Impact Of S1PR3 On The Initiation And Development Of Bacterial Sepsis And Its Molecular Mechanism

Part1 Impact of S1PR3 on the initiation and development of bacterial sepsisObjectives:Sphingosine 1-phosphate receptor 3(S1PR3)is a part of the seven transmembrane G protein coupled receptor family,which is connected with cell transmembrane signaling regulates cell proliferation,angiogenesis,and inflammation after uniting with Sphingosine 1-phosphate(SIP).Lately,it's believed that S1PR3 regulates peripheral and central innate immune cells proinflammatory response.But its role in modulating macrophage reaction to bacterial infection is still unknown.So,this section of the study was planed to research the expression and correlation of S1PR3 in sepsis.Methods:The protein levels of S1PR3 was investigated after lipopolysaccharide and heat-killed E.coli challenge bone marrow derived macrophage(BMDM).Sepsis was induced by cecum ligation and puncture(CLP)on WT(S1pr3+/+),S1pr3-/-and Slpr3+/-mice.Everyday we assessed mortality.To measure the bacterial burden,Important viscera homogenate,peritoneal lavage fluid,and blood samples were gained at 72 h after CLP challenge and were detected using agar plating method.Sepsis was induced x by E.coli-induced peritonitis.We assessed mortality hourly.To measure the bacterial burden,we obtain peritoneal lavage fluid at 24 h after E.coli-induced peritonitis challenge and were detected using agar plating method.Results:Using BMDMs,we found that the protein levels of S1PR3 increased approximately threefold after lipopolysaccharide and heat-killed E.coli challenge.Moreover,in peritoneal macrophages isolated from CLP-induced polymicrobial septic mice,the expression levels of S1PR3 were significantly elevated after sepsis onset.The induction of S1PR3 expression in peritoneal macrophages was further observed in E.coli-induced peritonitis.We found that 80.0%of the S1pr3-/-mice died within 10 days after CLP,whereas 23.8%of the WT mice and 20.8%of the S1pr3+/-mice died over the 10-day study period.We observed a systemically increased bacterial burden in the S1pr3-/-mice on both days 1 and 3.84.6%of the S1pr3-/-mice died 48 hours after the E.coli challenge,compared with 47.1%of the Slpr3+/-mice and 52.2%of the WT mice.Accordingly,a significantly higher bacterial load was observed in the S1pr3-/-mice.Conclusions:These results suggest a potential functional role for S1PR3 in the progression of sepsis.S1PR3 plays a protective role in the host defense against invading pathogens,ultimately improving the outcome.Part 2 Impact of S1PR3 on bacterial killing function of macrophageObjectives:Immune defense to remove invading E.coli was cut down because of the absence of S1PR3,induce the spread of pathogens and finally worsen the outcome.We then identify the different rates of bacterial clearance observed above were caused by which cells.Methods:Reciprocal BM transplantation was created between S1pr3-/-and WT mice as follows:S1pr3-/-?S1pr3-/-,WTT,S1pr3-/-?CWT and WT?S1pr3-/-.The chimeras then were under attacked by intraperitoneal E.coli.24h after infection,peritoneal lavage fluid(PLF)were collected and bacterial burdens were measured with agar plating method.Furthermore,the mortality of all chimeras were observed hourly.Recombinant adenovirus expressing S1PR3 and the control adenovirus expressing GFP were constructed and infected BMDM.In vivo,BMDM overexpressing S1PR3 or GFP were administrated to septic mice immediately after CLP,and 48h survival rates were evaluated.In the groups given with BMDM immediately after CLP,peritoneal lavage fluid was harvested at 24h after the cell transfusion.Specific agonist of S1PR3 was administrated to septic mice at 0.5 hours after CLP,and 48 h survival rates were evaluated.Specific agonist of S1PR3 was administrated to septic mice at 0.5,3 and 6 hours after E.coli injected in intraperitoneal,and 48h survival rates were calculated.In the groups,samples such asperitoneal lavage fluid,liver,lung and spleen were harvested at 3h and 6h after intraperitoneal injection of E.coli.Results:Slpr3-/-mice transplanted with WT bone marrows had significantly improved survival(33.3%)at 48 hours after intraperitoneal E.coli challenge.However,S1pr3-/-mice