Role And Mechanism Of PSGL-1 Involved In Neutrophil Recruitment And Antibacterial Function In Neonatal Sepsis

Background:Sepsis is one of the most common infectious diseases in neonates with high morbidity and mortality.Recent studies suggest that the increased susceptibility of neonates to various microbes is due to the defective function of their immune system.Neutrophils are the first line of defense against the invasion of microbes,and their rapid and effective recruitment to the infected site is the key step of the body’s innate immune response against microbial invasion.PSGL-1 is an important adhesion molecule expressed on the surface of neutrophils that plays a key role in mediating the initial adhesion phase of recruitment,and also acts as a signaling receptor that triggers intracellular leukocyte activation process.In our previous study,we found that the susceptibility of neonates and infants to microbial infection is increased,which is manifested in the dysregulated migration and recruitment ability of neutrophils in neonates,as well as impaired bacteria-clearing effect.However,the cause of impaired neutrophil recruitment and bacterial clearance in neonates is unclear.Given the important role of PSGL-1 in neutrophil recruitment as well as intracellular signal transduction,we further explored whether PSGL-1 is associated with delayed neutrophil recruitment and deficient antimicrobial capacity in neonatal sepsis.Therefore,this study intends to explore the expression changes of PSGL-1 in neonatal sepsis and its effect on neutrophil recruitment and antibacterial function in sepsis,preliminarily explore its regulatory mechanisms,in order to provide new immune targets and new ideas for clinical treatment of neonatal sepsis.Part Ⅰ Study of the relationship between defective PSGL-1 expression and increased susceptibility to microbes in neonatesObjective:To explore the relationship between PSGL-1 expression changes and increased susceptibility of neonates to microbes.Methods:Adult and neonatal sepsis mouse models were constructed to observe and compare the differences in survival and bacterial clearance of major organs after infection,thereby to clarify whether neonatal mice are more susceptive to bacterial infection than adult mice.The ratio of platelet-neutrophil complex and the expression levels of PSGL-1 and CXCR2 on peripheral blood neutrophils were measured using flow cytometry.Meanwhile,clinical blood samples were collected to determine the expression of PSGL-1,Mac-1,and CXCR2 on neutrophils.Results:The 7-day survival rate of neonatal mice was lower than that of adult mice in moderate and severe sepsis,and the difference between the two groups was statistically significant in the severe sepsis model(P<0.05).Bacterial counts at 12 h post severe septic challenge were significantly greater in the blood,lung,and spleen of neonatal mice compared with adult mice(P<0.05).The percentage of peripheral platelet-neutrophil complex(Ly6G+CD41+)was significantly decreased in neonatal mice compared with adult mice(P<0.05).The expression of PSGL-1 and CXCR2 on neutrophils was significantly decreased in neonatal mice compared with adult mice(P<0.01).In addition,the expression of PSGL-1 on neutrophils was significantly decreased in neonates than adults(P<0.01).There were no significant differences in Mac-1 and CXCR2 between neonates and adults,whereas CXCR2 showed some amount of attenuation in neonates.Conclusion:Neonatal neutrophils have defective PSGL-1 expression which may contribute to the increased susceptibility of neonates to microbial infection.Part Ⅱ Effect of PSGL-1 on neutrophil recruitment and antimicrobial function in sepsisObjective:To investigate the effect of PSGL-1 on neutrophil recruitment and antimicrobial function in sepsis using in vivo and in vitro experiments.Methods:In the in vivo experiment,newborn mice as well as adult mice were randomly divided into five groups:newborn control mice(Ctrl(N)),adult control mice(Ctrl(A)),newborn sepsis mice(Sepsis(N)),adult sepsis mice(Sepsis(A)),and adult sepsis intervention mice(Sepsis(A)+PSGL-1 Ab).Survival rate,major organ pathological damage and bacterial load were observe after septic challenge.Flow cytometry was used to detect the ratio of platelet-neutrophil complex in peripheral blood and the number of neutrophils in peritoneal irrigation fluid.Photoacoustic(PA)imaging was used to detect the levels of reactive oxygen species(ROS)in mice.Serum levels of NETs were measured by Elisa.In the in vitro experiments,mouse bone marrow neutrophils were extracted and divided into Ctrl group,PSGL-1 Ab group,LPS group and LPS+PSGL-1Ab group.Cells were collected for RNA-sequence to analyze whether blocking PSGL-1 affected neutrophil recruitment and antibacterial function after LPS challenge.Migration assay,bactericidal assay and Immunofluorescence of NETs were performed to