| BackgroundIn recent years,the prevalence of obesity has increased dramatically,which has become a serious global health problem.Obesity is a chronic inflammatory disease caused by excessive accumulation of lipid due to long-term energy intake higher than energy consumption,which can lead to metabolic syndromes such as insulin resistance,hypertension and dyslipidemia.Therefore,it is important to control obesity for the treatment of obesity-related diseases.Adipose tissue plays important roles in regulating energy balance and immune hemostasis.Adipose tissue can be classified into subcutaneous adipose tissue(SAT)and visceral adipose tissue(VAT)according to the different anatomical sites.There are significant differences in metabolic characteristics and functions between SAT and VAT.The accumulation of VAT around abdominal organs is closely related to insulin resistance,metabolic syndrome,cardiovascular disease and type 2 diabetes mellitus,while SAT in lower limbs and buttocks has protective energy storage effect,which can improve insulin sensitivity and reduce the risk of metabolic diseases.Obesity-induced adipose tissue inflammation is the main pathogenic factor of insulin resistance.Obesity-induced adipose tissue inflammation is mainly manifested by the increase of inflammatory cytokines such as TNF,IL-1?,IL-6,IL-8,MCP-1 and the infiltration and activation of related immune cells in VAT,which leads to chronic low-level inflammation in the whole body.The persistence of inflammation can further cause dysfunction of adipose tissue,and even lead to ectopic fat deposition in liver,skeletal muscle and pancreas,aggravating insulin resistance and metabolic syndrome.Therefore,understanding the regulation mechanism of adipose tissue immune microenvironment and controlling adipose tissue inflammation are of great significance for improving insulin resistance and preventing obesity-related diseases.Adipose tissue consists of adipocytes,immune cells,mesenchymal stem cells,fibroblasts and endothelial cells.Immune cells such as macrophages and B cells,are important components of adipose tissue immune microenvironment,which play critical roles in maintaining adipose tissue metabolic homeostasis or inducing obesity-related metabolic disorders.adipose-derived mesenchymal stem cells(ADSCs)are the precursors of adipocytes in adipose tissue,which also have immunoregulatory functions.According to the anatomical location,ADSCs can be divided into subcutaneous adipose tissue-derived ADSCs(S-ADSCs)and visceral adipose tissue-derived ADSCs(V-ADSCs).S-ADSCs and V-ADSCs have significant differences in gene expression,proliferation and differentiation and so on.So,it is worth to explore whether S-ADSCs and V-ADSCs participate in regulating the immune microenvironment of SAT and VAT,and leads to different metabolic characteristics and functions of VAT and SAT.Methods?.Isolation and culture of V-ADSCs and S-ADSCsC57BL/6J male mice at the age of 10-12 weeks were sacrificed.inguinal adipose tissue(subcutaneous adipose tissue)and epididymal adipose tissue(visceral adipose tissue)were extracted.Vascular stromal fractions were extracted using collagenase-containing Krebs-Ringer Buffer,and then were cultured in complete DMEM.After 24 hours,the supernatants were replaced with new culture medium.The adherent cells were cultured as ADSCs.The 3rd to 5th passage of ADSCs were used for the following experiments.?.Study on the differences of inflammatory phenotypes between S-ADSCs and V-ADSCs1.Differences in cell size and morphology between S-ADSCs and V-ADSCsS-ADSCs and V-ADSCs were planted in 6-well plate and were photographed under the microscope after 24 hours to compare the differences in size and