Study On The Regulatory Effect Of Macrophages On Phthalate Esters Induced Fatty Liver

Phthalates(PAEs)are widely used as industrial plasticizers,and their health hazards have caused increasing attentions.Diethylhexyl phthalate(DEHP)is the most common member of PAEs.Due to non-covalently bond to plastics,PAEs can easily migrate from plastics to environmental media.Therefore,humans can be exposed to PAEs via various manners.In addition,because plastics are widely used in food packaging materials,the oral route has become the main way for the general population to be exposed to DEHP.DEHP and some other phthalates have shown endocrine disrupting effects,which may interfere with the body’s metabolic homeostasis and cause various adverse health consequences such as non-alcoholic fatty liver(NAFLD).Although the effects of DEHP on human health have been studied for half a century,its toxic effect and mechanism on liver energy metabolism are still controversial.Previous studies mainly focused on the mechanism of DEHP on hepatocytes.It is believed that the peroxisome proliferators-activated receptor α(PPARα)of hepatocytes is the main target,but the activation of hepatocytes’ PPARα cannot fully explain DEHP-induced fatty liver.The reason for this complexity may be that in the occurrence and development of metabolic diseases indicated by system energy metabolism disorders,immune cells also play an important role in addition to stromal cells.Therefore,this research intends to use in vivolin vitro experiments,computational biology experiments,transcriptomics and metabolomics analysis,etc.to investigated cell-cell interactions based on cell-specific gene knockout mouse models and human-derived cell models.We explore the effect of cell-specific peroxisome proliferators-activated receptor γ(PPARγ,encoded by PPARG(human)/Pparg(mouse))on DEHP induced liver lipids metabolism disorder/fatty liver and the role of macrophages in regulating liver cell energy metabolism.In addition,we planned to establish a cell model for quickly screening out PAEs with similar effects and mechanisms to DEHP.Part One The role and mechanism of macrophages in phthalates-induced fatty liverObjective:To clarify the role and mechanism of macrophage differentiation/polarization in phthalates-induced fatty liver.Method:1.DEHP-induced fatty liver mice model.Male C57BL/6J mice,6-8 weeks old,were randomly divided into 4 groups,namely solvent control group and DEHP low,medium and high dose groups,each with 10 mice.DEHP low,medium and high dose groups were given 625,1250,and 2500 mg/kg bw of DEHP by gavage,respectively,for 28 consecutive days.The solvent control group(DEHP 0 mg/kg bw)was given 0.5%(wt/vol)carboxymethyl(CMC)by gavage.Animal were sacrificed at the endpoint of experiment.Plasma triglycerides(TG),cholesterol(CHO),cholinesterase(CHE),high-density lipoprotein cholesterol(HDL-C),low Low-density lipoprotein cholesterol(LDL-C),alkaline phosphatase(ALP)and Aspartate aminotransferase(AST)were detected via a biochemical analyzer.Oil red O staining was used to detect liver lipids;RNA sequencing and real-time quantitative PCR(RT-qPCR)were used to detect transcriptomic changes.2.DEHP-induced fatty liver model of hepatocyte-specific Pparg knockout mice.Male 6-8 week old C57BL/6J wild-type(WT)mice and hepatocyte-specific PPARy knockout(Hep-KO)mice were respectively divided into two groups.A total of 4 groups were obtained:wild-type control group(WT-CT,n=8),wild-type treated group(WT-DEHP,n=8),Hep-KO control group(Hep-KO-CT,n=5),Hep-KO treated group(Hep-KO-DEHP,n=5).The WT-DEHP and Hep-KO-DEHP groups were given 2500 mg/kg bw of DEHP by gavage for 28 consecutive days.0.5%(wt/vol)CMC was given to WT-CT and Hep-KO-CT via gavage.Animal were sacrificed and whole blood and liver were collected at the end point.Plasma TG,CHO,CHE,HDL-C,LDL-C and ALP were detected with the above method.Liver lipid accumulation and transcriptomic changes were investigated as above described.Liver