verify RNA-sequence results.Meanwhile,the polarization and phagocytosis of neutrophils as well as the formation of NETs were detected by immunofluorescence in overexpressing PSGL-1 dHL60 cells.Results:In in vivo experiments,blockade of PSGL-1 decreased the survival rate of septic mice in moderate sepsis.In severe sepsis model,survival rate was reduced in the Sepsis(N)group compared with the Sepsis(A)group and the survival rate was significantly lower in the Sepsis(A)+PSGL-1 Ab group compared with the Sepsis(A)group.Major organ damage was aggravated in the Sepsis(A)+PSGL-1 group compared with the Sepsis(A)group.The bacterial loads in blood,lung,liver and spleen were significantly lower in the Sepsis(A)group than in the Sepsis(N)group,while the bacterial loads in the Sepsis(A)+PSGL-1 Ab group were slightly higher than in the Sepsis(A)group.Platelet-neutrophil complex(CD41+Ly6G+)percentage was significantly higher in the Sepsis(A)group than in the Sepsis(N)group(P<0.05),whereas the percentage of platelet-neutrophil complex in the peripheral blood of mice in the Sepsis(A)+PSGL-1 Ab group was significantly lower than that in the Sepsis(A)group(P<0.01).The percentage of neutrophils in the peritoneal lavage fluid of the Sepsis(A)group was significantly higher than that in the Sepsis(N)group(P<0.01),whereas the percentage of neutrophils of Sepsis(A)+PSGL-1 Ab group had a lower proportion of neutrophils than the Sepsis(A)group(P<0.05).ROS levels were significantly higher in the Sepsis(A)group than in the Sepsis(N)group(P<0.05),while the ROS levels in the Sepsis(A)+PSGL-1 Ab group were lower than in the Sepsis(A)group(P<0.05).The levels of dsDNA and MPO-DNA complexes in plasma were significantly higher in the Sepsis(A)group than in the Sepsis(N)group(P<0.05),whereas dsDNA and MPO-DNA were lower in the Sepsis(A)+PSGL-1 Ab group than in the Sepsis(A)group(P<0.05).In in vitro experiments,RNA-sequence results revealed that after blocking PSGL-1,GO analysis was mainly enriched in leukocyte migration as well as in response to molecules of bacterial origin.Further migration experiments showed a significant increase in neutrophil migration after LPS stimulation compared with the Ctrl group(P<0.05),while LPS+PSGL-1 Ab showed a decrease in neutrophil migration compared with the LPS group.The bactericidal results showed a significant increase in the bactericidal level of neutrophils in the LPS group compared with the Ctrl group(P<0.01),while the bactericidal ability of neutrophils was reduced in the LPS+PSGL-1 Ab compared with the LPS group(P<0.05).Immunofluorescence results and Western Blot results showed that the LPS group showed a typical NETs reticulum and NETs formation was significantly induced,while NETs formation was significantly attenuated after intervention with PSGL-1.In the dHL-60 cell experiments,polarization was most pronounced in the LV-OE+LPS group and immunofluorescence results showed that cells in the LV-OE+LPS group phagocytosed more fluorescent E.coli compared with the NC+LPS group.NETs formation was most abundant in the LV-OE+LPS group.Conclusion:Blocking PSGL-1 in vivo and in vitro,both neutrophil recruitment and antimicrobial function were diminished in sepsis.Overexpression of PSGL-1 in dHL-60 cells increased neutrophil recruitment and antimicrobial function.Therefore,PSGL-1 plays an important role in neutrophil recruitment and antimicrobial function in sepsis.Part Ⅲ PSGL-1 regulates NETs formation through the cGAS-STING signaling pathwayObjective:To investigate the role of cGAS-STING signaling pathway in PSGL-1 regulation of NETs formation in sepsis.Methods:In in vitro experiment,mouse bone marrow neutrophils were extracted and divided into Ctrl group,PSGL-1 Ab group,LPS group and LPS+PSGL-1Ab group.The expression of cGAS,STING and IRF3 were detected by Western Blot after LPS stimulation.The NETs formation was detected by immunofluorescence,and the expression of STING and IRF3 was detected by Western Blot after further inhibition of STING using STING inhibitor.In dHL-60 cells,cells were collected for Western Blot to detect the expression of cGAS,STING,IRF3 after stimulation.Results:The protein expression of cGAS,STING,and IRF3 of bone marrow neutrophils gradually increased with the increase of LPS stimulation time,as well as the protein expression of PAD4.Compared with the LPS group,cGAS(P<0.05),STING(P<0.05)and IRF3 protein expression were significantly decreased in the LPS+PPSGL-1 Ab group.After further inhibition of STING,the NETs reticulation was reduced in the LPS+C-176 group compared with the LPS group,which is consistent with PAD4(P<0.05)and IRF3 protein.Western Blot results of dHL-60 cells revealed a significant increase in cGAS,STING,and IRF3 protein expression in the LV-OE+LPS group compared with the NC+LPS group(P<0.05).Conclusion:PSGL-1 promotes NETs formation via cGAS-STING signaling pathway in sepsis.