morphology between S-ADSCs and V-ADSCs2.Differences in expression and secretion of pro-inflammatory cytokines between S-ADSCs and V-ADSCs1)Detection of proinflammatory cytokines mRNA expressionS-ADSCs and V-ADSCs were planted in 6-well plate,and the expression levels of IL-1?,IL-6 and TNF-? were detected by quantitative real-time PCR in S-ADSCs and V-ADSCs2)Detection of the secretion of proinflammatory cytokines in the supernatantsS-ADSCs and V-ADSCs were planted in 6-well plate,and the supernatants were collected to detect the secretion of IL-6 and TNF-? by ELISA3.Differences in inflammation-related signaling pathways between S-ADSCs and V-ADSCsS-ADSCs and V-ADSCs were planted in 6-well plate.After extraction by protein lysate,the protein expression levels of p-ERK,p-JNK,p-p65,ERK and J-NK in S-ADSCs and V-ADSCs were detected by western-blot4.Differences in TLR4 and TLR2 between S-ADSCs and V-ADSCs1)Detection of TLR4 and TLR2 mRNA expression in SVFS-SVF and V-SVF were extracted from C57BL/6J male mice fed 10-12 weeks.The expression levels of TLR4 and TLR2 was detected by quantitative real-time PCR2)Detection of TLR4 and TLR2 mRNA expression in ADSCsThe expression levels of TLR4 and TLR2 in S-ADSCs and V-ADSCs were detected by quantitative real-time PCR.3)Detection of TLR4 and TLR2 protein expression in ADSCsThe protein expression levels of TLR4 and TLR2 in S-ADSCs and V-ADSCs were detected using western-blot and flow cytometry5.Effects of TLR4/TLR2 blockade on inflammatory phenotypes of V-ADSCs1)Detection of the expression of proinflammatory cytokines after blocking TLR4 or TLR2 in V-ADSCsV-ADSCs were treated with TLR4/TLR2 inhibitors.S-ADSCs and V-ADSCs were divided into the following groups:S-ADSC+DMSO,V-ADSC+DMSO,V-ADSC+CU CPT22(TLR2 inhibitor),V-ADSC+TAK242(TLR4 inhibitor)..The expression levels of IL-1?,IL-6 and TNF-? were detected by quantitative real-time PCR.2)Detection of the secretion of proinflammatory cytokines after blocking TLR4 or TLR2 in V-ADSCsS-ADSCs and V-ADSCs were treated as mentioned above.The supernatants were collected to detect the secretion of TNF-? by ELISA.3)Detection of inflammatory signaling pathways after blocking TLR4 or TLR2 in V-ADSCsS-ADSCs and V-ADSCs were treated as mentioned above.The protein levels of p-ERK,p-JNK and p-I?B were detected by western-blot after blocking TLR4 or TLR26.Effect of TLR4/TLR2 activation on inflammatory responses of ADSCs1)Detection the effect of TLR4 and TLR2 agonist on the expression of pro-inflammatory cytokinesS-ADSCs and V-ADSCs were treated with LPS,respectively,and were divided into the following groups:S-ADSC+PBS,V-ADSC+PBS,V-ADSC+LPS,V-ADSC+LPS.The expression levels of IL-1?,IL-6 and TNF-? were detected by quantitative real-time PCR.2)Detection the effect of TLR4 and TLR2 agonist on the secretion of proinflammatory cytokines in the supernatantsS-ADSCs and V-ADSCs were treated as mentioned above.The supernatants were collected to detect the secretion of IL-6 and TNF-? by ELISA.3)Detection the effect of TLR4 and TLR2 agonist on inflammatory signaling pathwaysS-ADSCs and V-ADSCs were treated as mentioned above.Proteins were extracted and the protein levels of p-ERK,p-JNK,p-p65,p-I?B,ERK,JNK,p65 and I?B was detected by western-blot.?.Study on the differences in macrophages and B cells between S-ADSCs and V-ADSCs1.Differences in macrophage percentage between SAT and VATS-SVF and V-SVF were extracted from C57BL/6J male mice at the age of 4,7 and 10 weeks,respectively.The percentages of CDllb+macrophages in SAT and VAT were detected by flow cytometry.2.Differences in B cell percentage between SAT and VATS-SVF and V-SVF were extracted from C57BL/6J male mice at the age of 4,7 and 10 weeks,respectively.The