The Regulatory Effect Of Cytochrome P450 1A1 On The Polarization And Phagocytosis Of Macrophages During Sepsis

Backgrounds:Sepsis is a systemic and excessive inflammatory reaction caused by various pathogenic bacteria,especially gram-negative bacteria,which enters the blood stream to grow and reproduce,and is often accompanied by severe complications such as sudden blood pressure drops,shiver,high fever and respiratory or(and)circulatory failure.Sepsis is one of the leading causes of death in intensive care unit,due to its diversity of causes,complexity of disease progression and uncertainty of pathogenic factors.It was believed that “cytokine storm” mediated by monocyte-macrophage system in the early-stage of sepsis was one of the most important factors that caused excessive inflammatory response and organ damage.Although numerous of studies investigated the regulatory effect of inflammatory cytokines in sepsis,the incidence and mortality of sepsis still remain high.Herein,we urgently need to find new targets to inhibit the excessive inflammatory response mediated by macrophage(Mj)and provide novel ideas and theoretical basis for the prevention and treatment of sepsis in the future.Cytochrome P450 1A1(CYP1A1)is a monooxygenase that mainly exists in the liver and intestine,and has the dual functions of cyclooxygenase and hydroxylase.Previous studies on CYP1A1 focused on its metabolic regulation of exogenous harmful substances.For example,CYP1A1 can metabolize dioxin,aflatoxin and other exogenous toxins to cytotoxic products,which may cause DNA damage and cancer.Recently,more and more studies investigated the regulatory effect of CYP1A1 on immunity.It has been shown that CYP1A1 knockout aggravated mice lung injury caused by hyperoxia,including edema,protein exudation and neutrophil infiltration.Another study reported that systematic overexpression of CYP1A1 increased the mortality of mice infected by Citrobacter,indicating that CYP1A1 plays an important role in regulating immune response.In addition to the systematic regulation,CYP1A1 can also affect the expression of inflammatory factors in different cells.It has been demonstrated that overexpression of CYP1A1 significantly reduced pro-inflammatory factors secreted by lipopolysaccharide(LPS)-induced bovine mammary epithelial cells,but the specific mechanism is unknown.It has been reported that pcb118 can up-regulate CYP1A1 by activating aromatic hydrocarbon receptor(Ah R)with a large number of pro-inflammatory factors release,such as tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6),by targeting mitogen activated protein kinase(MAPKs)pathway in rat thyroid frlt-5 cells.During the development of sepsis or inflammation,macrophage often transforms between type 1(M1)and type 2(M2).At the early-stage of inflammation,macrophage shifts to M1 type and promotes sterilization,while Interleukin-4(IL-4)is elevated at the mid-late-stage and shifts M1 to M2,which facilitates anti-inflammatory response.It has been reported that activation of signal transducer and activator of transcription 6(STAT6)augmented CYP1A1 and its hydroxylase-related metabolite expression,resulting in an increase of M2-related markers.However,the direct regulation of CYP1A1 on M2 or related transcriptional factors still remains unclear.As the first line of innate immune response,macrophage plays a critical role in the development of sepsis through its polarization and phagocytosis functions.Although the above studies have preliminarily revealed the relationship between CYP1A1 and immunity,its regulatory effect on macrophage polarization and phagocytosis during LPS or IL-4 stimulation,especially in sepsis,is still unknown.In this study,by using wild-type mouse peritoneal macrophage,Ah R knockout mouse