IL-27 Mediated Preterm Labor By Promoting Immuno-inflammation

Background and objective: Preterm labor（PL）is defined as the termination of pregnancy before 37 gestational weeks.It is a leading cause of infant morbidity and mortality.Immuno-inflammation is a critical trigger of preterm labor in the peripheral circulation and the maternal-fetal interface.Interleukin-27（IL-27）,a member of the IL-6/IL-12 family,is composed of IL-27p28 and EBI3 subunits.IL-27 receptor（IL-27R）is also a heterodimer composed of IL-27Rα subunit（IL-27Rα）and glycoprotein 130（gp130）,while IL-27Rα is unique to IL-27 R in the IL-6/IL-12 family.IL-27 could affect the differentiation,function,and infiltration of immune cells.It was found that the IL-27 could promote immuno-inflammation in various diseases.Our previous team-work had found that IL-27 protein level increased in the plasma of pregnant women with PL,which indicated that IL-27 was involved in PL.Bioinformatics analysis based on high-throughput sequencing technology can reveal the role and mechanism of molecules comprehensively.Therefore,we would investigate the role of IL-27 in regulating immuno-inflammation in peripheral blood and maternal-fetal interface in combination with bioinformatics analysis in PL.Methods:（1）The expression of IL-27 and its receptor in peripheral blood and at the maternal-fetal interface were investigated in preterm and term pregnancy by bioinformatics analysis and experiments.IL-27 related biological process was explored in peripheral blood to preliminarily determine whether it was related to immuno-inflammation of PL.PBMCs from pregnant women of term not in labor（TNL）were isolated and cultured in vitro.Using PBMCs stimulated by rhIL-27,the effects of rhIL-27 on PBMCs and the underlying mechanism were investigated.An indirect co-culture system of PBMCs and amniotic epithelial cell line（WISH）was established to detect whether rhIL-27 could promote the inflammatory effect of amniotic epithelial cells（WISH）by acting on PBMCs.（2）The expression of CD4+ T cells subgroups and Th1 cells infiltration-related molecules,IL-27 and its receptor in fetal membranes were compared between pregnant women of preterm labor and term labor by bioinformatics analysis and experiments.IL-27 related biological process was also investigated comprehensively.Amniotic epithelial cells（WISH）were treated by rhIL-27.The role of IL-27 in fetal membranes and the underlying mechanism were explored comprehensively.（3）Similarly,the expression of neutrophils at the maternal-fetal interface were compared between pregnant women of preterm pregnancy and term pregnancy by bioinformatics analysis and experiments.LPSinduced preterm labor mice model was established.Using IL-27Rα^-/-mice,neutrophils at the maternal-fetal interface between IL-27Rα^-/-mice and wild-type mice were compared.Results:（1）The expression of IL-27 and its receptor were higher in peripheral blood and chorion in preterm pregnancy than term pregnancy.In peripheral blood,IL-27 was mainly related to biological pathways associated with immuno-inflammation and pro-inflammatory cytokines.IL-27 could increase the expression of IL-27p28,EBI3 and IL-27Rα in PBMCs without affecting PBMCs’ proliferation and death.rhIL-27 could promote the expression of Th1 cells related molecules（T-bet,IFN-γ,ICAM-1）,and proinflammatory cytokines（IL-6 and IL-1β）in PBMCs,partially mediated by JAK2/STAT1 pathway.Furtherly,the expression of IL-6,IL-1β,and TNF-α in WISH cells were significantly enhanced by the conditional medium of IL-27-treated PBMCs.（2）The expression of infiltrating Th1 cells and related molecules as well as IL-27Rα were higher in human fetal membranes from preterm pregnancy than term pregnancy.In vitro,rhIL-27 could promote the expression of Th1 cells-infiltration related molecules in WISH cells through JAK2/STAT1/STAT3 signaling pathway.（3）The abundance of infiltrating neutrophils was higher at the maternal-fetal interface from preterm pregnancy than term pregnancy.Compared with PBS-treated mice,LPS-treated mice had more infiltrating neutrophils at the maternal-fetal interface than PBS-treated mice.Furthermore,LPS-induced IL-27Rα^-/-mice had fewer infiltrating neutrophils than LPS-induced WT mice.Conclusion:（1）In peripheral blood,IL-27 could activate PBMCs to upregulate the expression of Th1 cells related molecules and proinflammatory cytokines,partially through the JAK2/STAT1 pathway.Furtherly,IL-27 could enhance the inflammatory response of amniotic epithelial cells by acting on PBMCs.（2）At the maternal-fetal interface,IL-27 may promote Th1 cells infiltration in human FMs in PL,by promoting the expression of Th1 cellsinfiltration related molecules at least partly through JAK2/STAT1/STAT3 signaling pathway.Meanwhile,IL-27 could mediate neutrophils infiltration at the maternal-fetal interface in preterm pregnancy.In summary,IL-27 may be involved in PL by enhancing immunoinflammation in peripheral blood and maternal-fetal interface.IL-27 PROMOTED THE IMMUNO-INFLAMMATION OF PBMCS IN PRETERM LABORObjective: To investigate whether interleukin-27（IL-27）promotes the immuneinflammation of maternal peripheral blood mononuclear cells（PBMCs）and induces inflammatory response in amniotic epithelial cells in preterm labor（PL）.Methods:（1）The expression of IL-27 and its receptor in peripheral blood and tissues of maternal-fetal interface were analyzed by bioinformatics in preterm and term pregnancy.IL-27 related biological processes in maternal peripheral blood were investigated to determine the role of IL-27 in PL.The expression of specific transcription factors of CD4+ T cell subgroups and pro-inflammatory cytokines in maternal peripheral blood were also detected.