Study On The Effect And Mechanism Of Macrophage ILF3 In Atherosclerotic Vascular Disease

1.BackgroundAccording to the statistics of the World Health Organization(World Health Organization,WHO)in 2016,17.9 million people are killed by cardiovascular events(Cardiovascular disease,CVD)every year worldwide,accounting for 31%of the total annual mortality.Atherosclerosis,the major cause of CVD,was initially thought to be merely a degenerative vascular disease,but it is now recognized as a chronic lipid-driven inflammatory disease.An important initial event of atherosclerosis is the infiltration and accumulation of cholesterol lipoproteins in the arterial wall.The subsequent modification of these lipoproteins induces monocytes to infiltrate the vascular wall and differentiate into inflammatory cells,which secrete inflammatory factors to promote the local inflammatory response.According to the latest 2017 CANTOS clinical trials(Canakinumab Anti-Inflammatory Thrombosis Outcomes Study),Canakinumab treatment significantly prevented adverse cardiac events compared with the placebo group for patients with MI and elevated hs-CRP.In addition,the latest data from the 2019 COLCOT trial(Colchicine Cardiovascular Outcomes Trial)also showed that the 0.5mg colchicine treatment group significantly reduced the risk of initial and total ischemic cardiovascular events in patients with recent myocardial infarction compared with placebo.As Canakinumab and colchicine are both direct anti-inflammatory agents,these two Randomized,Parallel and Double-blind clinical trials directly demonstrated that the development of atherosclerosis is closely related to inflammation.The immune cells in atherosclerotic plaque include monocytes/macrophages,dendritic cells,T cells and B cells,among which macrophages are the most important for the development and progression of atherosclerosis.Macrophages differentiate into foam cells via uptake of oxidized low-density lipoprotein(oxLDL)during the process of atherosclerosis.The lipid-laden macrophages possessing reduced mobility secret pro-inflammatory cytokines and regulate the production of collagen and proteinases such as matrix metalloproteinases to influence plaque stability and form the complex atherosclerotic plaques.Interleukin-enhanced binding factor 3(ILF3),also known as NF90/NF110,encodes a double-stranded RNA binding protein that binds to other proteins,mRNA,and non-coding RNAs to regulate gene expression and mRNA stability,ILFS is involved in a variety of cellular functions.At present,more studies have focused on tumor genome and epigenome association studies and found that ILF3 may play a role in early dyslipidemia,thrombogenesis,and stroke subtypes.Fenofibrate,a lipid-lowering drug,has been reported to reduce inflammation by inhibiting ILF3 activity.Besides,ILF3 has been reported to promote angiogenesis by regulating the stability of VEGF mRNA in 21 cases of breast cancer.Although there is evidence that ILF3 plays a role in vascular disease,the effect of ILF3 on atherosclerosis has not been proven.Our previous investigation proved that the serum ILF3 level of ACS patients was significantly increased.In order to further clarify the relationship between ILF3 and atherosclerosis,we conducted this experiment to explore the specific effect of ILF3 on atherosclerosis and the underlying mechanism.2.Objectives1.To clarify the effect of macrophage ILF3 on the progression of atherosclerosis;2.To explore the mechanism of macrophage ILF3 in the progression of atherosclerotic disease.3.Method3.1 Clinical atherosclerosis specimensSamples of coronary arteries,carotid arteries and atherosclerotic diseases were collected from healthy people in Shandong Red Cross Society,The research protocol was approved by the Ethics Committee of Qilu Hospital,and all study participants provided written informed consent.3.2 Experimental animals(1)Animal strain:1)Eight-week-old male ApoE-/-mice in C57/BL6J strain background were purchased from Beijing Viewsolid Biotechnology Company and derived from Jackson Lab.2)Macrophage ILF3 conditional knockout mice(ILF3flox/flox)were generated with the combined use of CRISPR-Cas9 and Cre/LoxP in Beijing Viewsolid Biotechnology(Beijing).3)Macrophage ILF3 conditional transgenic mice(ILF3Rosa/+)were purchased from Beijing Viewsolid Company.4)Lyz2-Cre mouse is provided by Beijing Viewsolid Company.