that received S1pr3-/-bone marrow all died within 40 hours.There were significantly lower bacterial counts in the peritoneal lavage fluid from S1pr3-/-mice transplanted with WT bone marrows at 24 hours after E.coli infection.Also,in the converse experiment,replacement of WT mouse bone marrow with bone marrow from Slpr3-/-mice mirrored the phenotype observed in the Slpr3-/-mice.we constructed a recombinant adenovirus overexpressing S1PR3 and transfected it into WT BMDMs.The BMDMs were then adoptively transferred into systemic macrophage-depleted WT mice immediately after intraperitoneal E.coli injection.Notably,the administration of SlPR3-overexpressing BMDMs in the septic mice significantly improved their survival and enhanced bacterial clearance in the peritoneal cavity.A novel peptide named GPS-725.017,was used as an S1PR3-agonist candidate.The intracellular bactericidal effect of the agonist was further assessed.WT macrophages treated with GPS-725.017 showed a significant decrease in the intracellular bacteria count.Whereas in S1pr3-/-macrophages,treatment with GPS-725.017 did not improve the bactericidal effect.In WT mice with sepsis,the administration of GPS-725.017 significantly increased survival rates in both the CLP-induced model and the bacterial peritonitis model.Interestingly,in the bacterial peritonitis model,the S1PR3 agonist offered protection even when it was administered 6 hours after sepsis onset.The administration of GPS-725.017 did not rescue S1pr3-/-mice from CLP-induced lethal sepsis.In agreement with the improved survival rate,the bacterial loads in the peripheral organs/tissues were also significantly reduced in WT mice treated with GPS-725.017.In vitro phagocytosis experiment,there was no significant difference in phagocytic capacity of BMDM or AM of S1pr3-/-and WT mice at 15min,30min,60min and 120min.In gentamicin protection assay,the survival bacterial count of BMDM or AM of Slpr3-/-mice was significantly higher than that of WT mice at 3h and 6h,the number of viable bacteria in the peritoneal macrophages of WT mice was significantly lower than that of the control group at 3h and 6h after S1PR3 agonist treatment.Conclusions:S1PR3 exerts its antimicrobial activity through the ability of macrophages to kill bacteria,rather than phagocytosis.GPS-725.017 can efficiently activate S1PR3 signaling to fight against invading bacteria in macrophages.Activation of S1PR3 can cause a marked improvement in the outcome of sepsis based on its ability to improve the host bactericidal defense against pathogenic microbes.Part 3 The downsteam signaling of S1PR3 in promoting macrophage kill bacterialObjectives:Integrated with the first and second part of the study,we found S1PR3 gene knockout results in a deficit in the bacterial killing function of macrophages,thereby the bacterial load was increased and the survival rate was reduced.This part of the study,we want to explore the downsteam signaling of S1PR3 in promoting macrophage kill bacterial.Methods:The macrophages were incubated with heat-inactivated E.coli for 0,15,30,45,60 or 90 min.To measure the total intracellular ROS levels and Phagosomal pH,the cells were treated with the fluorogenic probe H2DFFDA.The activity of NOX2 was detected by chemiluminescence.The protein levels of markers at different maturation stages and Vps34 were detected by immunofluorescence and Western blot.The level of the coupling of Vps34 with Gi,Gq and G12 was detected by immunoprecipitation.Results:Using the chemiluminescent detector molecule lucigenin to track intracellular superoxide production,we observed that primary S1pr3-/-macrophages produced fewer ROS in response to E.coli than the WT macrophages.Consistent with this finding,the NOX2 activity induced by E.coli was significantly lower in the S1pr3-/-macrophages than in the WT macrophages.Activation of NOX2 also controls the phagosomal pH.Therefore,we compared the pH values of E.coli-containing phagosomes isolated from S1pr3-/-and WT macrophages.The E.coli-containing phagosomes tended to acidify more rapidly in the S1pr3-/-macrophages than in the WT macrophages.After phagocytosis was initiated by adding 3-mm polystyrene beads coated with E.coli outer membrane extracts to primary