD25+PD-1+ T cells and CD11c+MHC?+PD-L1+DC cells in spleens of infected mice was significantly higher from 90 days to 330 days after infection;2)HE staining revealed that the erosion of livers was getting worse during the infection.Lymphocyte infiltrated the livers,E.multilocularis cyst walls was formed.At the same time,the obvious inflammatory reaction zone can be seen between liver cells and E.mullilocularis cyst walls.Moreover,there are E.multilocularis;3)Immuhistochemistry showed that during early infectious stage PD-1 and PD-L1 remain low level and were observed in hepatic sinusoid and hyalomitome,however,at middle-late stage,they were both significantly increased and expressed mainly in fiber organization and germinal layer surrounding focus in infected group(p<0.05);4)Results of qRT-PCR demonstrated that in the early stages of the infection,the expressions of PD-1,IL-10 and PD-L1 mRNA were very low,but significantly increased in the late stages of the infection.The expression of IL-4 mRNA reached a peak after infection 90 days and then maintained high level(p<0.05);IFN-Y increased sharply in early stage only,then decreased to control level;5)C'BA results showed that the serum of TGF-p and IL-10 were significantly increased in middle and late stages post infection(p<0.05):and TGF-? and IL-10 decreased after PD-1 blockade.(2)Clinical trials:1)ELISA results showed that compared with healthy control,the serum of sPD-L1 was significantly increased in patients with Echinococcosis(p<0.05);2)Results of CBA showed that compared with healthy control the levels of IL-4,IL-6 and IL-10 increased obviously,however the levels of IFN-? decreased in patients with Echinococcosis;Results of stimulation in vitro also convinced serological results.Conclusion:CD4+CD25+PD-1+T cells and CD11c+MHC?+PD-L1+DC in the different periods of Echinococcus multilocularis persistent infection were different.In the middle and late stages,both cells in spleen of infected mice together with Tregs participated in Echinococcus multilocularis immune escape.In the infection,PD-1/PD-L1 took inhibitory effects through enhancing the secretion of TGF-? and IL-10 by CD4 CD25 regulatory T cells.sPD-L1 can promote the effects of PD-1/PD-Llsinaling pathway.Furthermore,PD-1/PD-L1 pathway play a role of immune suppression through regulating imbalance of Thl/Th2 inununo effects,Tregs proliferation and secretion of TGF-? and IL-10.These mechanisms are disadvantage for host dearancing livers Echinococcus multilocularis.PD-1/PD-L1 pathway and the expression of its related molecules may help Echinococcus multilocularis immune escape.