Regulatory Effect And Mechanism Of PD-L1 On Neutrophil Apoptosis During Sepsis

Sepsis was defined as life threatening organ dysfunction caused by dysregulated inflammatory response to infection.Septic shock refers to sepsis with particularly profound circulatory and metabolic dysfunction.Becasuse of a high incidence and mortality,sepsis urgently needs early intervention and treatment.According to recent data,the number of sepsis cases in the United States rose from 387,330 in 1996 to 1.1 million in 2011 and is expected to reach 2 million in 2020.The mortality of sepsis is close to heart attack and exceeds the stroke.Despite the great advances in therapeutic techniques such as fluid resuscitation and antimicrobial agents,sepsis remains the leading cause of death at ICU.Neutrophil are the most abundant cells involved in innate immune response.In the early phase of sepsis,neutrophil in the bone marrow rapidly migrate to infection focus in response to chemokines and kill microorganisms by phagocytosis,degranulation and forming neutrophil extracellular traps.Neutrophil migration and microbicidal efficacy are extremely important for the control of bacteria growth and prevention of sepsis.However,studies have confirmed that the increased lifespan of neutrophil during sepsis and the cascade of inflammatory can lead to tissue hypoxia and oragan dysfunction.Programmed cell death protein-ligand 1 is a marker of immune activation,and its interaction with programmed death receptor-1 can inhibit antigen-specific immune response,resulting in T cell dysfunction,activated immune cell depletion and immune tolerance.The expression of PD-L1 on antigen-presenting cells and tumor cells can be induced in tumor and inflammatory environments.Stusied have shown that PD-L1 level on neutrophil during sepsis can be up-regulated,and PD-L1-positive neutrophil could induce apoptosis of lymphocytes and their own migratory capacity was impaired.While another study has shown that PD-L1 can interact with B7-1 to regulate immune responses.Whether PD-L1 as a ligand would serve as receptors activating intracellular signaling pathways?In addition,protein sequence analysis of PD-L1 found three YXXM-like motif,which can bind to PI3K and activate p85 subunit.PI3K/Akt pathway activation can mediate cell apoptosis.whether PD-L1 would involved in the regutation of delayed neutrophil apoptosis in sepsis?In this project,the correlation of PD-L1 on neutrophil and PI3K/Akt pathway and delayed neutrophil aopotosis are the main focuses.Part I The regulatory effect of PD-L1 on neutrophil apoptosis during sepsisObjectiveTo explore the effect of PD-L1 expressed on neutrophil during sepsis on apoptosis of neutrophil.Methods1.Purified neutrophils from healthy volunteers were divided into 3 goup:control group,IFN-γstimulating group and IFN-γ+LPS stimulating group.After 21h of stimulation,the apoptosis rate of neutrophils was detected by flow cytometry and the expression of PD-L1 was evaluated by western blot.RA was used to induce the differentiation of HL-60 for 21 hours,and the apoptosis of HL-60 and the expression of PD-L1 were assessed by FC and WB,respectively.2.Flow cytometry was used to detecte PD-L1 exprssion of the purified neutrophil form sepsis patients and to assess apoptosis rate of neutrophil after 24h of culture.3.PD-L1 siRNA transfection was used to knockdown PD-L1 expression on neutrophil from sepsis patients and from healthy volunteer stimulated by IFNγ+LPS.Apoptosis rate was detected after 21h of transfection.Western blot was utilized to assess transfection efficiency.Results1.6 healthy volunteers were recruited in this experiment.The expression of PD-L1 on neutrophil was increased in IFN-γand IFN-γ+LPS stimulating groups,and the apoptosis rate was significantly decreased after 21h of culture(P<0.01).The expression of PD-L1 on HL-60 differentiated cells stimulated by RA was down-regulated and the apoptosis rate was lower than control group(P<0.01).2.A total of 14 sepsis patients were recruited in this study.The apoptosis rate of neutrophil cultured for 24h showed a negative linear relationship with the expressio of PD-L1(R~2=0.4417,P=0.01).3.Apoptosis rate was higter after PD-L1 siRNA transfection(P<0.01).The apoptosis rate of healthy neutrophils stimulated by IFNγ+LPS after transfected by PD-L1 siRNA was significantly higher than that of IFNγ+LPS group(P<0.01).ConclusionThe up regulated of PD-L1 on neutrophil during sepsis reduce the apoptosis rate of neutrophil.Part II The potential mechanism of PD-L1 regulating neutrophil apoptosis in sepsisObjectiveTo explore the potential mechanisan of PD-L1 regulating neutrophil neutrophil in sepsis.Methods 1.The constructed PD-L1 full-length plasmid(PD-L1FL/GFP)was used to transfect A549 cells,and the binding of PD-L1 to P85 in the transfected A549 cells was detected by immunofluorescence.2.PD-L1 full-length Plasmids and intracellular structure-konckout plasmids were transfected to HEK293 cells,respectively.The interaction between PD-L1 and p85 were assessed by Co-IP.3.Si RNA was used to knockdown PD-L1 in HL-60 cells.The Cotrol si RNA was used as nagetive control.Western Blot was used to detect p AKT/AKT.4.Si RNA was used to knockdown PD-L1 in neutrophils isolated from sepsis patients.Western Blot was used to assess p AKT/AKT.Results 1.PD-L1 full-length plasmid and PD-L1 intracellular-knockout plasmid carrying GFP gene were successfully constructed.2.Double antibody immunofluorescence method found that p85 can bind with PD-L1 in the transfected A549 cells.3.Co-Immunoprecipitation revealed that PD-L1 successfully pulled p85 down in PD-L1FL-GFP group and PD-L1TD/GFPgroup whereas no binding for p85 and phosphotyrosine was observed with GFP alone.The result of PD-L1TD/GFP plasmid group also indicated that PD-L1 interacted with p85 subunit,which excludes the role of PD-L1 intracellular sequences.4.The expression of p AKT was decreased in PD-L1 si RNA group compared with NC-si RNA group in transfected HL-60 cells.5.The expression of p AKT was decreased in PD-L1 si RNA group compared with NC-si RNA group in transfected neutrophil isolated from sepsis patients.Conclusion PD-L1 on neutrophil can regulate neutrophil apoptosis.PD-L1 bind to p85 subunit of PI3 K,which could affect phosphorylation level of AKT to inhibit neutrophil from apoptosis.The Anti-apoptosis effect of PD-L1 may be not relate to intracellar domain of PD-L1.