Font Size: a A A

CX3CR1hi Macrophages Relieve Adipose-derived Stem Cell Senescence To Sustain Visceral Adipose Tissue Homeostasis

Posted on:2024-02-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z X ZhouFull Text:PDF
GTID:1524307202994199Subject:Immunology
Abstract/Summary:PDF Full Text Request
BackgroundAdipose tissue is an energy storage and endocrine organ that regulates energy balance.According to its function,adipose tissue can be divided into white adipose tissue(WAT),which stores energy,and brown adipose tissue(BAT),which consumes energy in the form of heat.Depending on the anatomical location,WAT can be divided into subcutaneous adipose tissue(SAT)distributed throughout the body and visceral adipose tissue(VAT)around the internal organs.In the case of obesity,WAT,especially VAT,stores excess energy in the form of excessive triglycerides,leading to its pathological expansion and dysfunction,inducing adipose tissue inflammation,insulin resistance and metabolic disorders,thereby increasing the risk of diabetes,non-alcoholic fatty liver disease,cardiovascular disease and even cancer.Therefore,maintaining VAT homeostasis is a key link to maintain body energy balance and alleviate obesity-related metabolic disorders.Adipose tissue stem cells(ASC)are precursors of adipose cells that exist in the stromal vascular fraction(SVF)of adipose tissue.It has the potential of homing,proliferation and self-renewal.Under specific conditions in vitro,ASC can differentiate into adipocytes,chondrocytes,osteoblasts,cardiomyocytes and muscle cells.Its proliferation and adipogenesis differentiation potential is a prerequisite for maintaining the adaptive proliferation of adipocytes and preventing pathological hypertrophy.ASC plays an immunomodulatory role in adipose tissue and can promote the polarization of M2-type macrophages in adipose tissue.In addition,ASC plays an important role in the treatment of inflammatory diseases such as colitis and sepsis by inhibiting Th1-driven autoimmune and inflammatory responses,inducing Treg activation and secretion of IL-10 anti-inflammatory factor.In the adipose tissue of mice with metabolic disorders induced by obesity,especially VAT,the accumulation of ASC,adipocytes and T cells with senescence characteristics is often increased.However,ASC from obese individual or mice significantly weakened its proliferation and differentiation ability,resulting in adipose tissue dysfunction.Therefore,maintaining the function of ASC is essential for maintaining metabolic homeostasis in adipose tissue.There are many innate immune cells in adipose tissue SVF,such as macrophages,dendritic cells and eosinophils.Macrophages play an important regulatory role in adipose tissue.Adipose tissue macrophages can be divided into M2 anti-inflammatory macrophages and M1 pro-inflammatory macrophages.In addition to regulating inflammation,adipose tissue macrophages have different functions in obesity.For example,macrophages involved in the formation of " Crown-like structures" in adipose tissue of obese mice can recruit ASC to promote its adipogenesis differentiation and participate in the structural remodeling of adipose tissue.In high-fat diet-induced VAT in obese mice,macrophages are involved in regulating the adaptive immune response in adipose tissue,promoting the production and maintenance of memory T cells by promoting the proliferation of antigen-specific CD4+T cells and the production of IFN-γ.With further research,more subtypes of macrophages were found in adipose tissue under different immune microenvironments.By RNA single-cell sequencing,Chakarov et al.discovered two new unique subpopulations of Iacrophages,CX3CR1hi and CX3CR1lo,in adipose tissue,but their specific roles were not clear.CX3CR1+ macrophages have been found in a variety of tissues and play different roles.In the central nervous system,CX3CR1 is considered as a specific marker of microglia,which plays an anti-inflammatory and neuroprotective role.In neurodegenerative diseases,overactivated CX3CR1+ macrophages exacerbate the inflammatory response.In cardiovascular,CX3CR1+ macrophages are considered to be heart-resident macrophages,which can play a cardiomyocyte protective role during cardiac stress overload.In the liver,CX3CR1+macrophages play a role in alleviating liver inflammation and fibrosis in models of live inflammation.In the gut,CX3CR1+ macrophages can induce IL-10 production by intestinal flora and inhibit Thl cell activation in the late stage of colitis treatment,thus exerting antiinflammatory effects.In addition,in some tissue injury or tumor models,CX3CR1+macrophages also showed the ability to promote stem cell proliferation and repair or promote tumor cell growth.However,the function of CX3CR1+ macrophages in adipose tissue and the related metabolic regulatory mechanisms are still unclear and need to be further studied.The purpose of this study was to explore the regulatory function of CX3CR1+macrophages in VAT and reveal the role of CX3CR1+ macrophages and ASC.The aim is to find the intervention strategies to alleviate obesity-related metabolic disorders and provide valuable theoretical and experimental basis for the prevention and treatment of obesity-related diseases.Methods1.To study the recruitment of macrophages in VAT(1)The number of macrophages in mouse adipose tissue was detected by flow cytometry and immunofluorescence.