Role Of Formyl Peptide Receptor2 In Host Defense Against Bacterial Infection And Chorioamnionitis

Infiltration of inflammatory cells is an important feature of bacterial infectious diseases and plays an important role in pathogen removal and defense protection.Chemotaxis of inflammatory cells is mediated by G protein-coupled receptors(GPCRs).Formyl peptide receptor(FPR)is one of GPCRs,which includes FPR1,FPR2 and FPR3.FPR2(Fpr2 in mice)is widely concerned because it is expressed not only in inflammatory cells but also in many histiocyte and participates in many biological functions.The discovery of FPR2 expression in human placenta provides a possibility for the study of FPR2 in obstetrics.Chorioamnionitis(CAM)is the most common intrauterine infectious disease,which is the main cause of premature delivery,stillbirth,neonatal infection and septicemia.It is also related closely to fetal growth and development,occurrence and development of neonatal diseases and so on,which has drawn much attention by experts of obstetrics and pediatrics.Retrograde reproductive tract infection is an important infection route for CAM.In general,microorganisms existing in the reproductive tract of pregnant women do not cause amniotic membrane infection,but when the maternal immunity is low and the ability to remove pathogens is weakened,pathogens are easy to ascend and cause amniotic membrane and amniotic cavity infection,Escherichia coli(E.coli)is the main pathogenic bacteria which lead to CAM.The mechanism of FPR2 in E.coli induced infectious diseases will provide an evidence for the prevention and treatment of infectious diseases in pregnancy.In recent years,it has been attracted much attention since the exploration of the anti-inflammatory effect of FPR2 in combination with its ligands.Resolvin is an anti-inflammatory lipid medium derived from unsaturated fatty acids.Resolven D1(RvD1)is the ligand of FPR2.The elucidation of the role of FPR2/RvD1 in pregnancy infectious diseases will provide new ideas for the prevention and treatment of such diseases.This project carried out by using neutrophils derived from Fpr2 gene knockout mice,human embryonic kidney 293 cells(HEK 293 cells)transfected with Fpr2 gene and Fpr2 gene knockout infectious pregnant mice model,to clarify the function and mechanism of Fpr2 in neutrophil chemotaxis and killing E.coli,and explore the protective effect of Fpr2 on pregnancy infection.To collect the placental tissue samples,clarifying the difference of FPR2 expression in placenta tissue and Resolven D1 concentration in peripheral blood of CAM women,and proposed that FPR2 had effect on CAM,and the relative insufficiency of RvD1 may accelerate the development of inflammation.To confirm the anti-inflammation effect of RvD1 on trophoblast inflammation model induced by LPS through combining with FPR2 in vitro.To explore the therapeutic effect of RvD1/Fpr2 on infectious disease of pregnancy by observing the results of RvD1 application on infectious pregnant mice model.Part 1.The protective effect of Fpr2 in the early stage of E.coli infection.Materials and Methods1.Isolation and identification of mouse bone marrow neutrophils and intestinal mucosa E.coli.2.The chemotactic effect of E.coli supernatant on neutrophils derived from wild type(WT)and Fpr2 gene knockout(Fpr2-/-)mice and HEK293 cells transfected with Fpr2 was observed through chemotactic experiments.3.Weston Blotting was used to observe.the protein of MAPK signaling pathway of HEK293 cells and HEK293 cells transfected with Fpr2 after E.coli supernatant was applied.4.Bone marrow neutrophils derived from WT and Fpr2-/-mice were co-cultured with E.coli,then lysed and plated,and the phagocytosis and killing effects of Fpr2 on neutrophils were studied by counting colony colonies on agar plates.5.Hydrogen peroxide release was detected by Elisa to clarify the mechanism of neutrophil phagocytosis and killing bacteria affected by Fpr2.6.Neutrophils derived from WT and Fpr2-/-mice bone marrow were stimulated with the same concentration of E.coli for 1 hour to observe the formation of NETs.7.To establish a mouse peritonitis model(WT and Fpr2-/-)by intraperitoneal injection of inactivated E.coli,count the number of peritoneal neutrophils at different time by flow cytometry,and detect the expression of chemokines CXCL1 and CXCL2 in peritoneal fluid simultaneously.8.To establish a mouse bacteremia model(WT and Fpr2-/-)by intravenous injection of E.coli.Liver was collected 4 hours after injection to observe the accumulation of neutrophils in liver tissue.9.To establish the infection model of pregnant mice by intraperitoneal injection of LPS The preterm delivery time,number and weight of offspring,placental weight and congestion of WT and Fpr2-/-pregnant