Expression Of CD49d In Myeloid Derived Suppressor Cells And Its Immunosuppressive Function In Sepsis

Purpose:Myeloid-derived suppressor cells(MDSCs)are one of the mechanisms of immunosuppression during the late stage of sepsis.Different subsets have different immunosuppressive functions.Due to the heterogeneity of these cells,more detailed analysis and characterization of different subsets of MDSCs are needed.In this study,we established a mice sepsis model by cecal ligation and puncture,analyzed the expression level of CD49d on MDSCs in different immune organs at different time points after operation,and analyzed the expression level of CD49d on MDSCs in peripheral blood of sepsis patients,and explored the difference of immunosuppressive function and possible mechanism among MDSCs subsets with different expression level of CD49d,so as to provide theoretical basis for elucidating the immunosuppressive mechanism of the late stage of sepsis and discovering the molecular target of sepsis immunotherapy.Methods:1.Flow cytometry was used to analyze the proportion of CD49d~+MDSCs and CD49d~-MDSCs in live cells in peripheral blood of 15 healthy controls,15 early sepsis patients,and 10 late sepsis patients.2.Sepsis mice model was established by CLP,and the effect of sepsis model was verified by observing the general condition of mice after operation,the survival rate of mice after operation for 10 days and HE staining of pathological sections.3.To determine the expression of CD49d on the surface of MDSCs during the development of sepsis,the proportion and quantity of CD49d~+MDSCs and CD49d~-MDSCs in different immune organs(bone marrow and spleen)of mice at different time points(1d,4d,7d and 10d)after CLP were analyzed by flow cytometry.4.CD49d~+MDSCs and CD49d~-MDSCs from the bone marrow and spleen of late stage sepsis(CLP 10d)mice were separated by flow cytometry,and the co-culture experiment directly verified the immunosuppressive function of the two subgroups of MDSCs.The mRNA expression of immunosuppressive genes IDO-1,Arg-1,and NOS2 in two subgroups of MDSCs was detected by q-PCR,and the expression levels of Arg-1 and iNOS proteins in the two subgroups of MDSCs were detected by flow cytometry to determine the possible main immunosuppressive mechanisms of the two subgroups.Results:1.Compared with healthy controls,the expression of CD49d on peripheral blood MDSCs in patients with early stage of sepsis decreased,but increased in patients with late stage of sepsis.According to the expression of CD49d,MDSCs could be divided into two subsets:CD49d~+MDSCs and CD49d~-MDSCs.The proportion of CD49d~+MDSCs and CD49d~-MDSCs in peripheral blood cells in patients with the early stage of sepsis was lower than that in healthy controls,but significantly higher than that in patients with the late stage of sepsis(p<0.0001).2.The CLP sepsis mice model was established by ligating the cecum at the end of cecum about 1cm,puncturing with 22G needle and squeezing out a little feces.From the general performance of mice,CLP treated mice had less food and water intake,slow activity,wet hair,and abscess formation in the abdominal cavity of mice after dissection,which could form persistent infection.Pathology results indicated multi-organ damage and met the sepsis 3.0 definition.The mortality rate of mice stabilized at about 50%10 days post CLP,which is similar to the mortality rate of human sepsis3.The expression of CD49d in spleen and bone marrow of CLP mice was similar to that of sepsis patients.In early CLP groups it was transiently decreased compared with Sham groups,and increased in late CLP groups.The expression in spleen and bone marrow of CLP mice was significantly increased compared with Sham group on CLP 7d and CLP 10d(p<0.01).The proportion of CD49d~+MDSCs and CD49d~-MDSCs in bone marrow increased with time.The proportion of CD49d~+MDSCs in CLP 7d group was higher than that in CLP 1 d group(p<0.05),and reached the highest at CLP10d.The proportion of CD49d~-MDSCs in CLP 4d group was significantly different from that in CLP 1d group(p<0.0001),and the proportion of CLP 10d group was significantly higher than that in other groups.The proportion of CD49d~+MDSCs and CD49d~-MDSCs in the spleen showed the same trend over time as in the bone marrow,both increasing with the progression of sepsis.Splenic CD49d~+MDSCs,unlike bone marrow,were significantly elevated at the early stage of sepsis(p<0.001),continued to increase and reached a peak in the CLP 7d group,and did not change significantly in the CLP 10d and CLP 7d groups.The percentage of CD49d~-MDSCs in spleen was significantly increased on the 7th day post CLP and reached the peak on the 10th day post CLP.4.The results of co-culture of splenic cells and two subsets of MDSCs showed that CD49d~-MDSCs from bone marrow and spleen had stronger inhibitory effect on T cell proliferation than CD49d~+MDSCs,and the difference was statistically significant.5.There were significant differences in the mRNA expression levels of immunosuppressive genes between the two subsets of MDSCs in bone marrow.The mRNA expression levels of IDO-1,Arg-1 and NOS2 in CD49d~-MDSCs were much higher than those in CD49d~+MDSCs.Only the mRNA expression level of Arg-1 in CD49d~-MDSCs was higher than that in CD49d~+MDSCs in spleen.The protein expression of Arg-1 and iNOS in CD49d~-MDSCs was significantly higher than that in CD49d~+MDSCs in bone marrow,and only the protein expression of Arg-1 in CD49d~-MDSCs was higher than that in CD49d~+MDSCs in spleen.Conclusion:1.MDSCs express CD49d in sepsis patients;CD49d can serve as a marker for distinguishing the two subgroups of MDSCs.2.The expression of CD49d on the surface of MDSCs in bone marrow and spleen of septic mice was increased,and the inhibitory effect of CD49d~-MDSCs on T cells was stronger than that of CD49d~+MDSCs,which may play an important role in immunosuppression via Arg-1.