The Role And Mechanism Of Macrophage Polarization Regulated By IGF2BP2 In Inflammatory Diseases

Macrophages,as innate immune cells and antigen-presenting cells,are strategically distributed throughout the body.They participate in the regulation of host defense and immune homeostasis by ingesting and processing foreign substances,dead cells debris.Macrophages are highly heterogeneous cells with strong plasticity and can quickly transform its function according to organizational microenvironment changes,polarizing into different phenotypes:classical activation type(M1)and selective activation type(M2).Both M1 and M2 are closely related to inflammatory response.M1 macrophages are mainly involved in the pro-inflammatory response,and M2 macrophages are mainly involved in the anti-inflammatory response.Under physiological conditions,M1/M2 maintains a dynamic balance.Once the balance is impaired,diseases will occur.Therefore,adjusting M1/M2 balance of macrophages is an effective way to treat diseases.However,currently,little is known about the internal regulators of macrophages the polarization.N6-methyladenosine(m6A)modification is one of the most common internal modification of mRNA in eukaryotes and participates in developmental block and immune response of various biological processes.Insulin-like growth factor 2 messenger RNA-binding protein 2(IGF2BP2)is a highly conserved cancer-fetal RNAbinding protein that can regulate mRNA levels in many aspects such as localization,translation,and stability.Recent studies have found that IGF2BP2,as a m6A modified"readers",participates in the development of a variety of immune-related diseases,suggesting that it may regulate the body's immune homeostasis.Macrophage polarization state is a key factor in maintaining immune homeostasis.Does IGF2BP2 participate in the regulation of macrophage polarization?Does it participate in the maintenance of immune homeostasis by regulating macrophage polarization?It has not been reported.Based on this,we designed this project to study the regulatory effect and mechanism of IGF2BP2 on macrophage polarization and further study the role of IGF2BP2 in regulating macrophage polarization in inflammatory diseases.We first found that IGF2BP2 was involved in the regulation of macrophage polarization and clarified its molecular mechanism through a series of experiments:IGF2BP2-deleted macrophages exhibited enhanced M1 phenotype and promoted dextran sulfate sodium induced colitis development.However,the IGF2BP2-/macrophages were refractory to interleukin-4(IL4)induced M2 polarization and alleviated cockroach extract induced pulmonary allergic inflammation.MeRIP-qPCR,RIP-seq and RNA-pull down indicate that IGF2BP2 switched M1 macrophages to M2 activation by targeting tuberous sclerosis 1(Tsc1)via an m6A-dependent manner.Additionally,we also found that Signal Transducer and Activator of Transcription 6(STAT6)up-regulated the expression of IGF2BP2 through High Mobility Group AThook2(HMGA2),and dependent on m6A modification IGF2BP2 improved the stability of Peroxisome proliferator-activated receptor ?(Ppar?)and promoted the polarization of M2 macrophages.These findings fill the gap of IGF2BP2 in the regulation of macrophage polarization and provide a potential target of macrophages to treat inflammatory diseases.Part I The role of IGF2BP2 in macrophage polarizationObjective1.To analyze the expression of IGF2BP2 in macrophages2.To clarify the influence of IGF2BP2 on macrophage polarization in vivo and in vitroMethods1.The expression of IGF2BP2 in peritoneal macrophages and bone marrowinduced macrophages were measured by RT-qPCR and Western blot.2.The expression of surface markers on M1 and M2 macrophages were measured by RT-qPCR.3.The effect of IGF2BP2 on M2 macrophage polarization in vivo is tested by a Chitin injection mice model.Results1.The macrophages were stimulated with LPS(10Ong/ml)or IL4(20ng/ml).The results showed that after administration,the expression of Igf2bp2 in the macrophages was up-regulated.Meanwhile the level of IGF2BP2 protein was positively correlated with the time of LPS or IL4 stimulation.2.The expression of Il1?,Il12,Ifn-? and Tnf-? increased in IGF2BP2-/-M1 macrophages induced by LPS.However,the expressions of Argl,Tgf-?,Cd206,Ym1,and Fizz1 were reduced in IGF2BP2-/-M2 macrophages induced by IL4.3.Compared with WT macrophages,the expression of Arg1,Cd206,Fizz1,Pdl2,Tgf-? and Il10 in IGF2BP2-/-macrophages recruited by Chitin was significantly reduced.ConclusionsIGF2BP2 can promote the switch of M1 macrophages to M2 macrophages both in vivo and vitro.Part II The mechanism by which IGF2BP2 modulates macrophage polarizationObjective1.To identify the downstream targets genes of IGF2BP2 in macrophages2.To analyze the molecular mechanism how IGF2BP2 affects downstream targets genes3.To explore the mechanism of IGF2BP2-regulated metabolic reprogramming in macrophage polarizationMethods1.iRIP-seq method was used to screen out the downstream target genes of IGF2BP2 in bone marrow-induced macrophages.2.Determine the upstream and downstream effector of IGF2BP2 regulating macrophage polarization through RT-qPCR,Chip-qPCR and pharmacological inhibitor.3.Analyzed the phosphorylation level of the STAT1/MAPK/JNK/ERK/MEK/NFKB/AKT and TSC/mTORC1 in IGF2BP2-/-macrophages by Western blot.4.Through MeRIP-qPCR,RNA-pull down,RNA small interference and