(2)Construction of macrophage ILF3 specific expression mice1)The macrophage ILF3-specific knockout mouse(ILF3flox/flox Lyz2-Cre)was obtained by crossed ILF3flox/flox with tool mice Lyz2-Cre and named ILF3M'KO.2)Mice with ILF3ROSA/+and LYZ2-Cre were copulated to obtain macrophage ILF3-specific high expression mice(ILF3ROSA/+Lyz2-Cre),which were named ILF3M-TG.Genotypes of all the above mice were validated with DNA extracted from the tail of the mouse during reproduction.3.3 Establishment of atherosclerosis modelThe above macrophage ILF3-specific expression mice were mated with ApoE mice,and the ILF3M-KO/ApoE-/-and ILF3M-Tg/ApoE-/-model mice were obtained.After fed with the high-fat diet for 16 weeks,the aortic roots of mice were collected and made into sections to observe the formation of atherosclerotic plaques.3.4 Immunohistochemical staining of specimensThe aortic root sections of mice were stained with oil red O to observe the plaque area,and the collagen composition was detected by Sirus Red/Masson staining.The number of macrophages and smooth muscle cells in the sections was analyzed by staining of CD68 and ?-SMA immunohistochemical staining.The plaque vulnerability index was calculated based on the above staining data,and the vulnerability index was analyzed as follows:(macrophage positive area%+lipid positive area%)/(smooth muscle cell positive area%+collagen positive area%).The expression of inflammatory cytokines such as IL6 and TNF-? was detected by immunostaining.3.5 Abdominal macrophage extractionMice with different genotypes were intraperitoneally injected with 6%starch.Three days later,macrophages were extracted from mice by cell attachment method for subsequent cytological experiments.4.Results4.1 The expression of ILF3 increased with the severity of atherosclerosis(1)The ILF3 histochemical staining was performed on coronary arteries with different degrees of coronary stenosis in human specimens,and the detection showed that the expression level of ILF3 was significantly positively correlated with the degree of coronary stenosis.(2)ApoE-/-mice were fed with high fat for different periods to construct mouse models of atherosclerosis of different degrees.The expression of ILF3 was detected by immunohistochemical staining and Western blot,and it was found that the expression level of ILF3 increased significantly with the increase of high-fat feeding time.The above results indicated that the expression of ILF3 was significantly correlated with the degree of atherosclerosis in humans and animals,suggesting that the expression of ILF3 was involved in the progression of atherosclerosis.4.2 The expression of ILF3 in plaques showed obvious co-localization with macrophagesImmunofluorescence staining showed that ILF3 co-localized with macrophages in the plaque.In vitro experiments also proved that the expression level of ILF3 in macrophages was significantly increased after ox-LDL stimulation.4.3 Macrophage ILF3 promotes the progression of atherosclerosis and aggravates plaque instabilityAnalysis of the staining of the aortic root showed that macrophage ILF3-specific expression increased the severity of atherosclerosis and increased plaque instability,whereas knockdown of macrophage ILF3 significantly reversed this phenomenon.4.4 Combined transcriptome and proteome analysis suggested that macrophage ILF3 was closely related to lipid metabolism and inflammatory responseIntegrated transcriptome and proteome analysis showed that macrophage ILF3 is related to metabolic pathways such as lipid metabolism and inflammatory response,and is closely related to PI3K/Akt,MAPK and RAP1 signalling pathways.4.5 Macrophage ILF3 enhance the lipid-driven inflammation in plaquesThe deficiency of macrophage ILF3 can inhibit the formation of foam cells,while the overexpression of ILF3 can promote the formation of foam cells.Mouse aortic root staining confirmed that the high expression of ILF3 in macrophages could increase the expression of inflammatory factors in plaques,while the knockdown of ILF3 in macrophages could inhibit the production of inflammation.5.Conclusion(1)ILF3 expression is elevated during the progression of atherosclerotic disease,and it is closely related to macrophages.