Immune Regulation Of Myeloid-derived Suppressor Cells Via PD-L1/PD-1 In Septic Mouse

Part 1 Establishment of a septic mouse model induced by cecal ligation and punctureObjective: Sepsis 3.0 is defined as life-threatening organ dysfunction caused by a dysregulated immune response to infection.Though the cecal ligation and puncture(CLP)surgery is a classic model for sepsis,the degree of infection is affected by multiple factors.This part aims to establish a sepsis model with appropriate infection level for subsequent research on immune regulation,by observing survival rates,abdominal inflammation,and organ function after CLP.Methods: The 6-8 weeks old male C57BL/6 mouses were ligated at 0.5cm,1 cm or 1.5cm from the end of cecum,and punctured by a 22 G or 26 G needle,to induce abdominal infection.The mice were divided into 4 groups depending on different ligated lengths and needles.The sham surgery without CLP was used as a control surgery.The survival condition of mice in each group was observed for 7 consecutive days after operation.Five biomarkers related to the function of organs in periphery blood were measured 24 hours post surgery.Heart and liver were detected by hematoxylin-eosin(HE)staining to evaluate tissue damage.Results: The mice in the Sham group did not die within 7 days after the operation.By day 7,the survival rate of either the CLP 1 group with a small needle or the CLP 2 group with a short ligated length was not less than 80%,which represented a mild infection.In the CLP 4 group with a long ligated length,all mice died within 4 days,the survival rate was significantly less than that of Sham group(P < 0.001)and considered as a severe infection.In the CLP 3 group with a ligated length of 1cm and a 22 G needle,the survival rate on 7th days was 36.8%,which was significantly lower than that of the Sham group(P < 0.001)and considered as a median infection.In this group,cecal necrosis could be seen on the 1st day after CLP.While 7 days later,adhesive intestinal obstruction,peritoneal effusion and splenomegaly could be seen in the abdomen.The cecum was gradually covered by the surrounding tissue and formed an abscess that would cause chronic infection.Creatine kinase(CK),Creatine Kinase-Myocardial Band(CK-MB),Alanine aminotransferase(ALT),Aspartate aminotransferase(AST)and Lactate dehydrogenase(LDH)in the peripheral blood of mice in the CLP group were significantly higher than those in the Sham group.The HE staining showed that the myocardial fibers of the heart were broken,the hepatic cells were swollen,and the inflammatory cells were infiltrated in both heart and liver in the CLP group.Conclusions: The CLP surgery under the condition of 1cm ligation and a 22 G needle successfully established a sepsis model of median infection for subsequent research on immunosuppression experiments of sepsis.Part 2 Relationship of PD-1 and lymphocyte apoptosis in septic mouseObjective: Patients with sepsis are susceptible to life-threatening secondary infection,which is related to the existence of long-term chronic infection and sustained immunosuppression in the late stage of sepsis caused by lymphocyte exhaustion.Programmed death-1(PD-1),may play an important role in the process.This part of the experiments will focus on exploring the correlation between lymphocyte apoptosis and PD-1 expression in the spleen after sepsis.Methods: In this part,CLP surgery was used to establish a sepsis model,and the Sham group was used as a control group.The spleen was taken at 0,1,3 and 7 days after surgery,and the microstructure of the cells in the spleen was observed under a transmission electron microscope(TEM).Apoptosis of the spleen cells was assessed by TUNEL assay.After the splenic single cell suspension was prepared,the number of CD4+T cells and CD8+T cells,and the expression level of PD-1 were detected by flow cytometry.The expression of PD-1 in the spleen was evaluated by immunohistochemistry.Results: Seven days after CLP surgery,apoptotic lymphocytes with condensed nuclei could be seen in the spleen under TEM.TUNEL assay showed that apoptotic cells in the spleen continued to increase after CLP,and the increase became significant on the 7th day(P = 0.044).Immunohistochemical results showed that the number of PD-1 positive cells,which mainly located in the domain of periarterial lymphatic sheath(PALS)where T cells were abundant,increased significantly from the 1st day after CLP.Flow cytometry showed that CD4+T lymphocytes continued to decrease significantly from day 1 to day 7 after CLP,while the level of PD-1 expression on the cell surface increased significantly from day 1 and remained at a high level thereafter.CD8+T cells decreased slightly in the early stage after CLP,and decreased significantly on the 7th day,while the expression level of PD-1 on the surface of CD8+T cells increased significantly on the 7th day.There was a negative correlation between the number of CD8+T cell and the expression of PD-1(r =-0.99,P = 0.006).Conclusions: Cell Apoptosis in the spleen of septic mice gradually increased,the number of T cells gradually decreased,while the expression of PD-1 on the cell surface increased.There was a negative correlation between the number of T cells and the expression of PD-1.High PD-1 levels can cause a decrease in the number of T lymphocytes.Part 3 PMN-MDSC plays an immunosuppressive role via PD-L1 in septic mouseObjective: Myeloid-derived suppressor cells(MDSCs)are a heterogeneous group of immature myeloid cells.The long-term existence of MDSCs can lead to sepsis-related immunosuppression.This part aims to observe the number and distribution of MDSCs and the PD-L1 expression on MDSCs,and elucidate the immunosuppressive mechanism of MDSCs in sepsis.Methods: The cecal ligation and puncture(CLP)model was used to induce sepsis mouse model.The sham group was used as a control group.The spleen and the bone marrow(BM)were collected at 0,1,3,and 7 days after surgery.Flow cytometry was used to detect the number and proportion of MDSCs and their subpopulations in both BM and spleen.The morphology of cells in spleen was observed by transmission electron microscope(TEM).Flow cytometry was used to detect PD-L1 expression on MDSCs and the subpopulations from BM and spleen.Immunofluorescence staining and Western blot were used to detect the PD-L1 protein level in PMN-MDSCs from BM and spleen.Co-culture experiments in vitro were used to investigate the inhibitory effect of BM PMN-MDSC on T lymphocytes.Results: The number of BM MDSCs in the early sepsis was small,but it then continued to increase to 81.45% ± 2.70% of BM cells by day 7.BM PMN-MDSC was the predominant subgroup,which had the similar variation of MDSCs.The number of BM M-MDSCs was small and continued to increase after CLP,but BM M-MDSC also increased in the Sham group.Polymorphonucler and monocytic cells were seen in the spleen of the CLP mice under TEM.MDSCs in the spleen increased significantly on the 7th day,mainly due to the increase of M-MDSC.One day after CLP,the expression of PD-L1 on MDSCs from BM and spleen increased,but then decreased later.PMN-MDSC was the main subgroup that expressed PD-L1.Ly6 G and PD-L1 double-stained immunofluorescence of BM and spleen in situ showed that the fluorescent intensity of PD-L1 was higher on the 1st day after CLP,and Ly6 G and PD-L1 double-stained cells increased.The level of PD-L1 protein in isolated PMN-MDSC from BM and spleen were significantly increased on the 1st day after CLP.Co-culture result showed that BM PMN-MDSC with high level of PD-L1 could significantly inhibit the proliferation of T cells.Conclusions: Immunosuppressive role of BM PMN-MDSC in early sepsis was mediated by PD-L1 / PD-1 axis.