protein expression of PPARγ,F4/80 and CLEC4F were determined via immunohistochemistry(IHC)or multicolor immunofluorescence(mIF).3.DEHP-induced fatty liver model of macrophage-specific Pparg knockout mice.Male 6-8 weeks old C57BL/6J WT mice and monocyte/macrophage-specific PPARy knockout(Mac-KO)mice were respectively divided into two groups,and a total of 4 group including WT-CT(n=8),WT-DEHP(n=8),Mac-KO control group(Mac-KO-CT,n=5),Mac-KO treated group(Mac-KO-DEHP,n=5)were obtained.The WT-DEHP and Mac-KO-DEHP groups were given 2500 mg/kg bw of DEHP by gavage for 28 consecutive days.0.5%(wt/vol)CMC was given to WT-CT and Mac-KO-CT groups via gavage.Animal were sacrificed and whole blood and liver were collected at the end point.Plasma TG,CHO,CHE,HDL-C,LDL-C and ALP were detect with the above method.Liver lipid accumulation and transcriptomic changes were investigated as above described.Liver protein expression of CLEC4F,IL-1RA and COX7A2L were determined using above methods.In addition,changes in hepatocytes,monocytes and macrophages within mouse liver were detected via flow cytometry.Changes in liver lipid metabolomics were determined via untargeted lipid metabolomics analysis.Cytokines level within mouse liver were detected via a magnetic bead-based multiple cytokine detection kit.4.In vitro cell test.Human monocytic THP-1 cells were differentiated into MO macrophages,and mono-(2-ethylhexyl)phthalate(MEHP)and/or PPAR agonists,antagonists,inhibitors were treated during M1/M2 polarization.Live cell immunofluorescence combined with high-content imaging are used to analyze CD209 and CD36 expression.Total RNA were collected for routine transcriptomics sequencing analysis.Cell supernatant was collect to detect cytokines level.Monocytes taken from the bone marrow of WT and Mac-KO mice were differentiated into bone-marrow derived macrophages(BMDM)and polarized into M1/M2 type macrophages.DEHP or MEHP treatments were added during the polarization process.Live cell immunofluorescence and high-content imaging were used to analyze the proteins expression of CD206 and CD69.HepG2 cells were treated with MEHP in the presence or absence of M2 type macrophages,or directly treated with IL-1RA in co-culture with MEHP.Fluorescent probes(Tetramethylrhodamine,ethyl ester,TMRE),mitochondrial superoxide indicator(mitoSOX),BODIPY 493/503 were used to detect mitochondrial membrane potential,mitochondrial reactive oxygen species and neutral lipid droplets(LD),respectively.Total RNA was collected for routine transcriptomics sequencing analysis.5.Computational biology experiments.The interaction of PPAR subtypes with DEHP and MEHP were analyzed using surface plasmon resonance(SPR).Molecular docking and molecular dynamics simulation were used to study the binding force of PPARα/γ and MEHP.Results:1.DEHP-induced fatty liver model in mice,DEHP treatment significantly increased liver weight and liver index in mice.Plasma TG were significantly reduced,while CHO,ALP,HDLC,LDLC were significantly increased.Liver lipid accumulation was seen in each dose group.DEHP significantly activates the PPAR signaling pathway and lipid metabolism-related pathways at the transcription level,and the expression of Pparg,Cd36,Fabp1 and other genes are significantly increased.2.DEHP induced fatty liver model in hepatocyte-specific Pparg knockout mice,DEHP treatment makes the liver weight,liver index,plasma TG,CHE,CHO and other indexes of WT and Hep-KO mice change to a similar degree.At the transcriptome level,DEHP treatment caused similar genetic changes in WT and Hep-KO mice,and both significantly activated the PPAR signaling pathway.The expression of PPARy in animals of both genotypes increased after DEHP treatment,and non-hepatocytes(e.g.macrophages)were higher than hepatocytes.DEHP treatment also depletes liver Kupffer cells.3.DEHP induced fatty liver model in macrophage-specific Pparg knockout mice.Compared with WT,after DEHP treatment,the liver index,plasma