Effect Of Toll Like Receptor 2 On Macrophage Polarization And Its Correlation With Insulin Resistance

Insulin resistance（IR）is one of the important characteristics of obesity and metabolic syndrome.Its pathogenesis is complicated,and it is related to stress in the body’s reticulum,disturbance of intestinal flora,inflammation,etc.Among these factors,chronic low-grade inflammation is considered to be a key factor in the occurrence of insulin resistance.Insulin is secreted by isletβcells and exerts biological effects via the insulin signaling pathway.The inflammatory signaling pathways are activated as low-grade inflammation occurs in the body,which can interfere with the normal effect of insulin and its signaling molecules,leading to dysregulation of glycolipid metabolism and ultimately insulin resistance.The occurrence of low-grade inflammation is closely associated with Toll-like receptors（TLRs）.TLR2 is an important member of the TLRs family.It can not only recognize the lipoprotein,cell wall and other components of bacteria,but also combine with fatty acids in the body inducing an inflammatory response and depression of insulin sensitivity.Macrophages are involved in low-grade inflammatory responses in fat,liver and muscle tissue,and considered to be the triggers for inflammation-induced insulin resistance.Macrophages can be polarized into a pro-inflammatory-M1 phenotype and an anti-inflammatory-M2 phenotype responding to different microenvironments.TLR2 is abundantly expressed in macrophages,and it has been found that TLR2 signals can affect the polarization process of macrophages.So,is TLR2’s involvement in insulin resistance related to TLR2 regulating the polarization of macrophages and then regulating insulin resistance?It has not been reported.In this study,TLR2 knockout(TLR2^-/-)and wild-type（WT）male C57BL/6 mice were used for in vivo experiments,RAW264.7macrophages and 3T3-L1 adipocytes were used for in vitro experiments.The scientific hypothesis that TLR2 reduces insulin sensitivity by affecting macrophage polarization was explored,the molecular mechanism of insulin resistance is further enriched.This study provides experimental and theoretical basis for seeking new targets for the prevention and treatment of obesity and type 2 diabetes.Firstly,the genotypes of TLR2^-/-mice and WT mice were verified by PCR to ensure the rigor and reliability of the experiments.The results showed that none of the 12 TLR2^-/-mice had specific amplification bands of TLR2 gene,while the 12 WT mice had specific amplification bands of TLR2 gene.In order to prove the correlation between TLR2 and insulin resistance,a glucose tolerance test（GTT）and an insulin resistance test（ITT）were performed in TLR2^-/-and WT mice in youth and adult age,respectively.The results showed that compared with WT mice,glucose utilization was increased and insulin sensitivity was increased in TLR2^-/-mice,whether in youth or adult age,suggesting that TLR2 gene deletion could improve the glucose regulation ability and insulin sensitivity in mice.In order to determine whether TLR2 signal affects the polarization phenotype of macrophages and to understand the polarization of macrophages in mice,blood was collected from the iliac vein of the mouse eyes,and lymphocyte separation fluid is used to isolate peripheral blood mononuclear cells（PBMC）.Then the polarization to M1-type or M2-type macrophages was induced using granulocyte-macrophage colony stimulating factor（GM-CSF）combined with interferon-γ（IFN-γ）and lipopolysaccharide（LPS）,macrophage colony-stimulating factor（M-CSF）combined with interleukin 4（IL-4）and IL-13,respectively.Markers of M1 and M2 macrophages were detected by flow cytometry.And the amount of cytokine expression determined that the macrophage phenotype was detected by enzyme-linked immunosorbent assay（ELISA）.The results showed that compared with WT mice,the mean fluorescence intensity（MFI）of M1-like macrophages in TLR2^-/-mice was reduced in both youth and adult age,as well as the proinflammatory factors IL-6 and tumor necrosis factor-α（TNF-α）were also decreased;while the MFI in M2-like macrophages was increased,and the expression of anti-inflammatory factor IL-10 was increased in TLR2^-/-mice.The difference was statistically significant（P<0.05）.Furthermore,high-fat diet（HFD）was induced in mice.Both TLR2^-/-and WT mice were randomly divided into two groups,with 6 mice in each group,namely TLR2^-/-HFD group,TLR2^-/-normal diet（ND）group,WT HFD group and WT ND group.After feeding for 19 weeks,GTT and ITT experiments were performed in each group of mice.The results showed that there was no significant difference in GTT and ITT between TLR2^-/-mice fed with high-fat diet and basic feed,while GTT and ITT were significantly different between WT HFD group and WT ND group,and the utilization rate of glucose and insulin sensitivity were both reduced in WT HFD group（P<0.05）.GTT and ITT results suggest that TLR2 is involved in the insulin resistance and inhibition of TLR2 expression can prevent insulin resistance induced by a high-fat diet.The results of macrophage polarization in