RIPqPCR,further confirmed that IGF2BP2 regulates the level of downstream target genes by m6A modification.Use RNA half-life experiment to detect the effect of IGF2BP2 on downstream target genes stability.5.Analyze the effect of metabolic reprogramming on macrophage polarization by detecting the levels of OCR(O2 consumption rate),SRC(Spare respiratory capacity)and fatty acid metabolism-related genes in bone marrow-induced macrophages.Results1.In M2 macrophages,blocking the JAK/STAT6 signaling pathway reduced the expression of Igf2bp2,but blocking the PI3K/mTOR signaling pathway has no effect on the expression of Igf2bp2.We found that STAT6 protein can bind to Hmga2.The level of PPARy was almost vanished,and fatty acid metabolism-related genes was inhibited in IGF2BP2-/-M2 macrophages.2.IGF2BP2 selectively binding to the methylated bait of Ppary and down regulated Mett114,the expression of Ppary was decreased.We also found that compared with WT,the RNA expression and stability of Ppary was reduced in IGF2BP2-/-macrophages.3.In M2 macrophages,depleting IGF2BP2 reduced the level of TSC1,and the mTORC1 signaling pathway was enhanced.Once using the pharmacological inhibitors of mTORCl,the expressions of Fizz1,Tgf-?,Yml and Cd206 were reversed significantly.Consistent with absence of IGF2BP2,in M2 macrophages,loss of Tsc1 reduced the level of OCR.4.In IGF2BP2-/-M1 macrophages,the expression of Tscl and Tsc2 were also reduced,and the mTORC1 signaling was enhanced,but inhibiting mTORC1 does not affect the expression of pro-inflammatory factors.At the same time,loss of IGF2BP2,promoted the MEK/ERK signaling pathway,but the AKT signaling was inhibited,however,the P38/JNK/NF-KB/STAT1 signaling was not changed.Further studies found that inhibition of MEK1/2 significantly reversed the expression of proinflammatory factors such as Ifn-?,Il1? and 1112.5.In macrophages,IGF2BP2 protein selectively bind to the methylated bait of Tscl,and by down regulating Mett114,the expression of Tsc1 was decreased.Compared with WT,the RNA expression and stability of Tsc1 were reduced in IGF2BP2-/macrophages.Conclusion1.Ppary and Tsc1 were the downstream target gene of IGF2BP2 in macrophage polarization.2.In M2 macrophages,STAT6 regulates the expression of IGF2BP2 by acting on Hmga2.At the same time,IGF2BP2 enhances the stability of Ppary by m6A modification,which in turn affects fatty acid metabolism.3.IGF2BP2 enhances the stability of Tsc1 mRNA by m6A modification.In M1 macrophages,IGF2BP2 regulates the MEK/ERK signaling by acting on Tsc1,thereby affecting the release of pro-inflammatory cytokines.In M2 macrophages,IGF2BP2 modulates the mTORC1 signaling by acting on Tsc1,affecting the metabolic reprogramming of macrophages,and then affecting the expressions of antiinflammatory factors.Part III Role of IGF2BP2-modulated macrophage polarization in inflammatory diseasesObjectiveIGF2BP2 regulates macrophage phenotypic activation.The mutual transformation between macrophage phenotypes plays a key role in the occurrence and development of diseases such as inflammatory bowel disease,pneumonia,tumors,and viral infections.Studies have reported that dysregulation of IGF2BP2 is related to certain diseases such as diabetes and tumors,but its role in inflammation regulation is poorly understood.Therefore,we explored whether IGF2BP2 participates in the regulation of inflammatory diseases by macrophage polarization.1.To analyze the effect of macrophage polarization mediated by IGF2BP2 in ulcerative colitis2.To analyze the effect of macrophage polarization mediated by IGF2BP2 in asthmaMethods1.The expression of IGF2BP2,CD68,F4/80,CD206 and iNOS in samples by immunofluorescence.2.2.5%DSS salt was used to induced colitis in IGF2BP2-/-and wild-type littermates,Mice were sacrificed and calculated the severity of colitis by inflammationrelated indicators.Furthermore,IGF2BP2 chimera mice were used to confirm whether myeloid-derived IGF2BP2 could improve the progress of ulcerative colitis.3.Female IGF2BP2 chimera mice were sensitized with intranasal delivery of cockroach extract at a concentration of 20 ?g per mouse induced asthma model.Analyze the regulatory effect of bone marrow-derived IGF2BP2 in asthma by HE staining,flow cytometry and RT-qPCR.Results1.IGF2BP2 and CD68 proteins have been detected in human colitis tissues,and IGF2BP2 protein was expressed in CD68+macrophages.In DSS-induced ulcerative colitis,mice lacking myeloid IGF2BP2 showed more severe ulcerative colitis.Phenotype with faster weight loss,shorter colon length,higher DAI score and histological score,more expression of Il1?,Il6,Il17,Ifn-? and Il10 and CD68+iNOS+macrophages.Myeloid-derived IGF2BP2 could improve the progress of ulcerative colitis.2.IGF2BP2 protein was expression in CD68+macrophages of human asthma samples.In CRE-induced asthma,mice containing myeloid-derived IGF2BP2 chimera have more severe inflammation,higher number of macrophages,lymphocytes,neutrophils and eosinophils in alveolar lavage fluid and more expression of Argl,Cd206,Fizz1,Ym1,Il10 and F4/80+CD206+macrophages in lung tissue.Conclusion1.In DSS-induced ulcerative colitis,loss of myeloid IGF2BP2 can promote the polarization of M1 macrophages and aggravate the progression of inflammation.2.In CRE-induced asthma,myeloid-derived IGF2BP2 can aggravate the occurrence and development of asthma by promoting the macrophage polarization to M2.