(2)The knockout of macrophage ILF3 can slow down the progression of atherosclerosis,while the high expression of macrophage ILF3 can worsen the progression of atherosclerotic disease.(3)Combined transcriptome and proteome analysis and plaque histochemical staining demonstrated that macrophage ILF3 is closely related to the lipid-driven inflammatory response in plaques.1.BackgroundAtherosclerosis is a chronic inflammatory disease with high morbidity and mortality,which is characterized by the accumulation of foam cells in the arterial wall.It has long been thought that the formation of foam cells is caused by excessive lipid intake and abnormal cholesterol metabolism.There is increasing evidence that inhibition of foam cell formation is a promising method to inhibit the development of atherosclerotic lesions.In addition to excessive foam cell accumulation,inflammation is another major factor in atherosclerotic lesions.In recent years,macrophage polarization has been shown to play a crucial role in the regulation of inflammatory response.In general,macrophages are mainly differentiated into two phenotypes:pro-inflammatory macrophages activated by the classical pathway(M1 macrophages),and anti-inflammatory macrophages activated by the non-classical pathway(M2 macrophages).Recent data suggest that M2-type macrophages play a role in atherosclerotic regression in a mouse model.The presence of M2-type macrophages in human plaques may lead to plaque regression.Besides,experimental atherosclerosis models in mice have also shown decreased macrophages and enrichment of M2-type macrophages during plaque regression.Targeting inhibition of macrophage M1-type polarization is considered to be a feasible way to prevent the development of atherosclerosis.Our previous results demonstrated that macrophage ILF3 mediated the inflammatory microenvironment in plaque to participate in the progression of atherosclerotic plaques,but the specific mechanism remains unclear.Therefore,this study used the integrated transcriptome-proteomics and immunoprecipitation-mass spectrometry combined analysis to explore the mechanism of effect of macrophages ILF3 on inflammation as well as the specific mechanism for macrophage.Polarization.In the present study,we provide the experimental support for possible atherosclerosis therapeutic targets for clinical treatment.2.Objectives(1)To explore the mechanism of macrophage ILF3 affecting plaque instability;(2)To elucidate the mechanism of macrophage ILF3 on inflammatory response in plaques;(3)To reveal the molecular mechanism of macrophage ILF3 affecting the polarization of macrophages in plaques.3.Method3.1 Isolation and culture of macrophagesAfter intraperitoneal injection of 6%starch aseptic suspension,peritoneal cells were collected with phosphate buffer saline(PBS)and cultured in RPMI medium supplemented with 10%bovine serum,100 U/mL streptomycin and 100 U/mL penicillin.The peritoneal macrophages collected were subsequently used for lipid phagocytosis assay,flow cytometry,RT-PCR,or immunoprecipitation mass spectrometry.3.2 RNA immunoprecipitation(RIP)RIP detection was performed according to the Magna RIP RNA-Binding Protein Immunoprecipitation Kit(Millipore,Billerica,MA,USA).mRNA was precipitated with antibodies including anti-ILF3 antibody(ProteinTech,Chicago,IL,USA)and IgG antibody L.Finally,qRT-PCR was used for analysis,and the results showed the multiple changes of target mRNA between the immunoprecipitation group and the INPUT group.3.3 mRNA stability analysis and qRT-PCRMacrophages were treated with actinomycin D(10 mg/mL)for 0,2,4,8,12 and 24 h.The sample was suspended in Trizol reagent(Invitrogen#15596018).Total RNA was isolated from the cells according to the instructions for the RNEasy Mini kit(Qiagen,Hilden,Germany).Total RNA was retrotranscribed using the PrimeScript RT reagent kit with gDNA erase(Perfect Real Time)(Takara,Japan)and qRT-PCR was performed using LightCycler 480 SYBR Green I Master(Roche Life Science,NSW,Australia).The expression of each transcript was normalized using the control gene ?