TG,ALP,CHE and other indexes of Mac-KO mice did not significantly change.DEHP did not cause liver lipid accumulation.DEHP treatment caused the macrophages phenotype switch in Mac-KO mouse liver,and caused increase of M2 type macrophages.DEHP decreased inflammatory monocytes while increased patrolling monocytes and Kupffer cells.Oxidative phosphorylation(OXPHOS),arachidonic acid(AA)metabolism and other pathways were significant changed by DEHP in Mac-KO mice.4.In vitro cell test.DEHP/MEHP treatment activated macrophages PPARαand PPARy,and inhibited the M2 polarization of human and bone marrow-derived macrophages.DEHP/MEHP treatment caused the accumulation and enhancement of LD in HepG2 cells,and activated lipid metabolism as well as PPAR signaling pathway.M2 macrophage co-treatment significantly reduced LD,inhibited mitochondrial oxidative metabolism and activated the above pathways.PPAR ligand significantly changed the release of IL-1RA in human M2 macrophages.And IL-1RA significantly inhibits the increase of LD,mitoSOX,TMRE in HepG2 cells caused by DEHP.5.Computational biology experiment.DEHP did not bind to PPARα,PPARδ or PPARγ.MEHP can bind to both PPARα and PPARγ,and has a stronger affinity with PPARγ.MEHP did not bind to PPARδ.Conclusion:The synergistic activation of macrophage-specific PPARa and PPARy inhibits intracellular lipid metabolism including AA metabolism,inhibits M2 polarization,and promotes DEHP-induced liver lipid metabolism disorders and fatty liver formation.Macrophage-specific Pparg knockout causes PPARαactivation,which improves DEHP-induced liver lipid metabolism disorders by enhancing AA metabolism and promoting the polarization of M2 macrophages,therefore blocking the formation of fatty liver.Part Two:Mechanism of Arachidonic Acid Metabolism in Regulating Macrophage PolarizationObjective:Clarify the role of AA metabolism in regulating the M2 polarization of macrophages and explore the relationship between tumor associated M2 macrophages and AA metabolism.Methods:Human THP-1 cells were differentiated into M0 macrophages and polarized to M1,M2 type macrophages.Transcriptomics and metabolomics were used to analyze the characteristics of the energy metabolism-related pathways of human M0,M1,M2 type macrophages.And the protein expression of key enzymes in AA metabolism were detected via live cell immunofluorescence and high content imaging system(HCIS).Then by using specific inhibitors,AA and its metabolites during the M2 polarization process,followed by detection of macrophages markers(CD209,IL-4,TARC,etc.),the polarization effects of these compounds were investigated.Subsequently,using Mac-KO mouse bone marrow-derived monocytes to obtain BMDM and explore whether PPARy are involved in the regulation of polarization by AA metabolism.Finally,mouse esophageal squamous cell carcinoma(ESCC)tissues were collected for RNA sequencing.The obtained mice ESCC transcriptomics data,combined with the transcriptomics data of human ESCC from The Cancer Genome Atlas(TCGA)were used for analyzing the correlation between M2 type tumor-associated macrophages and AA metabolism.Results:Transcriptomics analysis showed that lipid metabolism is closely related to macrophage polarization,and inhibition of lipid metabolism-related pathways such as fatty acid biosynthesis(FAS)and OXPHOS can directly affect M2 macrophage polarization.AA metabolism is significantly different in human-derived M0,M1,and M2 macrophages.Compared with M1 macrophages,this metabolic pathway is significantly up-regulated in M2 macrophages and inhibition of this metabolic pathway can block M2 polarization.During the polarization process,AA treatment significantly inhibited the expression of M2 type macrophage markers(CD209,CD206,IL-4,TARC,etc.),while prostaglandin E2(PGE2)treatment significantly promoted the above-mentioned markers expression.PGE2 treatment significantly enhanced the OXPHOS pathway at both gene and protein levels,and PGE2 