four groups of mice showed that the polarization of macrophages in TLR2^-/-mice was not affected by high-fat diet induction,and there was no difference in the polarization of macrophages between TLR2^-/-HFD group and ND group.On the contrary,WT mice showed enhanced polarization of M1 macrophages,and M1-type polarization was more pronounced and pro-inflammatory factors were secreted more induced by a high-fat diet.These results suggest that the deletion of TLR2 gene can inhibit the polarization of macrophages induced by high lipid to M1 phenotype.In addition,the body weight and abdominal fat mass of TLR2^-/-mice were significantly lower than those of WT mice fed a high-fat diet.These results suggest that TLR2-mediated the polarization of M1 macrophages may be involved in the accumulation of fat in the body,leading to weight gain.The above experiments in mice suggest that macrophage polarization may be the key to TLR2 genes promoting insulin resistance.To verify the above conclusions,in vitro cytological experiments RAW264.7 macrophages and 3T3-L1 adipocytes were used.RAW264.7cells were induced with a high-fat diet model with palmitic acid（PA）.TLR2 agonist（Pam3CSK4）and TLR2 inhibitor（C29）were used to investigate the positive and negative effects of TLR2 on macrophage polarization and their effects on insulin sensitivity in adipose tissue.RAW264.7 macrophages were induced by TLR2 inhibitors singlely to increase M2 type polarization and increase the secretion of anti-inflammatory factor IL-10,and when TLR2 inhibitor and PA were applied jointly,they still showed increased M2 type polarization and more secretion of the anti-inflammatory factor IL-10.Under the action of TLR2 agonist,the polarization of cells to M2 type was reduced,while the polarization of cells to M1 type was increased and the secretion of proinflammatory cytokines IL-6 and TNF-αwas increased.Moreover,the ratio of M2 macrophage polarization decreased more significantly in combination of TLR2 agonist and PA group than in TLR2 agonist alone group.It is suggested that TLR2 gene deletion can inhibit the polarization of macrophages to M1 phenotypic,and can inhibit the polarization of macrophages to M1 phenotypic induced by PA.After co-culture of 3T3-L1 adipocytes and RAW264.7 macrophages,and then induced by TLR2 agonist,monocyte chemotactic protein-1（MCP-1）secreted by 3T3-L1 adipocytes was significantly increased,while the MCP-1 levels secreted by 3T3-L1 adipocytes was significantly reduced in the presence of TLR2 inhibitor induction.Moreover,there was no significant difference in the MCP-1 levels secreted by adipocytes when adipocytes were induced by TLR2 inhibitors alone and co-induced by TLR2 inhibitors and palmitic acid,indicating that TLR2 signal deletion can inhibit the secretion of MCP-1 in adipocytes induced by fatty acids,and thus inhibit the infiltration of M1 macrophages into adipocytes.The experimental results of glucose consumption by 3T3-L1adipocytes showed that there was no significant change in the glucose consumption rate after 3T3-L1 adipocytes and RAW264.7 macrophages cultured separately with or without TLR2 agonist or inhibitor induction.However,when 3T3-L1 were cultured with RAW264.7 macrophage culture medium of polarized M1 macrophage,the glucose consumption rate was reduced in the presence of TLR2 agonist,and the glucose consumption rate was increased in the presence of TLR2 inhibitor.And under the co-induction of TLR2 inhibitor and palmitic acid,the consumption rate of glucose by 3T3-L1 cells was the same as that when induced by TLR2 inhibitor alone.These results indicated that with the involvement of M1-polarized RAW264.7 macrophages,the sensitivity of 3T3-L1 cells to insulin was reduced by TLR2 agonists and the sensitivity of 3T3-L1 cells to insulin was increased by inhibitors.In short,with the cooperation of M1 macrophages,the sensitivity of adipocytes to insulin was reduced by TLR2.Bioinformatics technology was used to search the TLR2 signaling pathway and the insulin signaling pathway,and analyze the cross-linking of the two signaling pathways.The results showed that there was an interaction between the insulin signaling pathway and the TLR2 signaling pathway,and insulin receptor substrate（IRS）and c-jun N-terminal kinase（JNK）,protein kinase（Akt）,and nuclear factor-κB（NF-κB）,IRS and inhibitor of NF-κB kinase（IKK）,and Ras-related C3 botulinum toxin substrate（Rac1）and phosphatidylinositol 3 kinase（PI3K）can interact directly or indirectly,thereby affecting insulin metabolism.In summary,in vivo experiments and in vitro experiments revealed that the activated TLR2 signal can affect the polarization of macrophages,making macrophages inclined to the M1 phenotype;M1 macrophages infiltrate into adipose tissue and secrete more pro-inflammatory cytokines to block normal insulin signaling pathways,which reduce glucose tolerance and insulin sensitivity,and produce insulin resistance in the body.Therefore,the regulation of macrophage polarization through TLR2 signaling may be a new way to prevent and treat obesity,as well as type 2 diabetes,which is closely related to it.