-actin to show changes in expression relative to the control sample.3.4 Western blotProteins from tissue samples and cells were extracted,and the protein concentrations were determined by BCA method.The protein expression levels were detected by SDS-PAGE electrophoresis and corresponding antibody incubation.3.5 Flow cytometryAfter the peritoneal macrophages were treated with 50 ?g/mL oxLDL,the cells were labelled with markers of the M1 and M2 subtypes,and the labelled cells were cleaned,collected and analyzed by FACS flow cytometry system.3.6 Combined immunoprecipitation-mass spectrometry analysisAs previously described,macrophages were isolated from the abdominal cavity of C57BL/6 mice.Incubate in 1 mL medium with or without oxLDL(50 ?g/mL)for 24 h.Immunoprecipitation was performed using an immunoprecipitation kit.The gel blocks were collected by Western-blot analysis for rustic analysis.3.7 Statistical DataStatistical analysis was performed using GraphPad Prism 8(GraphPad Software),R v3.5.0(R Foundation for Statistical Computing,Vienna,Austria)and the GGplot2 Software package 9,10.After verifying the normal distribution of the data,t-test was performed on the two groups of data,and Tukey's Post Test was performed on three or more groups to evaluate the statistical differences.For data with two independent variables,two-way ANOVA and Bonferroni posttest were used.All tests were bilateral,and P<0.05 was considered statistically significant.4.Results4.1 Combined transcriptome and proteome analysis suggested that ILF3 in macrophages was closely related to lipid driven inflammatory responseIntegrated transcriptome-proteome analysis of macrophages confirmed that ILF3 is related to metabolic pathways such as fat digestion and absorption and inflammatory response,and is closely related to PI3K/Akt,MAPK and RAP1 signalling pathways.4.2 Inhibition of macrophage ILF3 effectively alleviates the formation of foam cells in plaquesIn animal experiments,serum from different groups of mice fed a high-fat diet for 16 weeks was detected.THE Macrophage ILF3 deficiency group had reduced serum cholesterol(CHO)and low-density lipoprotein(LDL)levels compared with control mice,while higher concentrations of CHO and LDL were found in mice with ILF3 overexpression.Moreover,we used Oil-red O staining to analyze the foam cell formation in different groups and verified that the ox-LDL uptake ability of macrophages was significantly decreased after ILF3 deletion,while the ox-LDL uptake of macrophages with overexpression of ILF3 was significantly increased compared with WT.4.2 Macrophage type ILF3 interferes with macrophage polarization and affects plaque inflammatory microenvironmentILF3 promotes the transformation of macrophages to pro-inflammatory macrophages(M1),and the lack of ILF3 in macrophages can significantly weaken the tendency of macrophages to M1 type polarization.At the same time,the change of ILF3 expression in macrophages can affect the inflammatory environment in plaques--the loss of ILF3 in macrophages can reduce the release of inflammatory factors such as interleukin-6(IL-6)and tumor necrosis factor ?(TNF?)in mouse plaques,while the overexpression of ILF3 in macrophages can promote the inflammatory response in mouse plaques.4.3 Macrophage ILF3 regulates the lipid-mediated inflammatory environment through the ILF3-RAP1A/ERK/AP-1 pathwayUsing IP-MS analysis of peritoneal macrophages,we confirmed that ILF3 specifically interacts with RAP1A under ox-LDL treatment and there is positive feedback between ILF3 and RAP1A.We also demonstrate for the first time that positive feedback between ILF3-RAP1A promotes the activation of the MAPK pathway and enhances the activity of c-fos and c-jun(two subunits of activating protein-1[AP-1]),thereby promoting inflammatory responses.4.4 Macrophage ILF3 affects macrophage polarization by regulating the stability of Argl mRNAThe interaction between ILF3 and Argl mRNA was confirmed by RNA immunoprecipitation.RIP-PCR analysis showed that ox-LDL could promote the binding of ILF3 to Argl mRNA.Moreover,the binding of ILF3 to Argl mRNA can promote the degradation of Argl mRNA in macrophages.5.Conclusion(1)It is revealed that the loss of ILF3 in macrophages inhibits the formation of foam cells;(2)We elucidate that macrophage ILF3 can promote the polarization of macrophages in plaques to Ml-type macrophages;(2)It was revealed that macrophage ILF3 affects the inflammatory response in macrophages through the ILF3-RAP1A/ERK/AP-1 pathway and the stability of Arg1 mRNA.