peritoneal macrophage,mouse peripheral blood monocytes(m PBMCs),human peripheral blood monocytes(h PBMCs),CYP1A1-overexpressed and-silent mouse macrophage,we explored the underlying mechanisms.Mice macrophages were shifted to M1 type by using LPS,Escherichia.Coli(E.coli)or cecal ligation and puncture(CLP)challenge and shifted to M2 type by using IL-4.On the basis of determined mechanisms,we used related inhibitors and CYP1A1 knockout mice to verify these mechanisms in vivo and in vitro,respectively.Through the above methods and experiments,we confirmed the changes of CYP1A1 in overactivated macrophage,explored the relationship between macrophage CYP1A1 and polarization or phagocytosis and investigated the underlying mechanisms.Our study suggests that CYP1A1 can act as a novel target to reverse the development of sepsis.Methods:Part Ⅰ: Effect of CYP1A1 on macrophage polarization.1.Mice were intraperitoneally injected with LPS or E.coli to establish septic model,mice peritoneal macrophages were extracted and subjected to western blot(WB)for CYP1A1 protein level analysis;2.Mice peritoneal macrophages were extracted,cultured and purified in vitro,which were treated with LPS for type 1 polarization or treated with IL-4 for type 2 polarization.The treated cells were used for CYP1A1 protein and m RNA measurements by WB and q RT-PCR;3.CYP1A1 overexpression RAW264.7 cell line(CYP1A1/RAW)and negative control RAW264.7 cell line(NC/RAW)were established and treated with LPS or IL-4.The treated cells and supernatants were harvested for TNF-α and IL-6 protein and m RNA exam by ELISA and q RT-PCR.The type 2 marker,arginase-1(Arg-1),was analyzed by WB and q RT-PCR;4.The peritoneal macrophages of Ah R knockout mice were extracted and used for CYP1A1 protein knockdown,and then treated with LPS.Supernatants were harvested for TNF-α and IL-6 protein examination by ELISA;5.RAW264.7 cells were pre-treated with ellipticine or rhapontigenin(CYP1A1inhibitors)and treated with LPS.Supernatants were harvested for TNF-α and IL-6 protein examination by ELISA.Part 2: The mechanism of CYP1A1 in regulating macrophage polarization.1.CYP1A1/RAW and NC/RAW cells were treated with LPS and collected for mitogen-activated protein kinases(MAPKs),nuclear factor kappa-B(NFκB)and c-Jun N-terminal kinase(JNK)/ activator protein-1(AP-1)detection by WB analysis.IL-4-treated cells were subjected to janus kinase 1(JAK1)/STAT6 detection;2.JNK,AP-1,JAK1 and STAT6 were knocked out in both CYP1A1/RAW and NC/RAW cells.The modified cells were treated with LPS or IL-4.The cells and supernatants were harvested for TNF-α and IL-6 detection by ELISA.The protein levels of Arg-1,JNK/AP-1 and JAK1/STAT6 were measured by WB.The nuclear protein was used for DNA-binding activity analysis by electrophoretic mobility shift assay(EMSA);3.CYP1A1/RAW and NC/RAW cells were treated with LPS or IL-4.The supernatants were collected for 12(S)-HYDROXY-5(Z),8(Z),10(E),14(Z)-EICOSATETRAENOICACID(12(S)-HETE)and 15(S)-HYDROXY-5(Z),8(Z),11(Z),13(E)-EICOSATETRAENOI CACID(15(S)-HETE)detection,cells were subjected to CYP1A1 hydroxylase activity analysis;4.RAW264.7 cells were treated with 12(S)-HETE plus LPS or 15(S)-HETE plus IL-4.The treated cells and supernatants were used for TNF-α and IL-6 protein and m RNA exam by ELISA and q RT-PCR.Arg-1,JNK/AP-1 and JAK1/STAT6 were measured by WB;5.The 12(S)-HETE in CYP1A1/RAW and NC/RAW were blocked by 12(S)-HETE antibody following LPS treatment.TNF-α and IL-6 levels were analyzed in supernatants by ELISA.The phosphorylation activity of JNK/AP-1 was detected by WB.The nuclear protein was used for DNA-binding activity analysis by EMSA;6.CYP1A1/RAW and NC/RAW cells were treated with LPS or IL-4,m RNA level of12-lipoxynase(12-LOX)or 15-lipoxynase(15-LOX)was then measured.CYP1A1/RAW