percentage of CD45R+ B cells in SAT and VAT were detected by flow cytometry.?.Study on chemotaxis of macrophages by S-ADSCs and V-ADSCs1.The expression and secretion levels of MCP-1 in S-ADSCs andV-ADSCs1)Detection of MCP-1 mRNA expression in S-ADSCs and V-ADSCsThe mRNA levels of MCP-1 in S-ADSCs and V-ADSCs was detected by quantitative real-time PCR.2)Detection of the secretion of MCP-1 in the supernatantsThe supernatants in S-ADSCs and V-ADSCs were collected to detect the secretion of MCP-1 by ELISA.2.Macrophage chemotaxis mediated by S-ADSCs and V-ADSCsS-ADSCs and V-ADSCs were planted in 12-well plate.Abdominal macrophages from C57BL/6J male mice were added into the upper chamber of transwell plate.The macrophage chemotaxis was detected by crystal violet staining and microscopic photography.3.Effect of TLR4/TLR2 blockade on the expression levels of MCP-1 in V-ADSCsV-ADSCs were treated with TLR4/TLR2 inhibitors.S-ADSCs and V-ADSCs were divided into the following groups:S-ADSC+DMSO,V-ADSC+DMSO,V-ADSCs+TAK242(TLR4 inhibitor),V-ADSCs+CU CPT22(TLR2 inhibitor).The expression of MCP-1 was detected by quantitative real-time PCR after blocking TLR4 or TLR2.4.Effect of TLR4/TLR2 activation in ADSCs on the expression and secretion levels of MCP-1 and chemotaxis to macrophagesS-ADSCs and V-ADSCs were treated with LPS,respectively,and divided into the following groups:S-ADSC+PBS,V-ADSC+PBS,V-ADSC+LPS,V-ADSC+LPS.The mRNA and secretion levels of MCP-1 were detected by quantitative real-time PCR;the of MCP-1 was detected by ELISA after TLR4 and TLR2 activation;and the chemotaxis to macrophages was detected by macrophage chemotaxis test.V.Study on chemotaxis of B cells mediated by S-ADSCs andV-ADSCs1.The expression levels of CCL91)Detection of CCL9 mRNA expression in SAT and VATThe expression levels of CCL9 in SAT and VAT were detected by quantitative real-time PCR.Detection of CCL9 mRNA expression in S-ADSCs and V-ADSCsThe expression levels of CCL9 in S-ADSCs and V-ADSCs were detected by quantitative real-time PCR.2.Detection of chemotaxis of B cells by S-ADSCs and V-ADSCsS-ADSCs and V-ADSCs were planted in 24-well plate.CD19+B cells from bone marrow of C57BL/6J male mice were collected by magnetic Beads sorting and were added into the upper chamber of transwell plate.The chemotaxis of B cells was detected by cell counting and flow cytometry.3.The expression levels of EBF11)Detection of EBF1 mRNA levels in SAT and VATThe mRNA levels of EBF1 in SAT and VAT were detected by quantitative real-time PCR.2)Detection of EBF1 mRNA levels in S-SVF and V-SVFThe mRNA levels of EBF1 in S-SVF and V-SVF were detected by quantitative real-time PCR.3)Detection of EBF1 mRNA levels in S-ADSCs and V-ADSCsThe expression levels of EBF1 in S-ADSCs and V-ACSCs were detected by quantitative real-time PCR.4.Effects of EBF1 gene silence on the expression of CCL9 in S-ADSCsS-ADSCs were planted in 12-well plate.After transfected with siEBFl for 48h,and the expression levels of EBF1 and CCL9 were detected by quantitative real-time PCR.5.Effects of EBF1 gene silence in S-ADSCs on chemotaxis to B cellsS-ADSCs were transfected with siEBF1,and the medium was changed after 48h.The chemotaxis to CD19+B cells was performed after 24h and and examined by cell counting and flow cytometry.Results1.V-ADSCs express higher levels of inflammatory phenotypes than S-ADSCsCompared with S-ADSCs,V-ADSCs have large size and irregular morphology with long and obvious pseudopods.The expression levels of IL-1?,IL-6 and TNF-?and the secretion levels of IL-6 in V-ADSCs were significantly higher than those in S-ADSCs,but there was no significant difference in the level of TNF-?.The expression levels of p-ERK,p-JNK and p-p65 in