（2）PBMCs were cultured and treated by rhIL-27 in vitro to detect whether IL-27 could promote Th1 cells related molecules and other inflammatory factors.（3）The phosphorylated and total protein of JAK2/STAT1 were detected in PBMCs stimulated by rhIL-27.AG490（JAK2 inhibitor）was added to PBMCs’ culture medium one hour before rhIL-27 to investigate the role of JAK2/STAT1 pathway in PBMCs under the stimulation of rhIL-27.（4）Amniotic epithelial cells（WISH）were stimulated with the conditioned medium of PBMCs,and the expression of IL-6,IL-1β and TNF-α were detected.Results:（1）The expression of IL-27 and its receptor were higher in peripheral blood and chorion in preterm pregnancy than term pregnancy.IL-27 was related to biological processes of immune-inflammation.The expression of Th1 cells related molecules and pro-inflammatory molecules in peripheral blood were higher in preterm pregnancy than term pregnancy.（2）rhIL-27 could promote the expression of Th1 cells related molecules,and proinflammatory cytokines in PBMCs and strengthen the positive feedback of its own.（3）IL-27 could increase the expression of phosphorylated JAK2/STAT1 in PBMCs.AG490（JAK2 inhibitor）could partially inhibit the pro-inflammatory effects of IL-27 on PBMCs.（4）IL-27 could increase the expression of IL-6,IL-1β and TNF-α in WISH cells by acting on PBMCs.Conclusion: IL-27 could activate PBMCs to upregulate the expression of Th1 cells related molecules and proinflammatory cytokines,partially through JAK2/STAT1 pathway.IL-27 could also strengthen the positive feedback of its own in PBMCs.Furtherly,IL-27 could amplify the inflammatory effects of amniotic epithelial cells by acting on PBMCs.IL-27 MEDIATED TH1 CELLS INFILTRATION AT THE MATERNAL-FETAL INTERFACE IN PRETERM LABORObjective: To identify the role of IL-27 on Th1 cells infiltration in human fetal membranes（FMs）and the underlying mechanisms in preterm labor.Methods:（1）The expression of Th1 cells,Th1 cells infiltration-related molecules,and IL-27Rα were studied in human fetal membranes from pregnant women with preterm labor and term labor by high throughput data and experiments.（2）IL-27 related genes,functional annotation,and enriched biological pathways were analyzed in preterm pregnancy and term pregnancy by high throughput data analysis and experiments.（3）Human amniotic cell cells（WISH）were treated by rhIL-27.The expression of Th1 cells related molecules were studied in human amniotic cell lineage（WISH）stimulated by rhIL-27.（4）The underlying mechanism of IL-27’s effects on Th1 cellsinfiltration was studied by high throughput data analysis and experiments.Results:（1）The level of Th1 cells,Th1 cells infiltration-related molecules,and IL-27Rα were higher in human FMs from PL group compared with TL group.（2）IL-27-correlated genes in preterm pregnancy were mainly related to immuno-inflammatory processes such as leukocyte degranulation,TNF-α signaling via NF-κB,and IFN-γ response.（3）rhIL-27 could up-regulate the expression of Th1 cells infiltrationrelated molecules in WISH cells.（4）IL-27 could increase the expression of phosphorylated JAK2/STAT1/STAT3 in WISH cells and AG-490（JAK2 specific inhibitor）could partly inhibit rhIL-27’s role.Conclusions: Our results suggested that IL-27 may promote Th1 cells infiltration in human FMs in PL,by promoting the expression of Th1 cells-infiltration related molecules at least partly through JAK2/STAT1/STAT3 signaling pathway.IL-27 MEDIATED NEUTROPHILS INFILTRATION AT THE MATERNAL-FETAL INTERFACE IN PRETERM LABORObjective: To reveal the role of IL-27 in neutrophils infiltration at the maternalfetal interface in preterm labor.Methods:（1）The expression of neutrophils and its relation with IL-27 were studied at the maternal-fetal interface from pregnant women with preterm labor and term labor by high throughput data analysis and experiments.（2）Using the LPS-induced preterm labor mice model,neutrophils infiltration was investigated at the maternal-fetal interface of IL-27Rα^-/-mice and WT mice.（3）The number of neutrophils in peripheral blood and their relationship with IL-27 were compared between preterm pregnancy and term pregnancy by bioinformatics analysis and experiments.Results:（1）The expression of neutrophils（Myeloperoxidase+,MPO+ cells）at the maternal-fetal interface in the PL group was more than the TL group.（2）Compared with PBS-treated mice,LPS-treated mice had increased infiltrating neutrophils at the maternal-fetal interface.Meanwhile,LPSinduced IL-27Rα^-/-mice had less neutrophil infiltration than LPS-induced WT mice.（3）The number of neutrophils in peripheral blood in preterm pregnancy was higher than those in term pregnancy,which was positively correlated with IL-27.Conclusion: IL-27 promoted neutrophils infiltration at the maternal-fetal interface in PL.