and NC/RAW were pre-treated with ML355(12-LOX inhibitor)or PD146176(15-LOX inhibitor)and then treated with LPS or IL-4,cells and supernatants were collected for CYP1A1 hydroxylase activity,Arg-1,TNF-α and IL-6 measurements.7.The hydroxylase activity of CYP1A1 was mutant in CYP1A1/RAW cells,which were then treated with LPS or IL-4.12(S)-HETE,15(S)-HETE,TNF-α and IL-6 levels were analyzed in supernatants by ELISA.Arg-1,JNK/AP-1 and JAK1/STAT6 were measured by WB.The nuclear protein was used for DNA-binding activity analysis by EMSA.Part Ⅲ: The effect of CYP1A1 in regulating phagocytosis of macrophage and its modulation in vivo.1.The mice peritoneal macrophages with CYP1A1 overexpression,CYP1A1 knockdown,12(S)-HETE treatment or rhapontigenin treatment were co-cultured with E.coli.The cells were collected for phagocytosis analysis and scavenger receptor A(SR-A)determination by q RT-PCR and WB.The function of SR-A was blocked by SR-A antibody in CYP1A1-knockdown macrophage following E.coli treatment.The treated cells were used for bacterial colony count.;2.Mice were intraperitoneally injected with E.coli to mimic sepsis.The PBMCs were isolated for CYP1A1 m RNA measurement by q RT-PCR.The activation of JNK/AP-1 was examined by laser confocal microscopy scanning in PBMCs and by WB in peritoneal macrophage;3.Mice were intraperitoneally pre-injected with CYP1A1 overexpressed peritoneal macrophage and CYP1A1 hydroxylase deficiency peritoneal macrophage,and then challenged by E.coli or CLP.The survival rates of mice were then monitored;4.Mice were intraperitoneally pre-injected with rhapontigenin and then challenged by E.coli or CLP.The survival rates of mice were then monitored;5.The IL-4 expression level was dynamically detected in mice PLF after LPS challenge.Mice were intraperitoneally injected with CYP1A1 overexpressed peritoneal macrophage at the peak of IL-4 release.The TNF-α level in mice PLF was measured;6.The peripheral blood from 30 septic patients and 30 healthy volunteers were isolated for plasma and PBMCs collection.The level of 12(S)-HETE in plasma was detected by ELISA.CYP1A1 m RNA level in PBMCs was determined by q RT-PCR.The correlation between septic patients’ Sequential Organ Failure Assessment(SOFA)scores and12(S)-HETE or CYP1A1 was also analyzed;7.CYP1A1 knockout mice and wild-type mice were injected with LPS for 15 h.The peritoneal lavage fluids were extracted for TNF-α and IL-6 protein analysis.The peritoneal macrophages were isolated for TNF-α and IL-6 m RNA determination.The lung tissues were harvested for overall observation and hematoxylin-eosin staining.Results:Part I: Effect of CYP1A1 on macrophage polarization.1.The protein level of CYP1A1 was increased in peritoneal macrophage of E.coli or LPS-challenged-mice with a time-dependent manner;2.Peritoneal macrophages were extracted and treated with LPS or IL-4.The protein and m RNA levels of CYP1A1 were significantly elevated by LPS or IL-4 treatment;3.CYP1A1 overexpression increased the protein expression of TNF-α,IL-6 and Arg-1in RAW264.7 cells in response to LPS or IL-4 treatment;4.Ah R knockout showed no effect on the reduction of TNF-α and IL-6 caused by CYP1A1 knockdown;5.Ellipticine or rhapontigenin treatment exhibited a decrease in TNF-α and IL-6 of overactivated macrophage.Part 2: The mechanism of CYP1A1 in regulating macrophage polarization.1.CYP1A1 overexpression elevated phosphorylation levels of JNK/AP-1 and JAK1/STAT6 in response to LPS and IL-4 treatments;2.Knocking out JNK/AP-1 and JAK1/STAT6 in CYP1A1-overexpressed RAW264.7cells significantly decreased the protein levels of TNF-α,IL-6 and Arg-1 which were induced by CYP1A1 overexpression in RAW264.7 cells;3.CYP1A1 overexpression increased CYP1A1 hydroxylase activity and LPS-induced12(S)-HETE