mice were observed.Meanwhile,peripheral plasma of pregnant mice was collected,and concentration of IL-1β,IL-6,IL-10 and TNFa were detected by Elisa.Results1.E.coli supernatant has chemotaxis effect on neutrophils derived from WT mouse bone marrow and HEK293 cells transfected with Fpr2,but significantly reduced on Fpr2-/-mouse neutrophils.2.E.coli supernatant can induce phosphorylation of MAP kinase in HEK293 cells transfected with Fpr2.3.Neutrophils derived from WT mice bone marrow can phagocytize and kill E.coli,while the ability of phagocytosis and killing of neutrophils from Fpr2-/-mice bone marrow decreased insignificantly accompanying by a decrease in hydrogen peroxide release.4.The ability of NETs formation of neutrophils derived from Fpr2-/-mouse decreased in vitro5.The mouse peritonitis model confirmed that the increasement of CXCL1 and CXCL2 lagged behind the recruitment of early neutrophils,and the deletion of Fpr2 resulted to the reduction of recruitment of mouse peritoneal neutrophils.6.The mouse bacteremia model confirmed that Fpr2 knockout reduced the aggregation of neutrophils in the liver.7.Fpr2-/-pregnant mice had more severe inflammatory reaction than WT mice with the same dose of LPS peritoneal injection,premature delivery time was advanced,weight of fetal mice and placenta decreased,and more obvious infarction in placenta was observed.Part 2.The changes of FPR2 in placenta of CAM women Materials and Methods1.Peripheral blood and placental tissues were collected derived from patients with PROM>28 weeks and<37 weeks,who were delivered by caesarean section in our hospital,and were diagnosed as CAM by placental pathology.The women without pregnancy complications such as hypertension and diabetes as control.Concentration of IL-1β,IL-6,IL-10,TNFa and RvDl in plasma were detected by Elisa.2.HE staining was used to observe neutrophil infiltration in CAM patients.3.The expression and localization of FPR2 and PPARy were observed by immunohistochemical staining.4.Weston Blotting was used to detect the changes of FPR2,PPARγ,NFκB-p65,IκB and RvDl synthetase 5-lipoxygenase(5-LOX)and 15-lipoxygenase(15-LOX)in placenta tissues of CAM patients and women giving birth normally.Results1.Concentration of IL-1β,IL-6,IL-10 and TNFa in CAM plasma increased while RvD1 decreased.2.Large amount of neutrophils infiltration was examined in decidua tissue.3.The expression of FPR2 and PPARy in the placenta of CAM patients increased,while no change of PPARγ in nucleus of tissue cells.There was no difference of RvD1 synthetase 15-LOX and 5-LOX.The expression of NF-KBp65 in nucleus of tissue cells of CAM patients increased and IκB decreased significantly.Part 3 The effect of RvDl/FPR2 on the biological function changes of human trophoblast cells induced by lipopolysaccharideMaterials and Methods1.HTR8-Svneo human trophoblast cells cultured in vitro were stimulated by LPS with a final concentration of lOug/mL.The experiment was divided into 5 groups:①control group,②LPS group,③LPS+RvD1 group,④WRW4(FPR2 antagonist)+LPS+RvD1 group,and ⑤GW9662(PPARy antagonist)+LPS+RvD1 group.1.The concentration of IL-1β,IL-6,TNFa and IL-10 in cell supernatant was detected by ELISA.2.The ability of cell migration,tube formation,proliferation and apoptosis were detected by cell scratch test,tube forming test,CCK8 test and flow cytometry.3.Weston Blotting was used to detect the cell expression of FPR2 and IκB in each group,and the nuclear translocation of PPARγ and NF-κBp65 were observed.4.The infection model of pregnant mouse was established by intraperitoneal injection of LPS.The effects of RvD1 on the inflammation of uterus,the proportion of premature delivery and the stillbirth of offspring were observed.Results1.HTR8-Svneo human trophoblast cells express TLR4,FPR2 and PPARy.2.RvD1/FPR2 saved the dysfunction of trophoblast cells induced by LPS,reduced the secretion of inflammatory factors such as IL-1β,IL-6,TNFa in cell supernatant.3.The improvement of trophoblast function by RvD1/FPR2 was related to the inhibition of NF-κB signaling pathway,which may be associated with the activation of PPARγ.4.RvD1 pretreatment alleviated LPS-induced inflammatory response in pregnant mice and increased the live rate of offspring.Conclusion1.Fpr2 plays an important protective role in the early stage of E.coli infection.2.The relative deficiency of RvD1 may accelerate the progress of inflammation in CAM patients.3.RvD1 can inhibit inflammation induced by LPS by binding with FPR2 expressed on human placental trophoblasts.4.RvD1/FPR2 can inhibit inflammation by activating PPARy and blocking NF-κB signal pathway.5.Preventive application of RvD1 can improve the outcome of premature delivery and stillbirth in pregnant mice caused by severe inflammation.