The Mechanism Of Neutrophil-mediated T Lymphocyte Dysfunction In Sepsis

Background and ObjectiveSepsis is a condition with high mortality and morbidity rates and is common in bacteria-causing infections especially in intensive care unit.With the research to the depth,the pathophysiology of sepsis has been gradually unveiled.Maintaining the balance of the immunity system is essential in sepsis.At the early stage of infection,the immune system is in an activated condition,which can effectively defend against various pathogenic factors including microorganisms.However,with the progress of the disease,immune system dysfunction gradually occurs and causes immunosuppression.Immunosuppression is rather complicated and happens in both cellular and humoral immunity.Neutrophil plays a critical role in eliminating invading bacteria,which is considered as the main defense at the early stage of infection.Extensive basic and clinical studies have identified that neutrophil dysfunction damages the immune system.Notably,as neutrophils are capable of phagocytizing bacteria,why significant and persistent increasement in neutrophils means disease progression?We intend to investigate the changes of neutrophil function and the mechanism of neutrophil immunosuppression in sepsis through in vitro and in vivo experiments.These results will provide reliable theoretical basis for the immunotherapy of sepsis.MethodsIn in vivo experiments,we use CLP mouse model to simulate clinical scenario of patients with sepsis,Survival rate of mice is observed for 72 hours.Immunohistochemistry is used to observe the infiltration of neutrophils and the expression of PD-L1.We measured the proportion and karyotype of neutrophils with different degrees of maturation in peripheral blood.Flow cytometry is used to detect CD80,CD86 and MHC II of neutrophils,the expression of PD-1 and CTLA-4 in spleen lymphocytes,lymphocytes apoptosis and Tregs proportion.In in vitro experiments,lipopolysaccharide is employed to stimulate mouse bone marrow neutrophil.Flow cytometry is used to detect the apoptosis of neutrophils and the expression of PD-L1 at different time points.Neutrophils from wild type（WT）and PD-L1 deficient(PD-L1^-/-)mice are used to co-culture with lymphocytes.Flow cytometry is used to detect lymphocytes apoptosis and Tregs proportion.ELISA detecting kit is used to determine IL-2.ResultsPathological examination showed that lung of CLP mice incurred significant inflammation response.Neutrophils infiltration and the expression of PD-L1 in neutrophils was significantly increased in lung of septic mice compared with those of control mice.Immature neutrophils were significantly increased in peripheral blood.LPS stimulation could inhibit neutrophils apoptosis significantly.In sepsis,neutrophils had high expression of antigen presenting molecules（CD80,CD86,MHC II）and immunosuppressive molecules（PD-L1）.In sepsis,Tregs number and the apoptosis of T cells increased significantly.Co-culture of neutrophils with T cells increased the proportion of CD4⁺CD25⁺FOXP3⁺Tregs.Co-culture of LPS-treated neutrophils with the lymphocytes suppressed the secretion of IL-2 and increased T cell apoptosis significantly.The effect was diminished in the PD-L1 deficient neutrophils.ConclusionsAt different stages of sepsis,neutrophils showed dual functions of antigen presentation and immunosuppression.Neutrophils negatively regulate lymphocyte function by expressing immunosuppressive molecules,which may be the mechanism of immune suppression in sepsis.