(2)The expressions of F4/80 and MCP-1 in adipose tissue were analyzed by GEO database and quantitative PCR.(3)Using MCP-1 neutralizing antibodies and CCR2 inhibitors,the effect and mechanism of recruitment of macrophages by ASC via the MCP-1/CCR2 axis were verified by chemotactic and in vivo experiments.2.To study the effects of macrophage deletion on VAT and metabolic status of shortterm high diet mice(SHFD)(1)The changes in body weight and glucose tolerance of mice were detected by macrophage deletion experiment,and the effects of macrophage clearance on metabolic indexes of SHFD mice were evaluated.(2)H&E staining of adipose tissue,quantitative PCR,β-galactosidase activity staining were used to evaluate the effect of macrophage deletion on VAT function in s SHFD mice.(3)ASC senescence and the number of CX3CR1hi macrophages in VAT after macrophage deletion were detected by flow cytometry,immunofluorescence staining of adipose tissue and immunohistochemistry.3.The role of CX3CR1hi macrophages and ASC was studied by cell experiment(1)Through Transwell co-culture experiment of macrophages and ASC,western blot,quantitative PCR,β-galactosidase activity staining and other techniques were used to explore the anti-senescence effect of CX3CR1hi macrophages and the molding effect of ASC.(2)The phenotypic changes of macrophages in the co-culture system were detected by quantitative PCR and flow cytometry using exosomes derived from ASC and GW4869,an inhibitor of exosome secretion.(3)Using the small interference of Cx3CR1 and Argl(arginase 1),quantitative PCR and western-blot were used to detect the effect of Cx3CR1 and Argl gene knocked out macrophages on ASC senescence in co-culture system.(4)Mouse peritoneal macrophages derived from WT,Cx3CR1-/-and Cx3CR1+/-were treated with exosomes,calcium inhibitors EGTA and BAPTA,and a cytotropic calcium probe Fluo-4AM.The molecular mechanism of CX3CR1 regulation of Argl was detected by western blot,quantitative PCR,flow cytometry and laser confocal technique.(5)GC7,an eIF5A small interference and eIF5A hypusination modified inhibitor,was used in the co-culture system to study the molecular mechanism of CX3CR1hi macrophages inhibiting ASC senescence by western-blot and β-galactosidase activity.4.To study the effects of transferring CX3CR1hi macrophages on VAT and metabolic status of long-term high-fat mice(LHFD)(1)By transferring CX3CR1hi macrophages,in vivo imaging system,flow cytometry,immunofluorescence,glucose tolerance test and insulin tolerance test were used to detect the VAT function and body metabolism indexes of LHFD mice.(2)The effect of CX3CR1hi macrophages on ASC senescence was detected by immunofluorescence,immunohistochemistry and flow cytometry.(3)Quantitative PCR,western blot,small animal metabolism monitoring and other technologies were used to detect gene expression,anal rectal temperature,O2 consumption,CO2 production and heat production in VAT,and to evaluate the effects of CX3CR1hi macrophages on adipose tissue function and energy consumption in LHFD mice.(4)LHFD mice were transferred into Cx3Cr1-/-and CXxCr1+/-macrophages by glucose tolerance test,insulin tolerance test,adipose-tissue β-galactosidase activity staining,immunofluorescence and flow cytometry.The metabolic indexes,ASC senescence in VAT and the number of CX3CR1hi macrophages were detected.Results1.Increased infiltration of macrophages in VAT(1)Macrophage infiltration in mouse VAT was detected by flow cytometry,and the results showed that macrophage infiltration increased in VAT;Immunofluorescence results showed that VAT caused macrophage infiltration through recruitment chemotaxis.(2)GEO database and quantitative PCR showed that the expression of F4/80 in VAT was correlated with MCP-1 expressed in ASC.(3)Chemotactic experiment results showed that blocking the MCP-1/CCR2 axis in vitro resulted in a decrease in the recruitment of chemotactic macrophages by ASC;In vivo injection of CCR2 inhibitors blocked the MCP-1/CCR2 axis,and flow cytometry showed a decrease in the number of CCR2-dependent CX3CR1hi macrophages in VAT.2.Macrophage deletion leads to VAT senescence and metabolic disorders in SHFD mice(1)Weight changes and glucose tolerance experiments in mice showed that macrophage deletion led to weight loss,impaired glucose tolerance and metabolic disorders in SHFD mice.