treatment could inhibit the expression and activity of PPARy.In addition,there is a correlation between AA metabolism and M2 polarization in human and mouse ESCC.Conclusion:AA metabolism can directly promote the M2 polarization of macrophages through PGE2 biosynthesis,which involves the PPAR signaling pathway and OXPHOS.PGE2 activates PPAR signaling pathway and enhance OXPHOS.Part Three:Application of human-derived PPRE-macrophage model in the identification of phthalates hazardsObjective:Establish and verify a human-derived macrophage model containing PPAR response element(PPRE).Use this model to identify PAEs that regulate the M2 polarization of macrophages,and explore the potential mechanism of PPAR signaling pathway activation.Methods:Human macrophages,liver cancer cell line HepG2 cells and esophageal cancer cell line TE-1 cells were used to compare the differential response of PPARy ligands by determining gene or protein expression.The PPRE promoter-H2B-eGFP sequence was stably transferred into THP-1 cells to construct PPRE-eGFP-THP1 cells.And the obtained cells were validated by RNA sequencing and RT-qPCR.Using the successfully constructed PPRE-eGFP-THP1 cells to induce macrophages,i.e.PPRE-M0 cells and high-content imaging analysis,the PPRE response of PPARs specific activators,inhibitors,antagonists and DEHP,MEHP was determined.In parallel,live cell immunofluorescence was used to detect the protein level of CD36(PPARs target).DEHP and MEHP were added to macrophages during the M2 polarization process and their polarization effects were determined by CD209 protein expression.Furthermore,butyl benzyl phthalate(BBP),di-n-butyl phthalate(DBP),di-methyl phthalate(DMP),diisodecyl phthalate(DIDP),diisononyl phthalate(DINP),diisobutyl phthalate(DIBP),dicyclohexyl phthalate(DCHP)and diethyl phthalate(DEP)were applied on the above cell model for identifying the polarization of M2 macrophages and exploring potential PPAR signaling pathway activation.Results:The response of macrophages to PPARy ligands was much stronger than that of HepG2 or TE-1 cells.In macrophages induced by PPRE-eGFP-THP1 cells,PPARα/γ agonists and DEHP and MEHP can activate PPRE and promote the expression of CD36.PPARα/γ antagonists or inhibitors have the opposite effect.The activation of PPRE and the promotion of CD36 expression induced by DEHP and MEHP can be blocked by PPARα/γ inhibitors and antagonists.In addition,in the process of inducing M2 polarization,DEHP and MEHP can inhibit the expression of CD209,CD36 and LD level of M2 type macrophages.The tested PAEs(DBP,DEP,DMP,DINP,DIDP,DIBP and BBP)significantly inhibited the expression of CD209 during M2 polarization while DCHP did not significantly impact this process.Among them,DIBP,BBP and DIDP activated PPRE and promoted the expression of CD36.Conclusion:The macrophages induced by PPRE-eGFP-THP1 cells have the potential to recognize PPAR signaling pathway activation and the M2 polarization of macrophages.The tested PAEs significantly inhibited the M2 polarization of macrophages except for DCHP.DIBP,BBP and DIDP may function by activating the PPAR signaling pathway.SummaryDuring the formation of DEHP induced fatty liver in mice,MEHP not only activates hepatocytes’ PPARα,leading to accelerated lipid mobilization,but also activates macrophage-specific PPARy,inhibits macrophages intracellular AA metabolism,thereby inhibiting M2 polarization.After macrophage-specific Pparg is knocked out,MEHP activates macrophages PPARα,and promotes the production of PGE2 by enhancing AA metabolism,thereby promoting M2 polarization.M2 type macrophages inhibit hepatocytes’ OXPHOS in a paracrine manner through cytokines(such as IL-1RA),thereby reducing lipid accumulation in hepatocytes and inhibiting the formation of fatty liver.Similar to DEHP,DIBP,BBP and DIDP may inhibit the M2 polarization of macrophages by activating the PPAR signaling pathway,and have the potential to induce the formation of liver fatty liver.