The Roles Of ADSC-derived Exosomes In Macrophage Polarization And Diet-induced Obesity

BackgroundObesity is chronic metabolic disease caused by energy excess.There are two different types of adipose tissues in mammals,white adipose tissues(WAT)and brown adipose tissues(BAT).WAT inflammation characterized by infiltration of inflammatory cells can lead to insulin resistance and metabolic syndromes,thus increasing the risk of many life-threatening diseases such as type 2 diabetes,cardiovascular events,even cancer.Obesity and related pathologies are becoming global diseases,and have received more and more attention in the whole world.Therefore,the intervention for obesity is important to treat obesity-related diseases.Adipose macrophages are the important immune cells in the stromal-vascular cell fraction(SVF)of adipose tissues.Currently,macrophages are defined as two subtypes,"classically activated" M1 and "alternatively activated" M2.In lean WAT,alternatively activated M2 macrophages expressing interleukin(IL)-10 and arginase-1(Arg-1)are the main resident macrophages,which help to maintain the local immune and metabolic homeostasis.During obesity,M1 polarization in WAT contributes to WAT inflammation.M1 macrophages produce proinflammatory cytokines such as TNF-? and contribute to the development of insulin resistance.Adipose-derived stem cells(ADSCs)are mesenchymal stem cells(MSCs)that reside in SVF of adipose tissues.ADSCs possess multipotency to differentiate into different types of cells,such as adipocytes and osteoblasts.In our previous work,we have demonstrated that ADSCs from lean mice have the ability to polarize macrophages into anti-inflammatory M2-like phenotypes,thus remodeling the immune and metabolic homeostasis in WAT.But the precise modes and regulatory mechanisms remain to be unveiled.Exosomes are nanoscale vesicles secreted by cells,with a diameter range from 30 to 150 nm.Exosomes bear various biological molecules including proteins,lipids,RNAs,and transport these cargos between different cell types,thus mediating intercellular communication.However,there is no evidence showing the effects of ADSC-derived exosomes on macrophages.In this study,we established diet-induced obesity models in mice,and treated obese mice with ADSC-derived exosomes.We found that ADSC-derived exosomes efficiently attenuates WAT inflammation,insulin resistance and obesity development.In addition,we revealed that ADSC-derived exosomes induce the polarization of macrophages toward M2 phenotypes.Our finding may provide a new therapeutic approach for obesity and obesity-related diseases.Methods1.Isolation and culture of ADSCsADSCs were derived from lean mice.The epididymal fat pads were minced and digested with type I collagenase for 45 minutes at 37?.After filtration and centrifugation,SVF was obtained and cultured in completed Dulbecco's modified Eagle's medium(DMEM)containing 10%fetal bovine serum(FBS)and 5 ng/ml basic fibroblast growth factor.Adherent cells are ADSCs.The 4th passages of ADSCs were used for the experiments.2.Culture of peritoneal macrophagesMouse peritoneal macrophages were isolated from peritoneal cavity of C57BL/6 male mice,which were injected with 6%sterile starch solution 3 days ago.After overnight culture in DMEM supplemented with 10%FBS,the adhered cells were used as macrophages.3.Isolation of ADSC-derived exosomesADSCs were cultured with exosome-free culture medium.Supernatants from ADSCs were filtrated.The filtered supernatants were used to precipitate exosomes using Exoquick-TCTM.The exosomes were stored at-80?.Exosomes were identified by Zetasizer Nano ZSP,transmission electron microscopy,and western blot assay for specific markers.4.The effects of ADSC-derived exosomes on the polarization of macrophages in vitroMacrophages were co-cultured with ADSC-derived exosomes,and then the M1-related markers iNOS,TNF-?,IL-12 and M2-related markers Arg-1,IL-10 were detected using real-time quantitative PCR.Western-blot was used to detect the expression of Arg-1.5.The establishment of diet-induced obesity models in miceC57BL/6 male mice at the age of 8 weeks were fed on high-fat diet(HFD)(60%of total calories)for 20-26 weeks to induce obesity;mice fed on normal chow diet(NCD)were used as control.During the last 6-8 weeks of HFD feeding,mice were intraperitoneally administrated with ADSC-derived exosomes(30 ?g/mouse)once every 3 days.6.The effects of ADSC-derived exosomes on body weight in