macrophages,the phagosomes from S1pr3-/-macrophages displayed lower levels of lysosomal-associated membrane protein 1(a lysosomal marker)and early endosome antigen 1(an early endosomal marker that is critical for phagosome maturation)than the WT macrophages.Thirty minutes after the phagocytosis of beads coated with E.coli outer membrane extract,the accumulation of Vps34 was significantly reduced in the S1pr3-/-macrophages.Consistent with this finding,the amount of PtdIns(3)P was significantly lower in the S1pr3-/-macrophages than in the WT macrophages at 30 and 60 min after phagocytosis.Using a coimmunoprecipitation assay,we found that more heterotrimeric G proteins(Gai,Gaq,and Gal 2)coimmunoprecipitated with Vps34 in the phagosomes isolated from WT macrophages treated with beads coated with E.coli outer membrane extract.Administration of GPS-725.017 neither increased the survival rate nor decreased the local and systemic bacterial burden in macrophage-specific Vps34-/-mice subjected to E.coli sepsis.Conclusions:S1PR3 promotes the enrichment of Vps34 in phagosome,on the one hand,up-regulates the activity of phagosome NOX2 and promotes the removal of oxygen-dependent bacteria.On the other hand,the process of phagosome maturation is accelerated,and the removal of non-oxygen-dependent bacteria is promoted,thus participating in maintaining the immune homeostasis of sepsis.Part 4 S1PR3 expression levels and its correlation with severity of sepsisObjectives:Integrated with the above three parts' study,S1PR3 enhances the phagocytosis of macrophages,ultimately improving the outcome.We next to identify whether S1PR3 signaling may offer a promising therapeutic approach for the prevention and/or treatment of sepsis.S1PR3 expression levels and its correlation with severity of sepsis were determined in critical ill patients.The germicidal function of monocytes was also tested.Methods:From September 2016 to December 2016,44 patients were admitted to the intensive care unit of the First Affiliated Hospital,College of Medicine,Zhejiang University and were considered for inclusion in this study if they met the criteria for severe sepsis or septic shock.Exclusion criteria for the current study included the following:with human immunodeficiency virus infection,treatment with corticosteroids or chemotherapy.26 patients in the intensive care unit who did not show features of systemic inflammatory response syndrome or any evidence of infection and 31 Healthy volunteers were enrolled as nonseptic control patients.Clinical and demographic data were recorded within the first 24 h after diagnosis of severe sep'sis or septic shock by two senior intensivists.Sequential Organ Failure Assessment(SOFA)scores,Acute Physiologic and Chronic Health Evaluation II(APACHE II)scores,and 28-day mortalities were recorded for all patients.The PBMCs were isolated by Ficoll,and the levels of S1PR3 mRNA and human leukocyte antigen-DR(HLA-DR)were detected by quantitative PCR.The bactericidal effect of S1PR3 mRNA expression levels on E.coli was observed by fluorescence microscopy.Results:Compared with the healthy controls,the S1PR3 mRNA levels were significantly increased in ICU controls and patients with sepsis,and the ICU controls showed much higher S1PR3 mRNA levels than the patients with sepsis.Furthermore,the septic nonsurvivors had significantly decreased S1PR3 mRNA levels compared with the septic survivors.Moreover,the S1PR3 level was negatively associated with the SOFA score of the patients with sepsis.We found that the ICU controls had much higher HLA-DR expression levels than the patients with sepsis.We further divided the patients with sepsis into two groups according to the expression of HLA-DR on circulating monocytes.Interestingly,the S1PR3 levels were significantly reduced in patients with decreased expression of HLA-DR(<30%)compared with those with higher HLA-DR expression(>30%).In a subgroup of patients with sepsis,the S1PR3 level was significantly correlated with the bactericidal activity of the monocytes.Conclusions:In septic patients,lowered S1PR3 levels in PBMCs were positively correlated with the severity of sepsis.Interventions targeting S1PR3 signaling may serve as promising therapeutic approaches for sepsis.