secretion of RAW264.7 cell,as well as IL-4-induced 15(S)-HETE and CYP1A1 hydroxylase activity;4.LPS plus 12(S)-HETE treatment significantly increased TNF-α and IL-6 levels,as well as the activation of JNK/AP-1 pathway,in RAW264.7 cells,compared to that in LPS-treated group.IL-4 plus 15(S)-HETE treatment elevated Arg-1 expression and JAK1/STAT6 phosphorylation level in RAW264.7 cells,compared to that in IL-4-treated group;5.By using 12(S)-HETE antibody,the 12(S)-HETE was neutralized in LPS-induced CYP1A1/RAW cell,which resulted in a decrease of TNF-α and IL-6 augmented by CYP1A1up-regulation.12(S)-HETE antibody also decreased the activation of JNK/AP-1 pathway,as well as the DNA-binding activity of AP-1;6.CYP1A1 overexpression had no effect on expression of 12-LOX or 15-LOX.The inhibitor of 12-LOX or 15-LOX was unable to reverse the pro-inflammatory response caused by CYP1A1 overexpression;7.The deficiency of CYP1A1 hydroxylase activity significantly reduced CYP1A1overexpression-resulted up-regulation of 12(S)-HETE,15(S)-HETE,Arg-1,TNF-α and IL-6,and overactivation of JNK/AP-1 pathway and JAK1/STAT6 pathway in response to LPS or IL-4,respectively.Part Ⅲ: The effect of CYP1A1 in regulating phagocytosis of macrophage and its modulation in vivo.1.CYP1A1 overexpression impeded the phagocytosis function of macrophage by targeting SR-A,while CYP1A1 knockdown or rhapontigenin treatment augmented macrophage phagocytosis ability.12(S)-HETE treatment showed no effect on phagocytosis;2.The protein level of CYP1A1 was increased in PBMCs of E.coli-induced mice,while the activation levels of JNK/AP-1 were both elevated in peritoneal macrophage and PBMCs;3.The mortality of septic mice which were intraperitoneally transferred with CYP1A1-overexpressed peritoneal macrophage was increased,while CYP1A1-hydroxylase-deficiency macrophage transferring showed no effect on the mortality;4.Rhapontigenin treatment reduced the mortality of septic mice;5.The TNF-α level in PLF of mice which were intraperitoneally transferred with CYP1A1-overexpressed peritoneal macrophage was decreased at the late-stage of peritonitis;6.The levels of 12(S)-HETE(palsma)and CYP1A1(PBMCs)were increased in septic patients,which also have a positive correlation with SOFA score;7.CYP1A1 knockout reduced the expression levels of TNF-α and IL-6 in peritoneal lavage fluids and macrophages of LPS-induced mice,while the injury of lung tissue was augmented.Conclusions:1.LPS and IL-4 can both up-regulate CYP1A1 expression in mouse macrophage,while LPS-induced CYP1A1 expression was Ah R-independent.2.CYP1A1 overexpression enhanced JNK/AP-1 activation with an increase of TNF-αand IL-6,which was mediated by a CYP1A1-hydroxylase-metabolite,12(S)-HETE,during LPS-induced type 1 polarization(12-LOX-independent).CYP1A1 overexpression also enhanced JAK1/STAT6 activation with an increase of Arg-1,which was mediated by a CYP1A1-hydroxylase-metabolite,15(S)-HETE,during IL-4-induced type 2 polarization(15-LOX-independent).3.At the early-stage of sepsis(type 1 polarization),CYP1A1 augmented the secretion of pro-inflammatory cytokines of macrophage resulted in an increase of mortality of septic mice,while rhapontigenin(a CYP1A1 inhibitor)decreased the mortality of septic mice.CYP1A1 also elevated Arg-1 expression and promoted type 2 macrophage polarization at the late-stage of inflammation.Additionally,CYP1A1 controls macrophage phagocytosis ability by targeting SR-A.4.Consistent with the in vitro results,CYP1A1-12(S)-HETE-JNK/AP-1 axis also exists in septic patients.CYP1A1 and 12(S)-HETE levels are positively correlated with SOFA score.5.CYP1A1 knockout relieved mice peritonitis and enhanced lung injury,suggesting the regulation of CYP1A1 may be in a tissue-or cell type-specific manner.