HFD-fed miceBody weight was recorded every 7 days,and food intake was evaluated during the intervention.At the end of intervention,the weight of adipose tissues was measured.7.Metabolic parameters measurementGlucose tolerance test(GTT)was performed in mice after an overnight fast,blood glucose levels were determined at 0,15,30,45,60,90,120 min after an intraperitoneal injection of glucose.The insulin tolerance test(ITT)was performed in mice after intraperitoneal administration of human insulin.Serum triglyceride(TG)and TC(total cholesterol)were also detected by Triglyceride or Cholesterol Test Kits.8.The influence of ADSC-derived exosomes on the beiging of WAT from HFD-fed miceThe epididymal adipose tissues and subcutaneus adipose tissues were isolated,and the total RNA were extracted using Trizol.The brown adipocyte and beige adipocyte markers(UCP1,PGC1?,TBX1,TMEM26)were detected by real-time quantitative PCR.Western-blot was used to detect the expression of UCP1.9.The effects of ADSC-derived exosomes on WAT inflammation in HFD-fed miceThe supernatants from cultured adipose explants were collected for the detection of TNF-? and IL-10.The mRNA levels of the M1/M2 relative cytokines(TNF-?,IL-6,IL-12 and Arg-1)were detected in SVF from WAT by real-time quantitative PCR.The expressions of F4/80 and Arg-1 in macrophages were detected in SVF by Flow Cytometry.Results1.ADSC-derived exosomes induce M2 polarizationMacrophages educated by ADSC-derived exosomes showed an increase in mRNA levels of M2-related Arg-1 and IL-10,while no significant changes in those of M1-related iNOS,TNF-? and IL-12.Meanwhile,ADSC-derived exosomes led to an increase of Arg-1 in macrophages.In addition,ADSC-derived exosomes mitigated the inflammatory response of macrophages stimulated by LPS plus IFN-?,as evidenced by a marked decrease in mRNA levels of iNOS,TNF-? and IL-12 in macrophages.Results from ELISA further confirmed that education by ADSC-derived exosomes markedly reduced the production of pro-inflammatory cytokines TNF-?and IL-12 from macrophages stimulated with LPS plus IFN-?.2.ADSC-derived exosomes attenuate diet-induced obesityHFD-fed mice were divided into two groups,one group treated with exosomes,and another group treated with normal saline(NS).NCD-fed mice treated with NS served as control group.Compared with NCD-fed mice,HFD-fed obese mice showed a significant increase of body weight and fat mass,while exosome treatment in HFD-fed mice caused a significant decrease in body weight and fat mass.3.ADSC-derived exosomes improve metabolic disorders in HFD-fed miceCompared with HFD-fed control mice,exosome-treated HFD-fed mice showed a significant decrease of glucose levels after glucose or insulin injection during GTT and ITT.In addition,multiple treatments with ADSC-derived exosomes caused a dramatic decrease in plasma levels of TG and TC in HFD-fed mice.4.ADSC-derived exosomes drive WAT beiging in HFD-fed miceAt the end of intervention of exosomes,epididymal visceral fat and inguinal subcutaneous fat were acquired.As compared to NCD feeding,long-term of HFD feeding markedly inhibited the expression of brown,beige adipocyte markers(UCP1,PGC1?,TMEM26,TBX1)in epididymal and inguinal WAT,while multiple deliveries of ADSC-derived exosomes significantly reversed their expression.The protein levels of UCP1 in epididymal and inguinal WAT were repressed by HFD feeding,while restored by exosome treatment.5.ADSC-derived exosomes inhibit WAT inflammation in HFD-fed miceAfter multiple deliveries of ADSC-derived exosomes into HFD-fed mice,the epididymal adipose explants secreted reduced levels of pro-inflammatory cytokine TNF-?,but enhanced levels of anti-inflammatory cytokine IL-10.The mRNA levels of TNF-?,IL-12 and IL-6 decreased significantly in SVF from HFD-fed mice after multiple treatments with ADSC-derived exosomes,but the mRNA level of Arg-1 was increased.Flow cytometry data demonstrated that the infiltration of F4/80+macrophages in WAT,which were higher in HFD-fed obese mice than those in NCD-fed lean mice,significantly decreased after exosome treatment.But Arg-1+macrophages in WAT from HFD-fed mice sginificantly increased after multiple deliveries of ADSC-derived exosomes.Conclusion1.ADSC-derived exosomes induce M2 polarization.2.ADSC-derived exosomes attenuate diet-induced obesity.3.ADSC-derived exosomes improve metabolic disorders in HFD-fed mice.4.ADSC-derived exosomes inhibit WAT inflammation in HFD-fed mice.