BLP Tolerance Improves The Prognosis Of Septic Mice By Promoting The Early Recruitment Of Local Neutrophils

Sepsis is a life-threatening organ dysfunction of host imbalance response caused by infection.Because of its high incidence,high mortality and high medical costs,sepsis attracts the attention of the medical profession.The pathogenesis of sepsis is very complex and involves various cell types and crucial systems.The cause of sepsis is heterogeneous and its clinical features are diverse,making it one of the most challenging syndromes both to diagnose and to cure.Therefore,sepsis is also a hot topic for researchers in recent years.Bacterial lipoprotein(BLP)is a kind of protein in the outer membrane of bacterial.It is found that pretreatment of small doses of BLP to mice or mononuclears/macrophages in vitro can induce a hyporesponsive state to a second BLP challenge,characterized by enhanced clearance of bacteria,reduced release of inflammatory cytokines and increased survival rate,which is called BLP tolerance.But up to now the mechanism of BLP tolerance has not been fully understood.Neutrophils constitute the dominant leukocyte population in the circulation,and they are powerful effector cells that destroy infectious threats through phagocytosis,degranulation,reactive oxygen species(ROS)and neutrophil extracellutraps.Therefore,neutrophils mediate the earliest innate immune responses to infection,and play a key role in the resolution of infections.In the pastt,most of the studies on BLP tolerance were in vitro experiments,with mononuclear/macrophage as the main research subjects,seldom about neutrophil.And they all focused on the changes of cells or mice after the second BLP challenge,never paying attention to the period during BLP pretreatment.Therefore,the main purpose of this study was to explore the role of neutrophils in the protective mechanism of BLP tolerance and the changes in the body during BLP pretreatment.We have used various animal models with different drug intervention to observed the dynamic changes of neutrophil including counts and percentages,the bacterial loads and the cytokine levels in abdominal cavity and circulation at different time points.We also observed the changes in important organ function and 7-day survival rate of the septic mice.This experiment is divided into three parts:Part 1:The effect of BLP tolerance on neutrophil early recruitment.Part 2:The role of enhanced early local neutrophil recruitment in the protective effect of BLP tolerance.Part 3:The mechanism of enhanced early recruitment of neutrophils to abdominal cavity induced by BLP pretreatmentThe results are as follows:1.Pretreatment with BLP can enhance neutrophil recruitment to the site of infection in sepsis induced by Salmonella Typhimurium or cecal ligation puncture(CLP).At the time points of 1h,3h,6h and 24h after CLP,the percentage of peritoneal neutrophils in control group and BLP tolerance group were 3.1%and 79.6%,85.6%and 65.8%,65.8%and 65.3%,respectively.And the number of neutrophils in control group and BLP tolerance group were 0.28×106 and 1.2×106,0.76×106 and 4.59×106,2.31×106 and 7.87×106,12.06×106and 14.33×106,respectively.Moreover,the clearance of bacteria was significantly increased in the BLP tolerance group than in the control group at each time point.2.In CLP model,when the enhanced early recruitment of neutrophils was inhibited by CXCR2 inhibitor SB225002,the 7-day survival rate of BLP tolerant mice decreased from 46.8%to 12.5%(n=30).Furthermore,the protective effects of BLP tolerance on mice such as increasing bacterial clearance,reducing the release of inflammatory cytokines,protecting the function of important organs was significantly weakened.3.The chemokine levels of BLP tolerized mice including KC,MIP-2,IP-10 in the peritoneal lavage fluid were significantly higher than that in control group in the early stage of sepsis,and the down-regulation of CXCR2 on peripheral neutrophils was reduced after sepsis in BLP tolerized mice.4.Three hours after BLP peritoneal injection,the levels of KC,MIP-2,IP-10,IL-6 and G-CSF in the peritoneal lavage fluid increased significantly,and neutrophils began to be recruited to the abdominal cavity.Conclusions:1.Early enhanced recruitment of neutrophils plays a key role in the protective mechanism of BLP tolerance in sepsis.2.The early enhanced recruitment of neutrophils in BLP tolerized septic mice is closely related to the increased release of chemokine and the reduced down-regulation of CXCR2 on peripheral neutrophils.3.During BLP pretreatment,the levels of KC,MIP-2,IP-10 in the peritoneal lavage fluid increase significantly,and neutrophils are recruited to the abdominal cavity.