V-ADSCs were significantly higher than those in S-ADSCs.These results suggest that the expression levels of inflammatory phenotypes in V-ADSCs were significantly higher than those in S-ADSCs.In addition,the expression levels of TLR4 and TLR2 in V-ADSCs were significantly higher than those in S-ADSCs,and the expression of TLR4 and TLR2 in V-SVFs was also higher than that in S-SVFs.TLR4 and TLR2 inhibitors significantly inhibited the expression of p-ERK,p-JNK and p-I?B and the mRNA levels of proinflammatory cytokines in V-ADSCs,suggesting that TLR4 and TLR2 are involved in regulating the inflammatory phenotypes of V-ADSCs.Besides,TLR4 and TLR2 activation by LPS markedly upregulated the expression of IL-1?,IL-6,TNF-? and the secretion of IL-6 and TNF-? in both S-ADSCs and V-ADSCs.2.VAT contains high percentage of macrophages,while SAT contains high percentage of B cellsThe percentages of macrophages and B cells in SAT and VAT were detected by flow cytometry.The results showed VAT contains high percentage of CD 11b macrophages,while SAT contains high percentage of CD45R B cells.3.V-ADSCs recruits more macrophages than S-ADSCsThe expression and secretion of MCP-1 in V-ADSCs were significantly higher than those in S-ADSCs.In addition,the chemotaxis ability of V-ADSCs on macrophages was significantly stronger than that of S-ADSCs.TLR4/TLR2 inhibitors significantly inhibited the expression levels of MCP-1 in V-ADSCs,while TLR4/TLR2 activation significantly promoted the expression and secretion of MCP-1 in both S-ADSCs and V-ADSCs,thereby enhancing the chemotaxis to macrophages.Particularly,after TLR4/TLR2 activation,the expression and secretion levels of MCP-1 in V-ADSCs were significantly higher than those in S-ADSC,B cell chemotaxis mediated by S-ADSCs were also remarkably increased compared with V-ADSCs.These results suggest that V-ADSCs express high levels of TLR4 and TLR2,which further promote the expression and secretion of MCP-1,resulting in the enhancement of chemotaxis to macrophages and the infiltration of macrophages in VAT.4.S-ADSCs recruits more B cells than V-ADSCsThe expression levels of CCL9 in S-ADSCs were significantly higher than those in V-ADSCs,consistently the expression levels of CCL9 in SAT significantly higher than those in VAT.In addition,S-ADSCs recruits more CD19 B cells,especially CD19+CD45RhighB cells,than V-ADSCsWe further examined the expression of EBF1 in adipose tissue,SVF and ADSCs,and found that the expressions of EBFf un SAT,S-ShF and S-ADSCs were significantly higher than those in VAT,V-SVF and V-ADSCs,respectively.To clarify the regulatory effect of EBF1 on CCL9,the expression of EBF1 gene was silenced by siEBF1 in S-ADSCs,and then the expression levels of CCL9 in S-ADSCs and B cell chemotaxis were detected.The results showed that the expression of CCL9 was significantly decreased after EBF1 gene silence,and the chemotactic effect on CD19+B cells,especially CD19+CD45RhighB cells,was significantly reduced.These data suggest that,S-ADSCs express high levels of EBF1,which promote the expression of CCL9 and recruit CD19+B cells,especially CD19+CD45Rh'ghB cells,leading to the infiltration of B cells in SAT.Conclusion1.Compared with S-ADSCs,V-ADSCs express high levels of TLR4,TLR2 and inflammatory phenotype,which are prone to induce inflammatory response.2.V-ADSCs express TLR4 and TLR2 that promote the expression and secretion of MCP-1,mediating macrophage chemotaxis and resulting in a large number of macrophages infiltrated in VATs.3.S-ADSCs express EBF1 that promotes the expression of CCL9,mediating CD19 B cell chemotaxis and resulting in a large number of B cells infiltrated in SATs. |
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