(2)H&E staining of adipose tissue showed that macrophage deletion led to hypertrophy of adipocytes in SHFD mice VAT;The results of β-galactosidase activity staining showed that VAT senescence was caused by macrophage deletion.Quantitative PCR results showed that macrophage deletion led to the downregulation of lipid differentiation related gene expression in SHFD mice VAT.(3)The results of immunofluorescence staining of adipose tissue showed that macrophage deletion led to ASC senescence;Flow cytometry results showed that the number of ASC and CX3CR1hi macrophages decreased due to macrophage deletion.Immunohistochemical results showed that CX3CR1 expression decreased in VAT sections after macrophage deletion.3.CX3CR1hi macrophages inhibit ASC senescence through Argl-eIF5A hypusination axis(1)After Transwell co-culture of macrophages and ASC,western-blot and βgalactosidase activity staining results showed that co-culture of macrophages alleviated ASC senescence;Quantitative PCR results showed that ASC promoted macrophages to CX3CR1hi Arg1hi phenotype.(2)After stimulation of macrophages by ASC derived-exosomes,quantitative PCR and flow cytometry results showed that exosomes promoted the expression of CX3CR1 and Argl in macrophages;After the addition of exosome inhibitors in the co-culture system,ASC could not up-regulate the expression of CX3CR1 and Argl in macrophages,and the results of western-blot and β-galactosidase activity staining showed that the effect of macrophages on ASC senescence disappeared.(3)Quantitative PCR and western-blot results showed that macrophages with Cx3cr1 and Argl gene knocked out in co-culture system could not inhibit ASC senescence;Quantitative PCR results showed that the expression of Argl in macrophages decreased after CX3CR1 knockout.(4)Western-blot and quantitative PCR results showed that the expression of p-Stat3 and Argl in Cx3cr1-knocked out macrophages decreased;western-blot and flow cytometry results showed that exosome stimulation could not improve the expression of Argl in Cx3cr1knocked macrophages.Flow cytometry and confocal laser results showed reduced Ca2+content in Cx3cr1-knocked out macrophages.(5)Western-blot and β-galactosidase activity staining results showed that macrophage co-culture promoted eIF5A hypusination of ASC,and increased the expression of mitochondrial and lysosomal related proteins;After GC7 and small interference of eIF5A inhibited eIF5A hypusination in ASC,western-blot results showed that macrophage co-culture lost the effect of inhibiting ASC senescence.4.Transferring CX3CRhi macrophages can inhibit VAT senescence of LHFD mice and alleviate metabolic disorders(1)in vivo imaging system and flow cytometry results showed that transferred CX3CR1hi macrophages were mainly infiltrated in VAT;The results of glucose tolerance test and insulin tolerance test showed that transferred CX3CR1hi macrophage promoted glucose and insulin sensitivity in LHFD mice.(2)H&E staining of adipose tissue showed that the transfer of CX3CR1hi macrophages alleviated the adipocyte hypertrophy caused by LHFD;Immunofluorescence results showed that CX3CR1hi macrophages inhibited ASC senescence in VAT.Flow cytometry results showed that CX3CR1hi macrophage transfer increased the number of ASC and CX3CR1hi macrophages in VAT.(3)Quantitative PCR results showed that CX3CR1hi macrophages promoted the expression of brown gene in VAT in LHFD mice;The transfer of CX3CR1hi macrophages promoted the increase of rectal temperature in mice with LHFD.Small animal metabolic cage results showed that CX3CR1hi macrophage transfer promoted the energy consumption of adipose tissue in LHFD mice.(4)LHFD mice were transferred with Cx3cr1-/-and Cx3cr1+/-macrophages,and the results of glucose tolerance test and insulin tolerance test showed that Cx3cr1-/-macrophages could not alleviate the metabolic disorders of LHFD mice;Adiposis-tissue β-galactosidase activity staining,immunofluorescence and flow cytometry showed that Cx3cr1-/-macrophages could not inhibit and maintain the senescence and quantity of ASC in LHFD mice VAT.Conclusion1.ASC recruits chemotactic macrophages through the MCP-1/CCR2 axis to infiltrate in VAT.2.In VAT from SHFD mouse,CX3CRhi macrophages maintain VAT adaptive expansion and promote metabolic homeostasis by alleviating ASC senescence.3.ASC shapes macrophages into Arg1hiCX3CR1hi phenotype through exosomes,and CX3CR1hi macrophages alleviate ASC senescence through Argl-eIF5A hypusination axis.4.Transferring CX3CR1hi macrophages to LHFD mice maintained the amount of ASC in VAT and inhibited its senescence,thereby promoting VAT beige and heat production,and alleviating metabolic disorders in mice.
Keywords/Search Tags:macrophage, adipose-derived stem cell, visceral adipose tissue, senescence, obesity, CX3CR1
PDF Full Text Request
Related items