TLR4 Knockout Mediates Spontaneous Obesity In Aged Mice Via Chronic Low-grade Inflammation

Objective:To explore the effect of TLR4 knockout on obesity in aged mice and its mecha-nism.Methods:In this study,sixty male wild type（WT;C57BL/10Sc N background）mice,and sixty male TLR4 knockout(TLR4^-/-;C57BL/10Sc N background)mice were divided into young（6-month-old）mice,middle-aged（12-month-old）mice and aged（18-month-old）mice according to age.Each age group was divided into two groups:WT group and TLR4^-/-group,with 20 mice in each group.All mice were weaned 21days after birth and fed with standard chow.1.To investigate the effect of TLR4 knockout on body weight in aged mice:Record the body weight,subcutaneous fat weight,perirenal fat weight,and body length of the mice at the age of 6 months,12 months,and 18 months,and calculate the subcutaneous fat index,perirenal fat index and Lee’s index（reflecting the degree of obesity in mice）.HE staining was used to observe the tissue structure in liver and adipose tissue;Oil red O staining was used to observe fat accumulation in liver.2.To investigate the effect of TLR4 knockout on lipid metabolism in aged mice:Real-time quantitative PCR was used to detect the mRNA expression of glucose and lipid metabolism genes Leptin,Adipoq,Srebp1c,and carbohydrate-responsive ele-ment-binding protein（Ch REBP）in liver and adipose tissue;Western blot was used to detect the protein expression of Adipoq and Ch REBP in liver.3.To investigate the effect of TLR4 knockout on liver inflammation in aged mice:Real-time quantitative PCR was used to detect mRNA expression of IL-1β,IL6,IL10,CD86（M1 type macrophage marker）,and CD163（M2 type macrophage mark-er）;Immunohistochemical was used to detect immunoreactivity of CD86,CD163 in the liver.4.To investigate the effect of TLR4 knockout on My D88 and TRIF signaling pathways in the liver of aged mice:Real-time quantitative PCR was used to detect the mRNA expression of S100A8,S100A9,My D88,TRIF and IRF3 in the liver;Western blot was used to detect the protein expression of TRIF and p IRF3 in the liver;Im-munohistochemistry was used to detect the immunoreactivity of p IRF3 in the liver.5.To investigate the effect of TLR4 knockout on Th cell differentiation in the spleen of aged mice:Record the spleen weight of the mice at the age of 6 months,12months,and 18 months,and calculate the spleen index;Real-time PCR was used to detect the mRNA expressions of Tbet,Gata3,Rorγt,Foxp3,IL2,IL4,IL17a and IL10in the spleen;Western blot was used to detect the protein expressions of Tbet,Gata3,Rorγt and Foxp3;Immunohistochemistry was used to detect the immunoreactivity of Tbet,Gata3,IL17a and Foxp3 in the spleen.6.To investigate the effect of TLR4 knockout on systemic inflammation and lep-tin in aged mice:ELISA was used to detect the protein expression of IL1β,IL6,IL10and Leptin in serum.Result:1.6-months TLR4^-/-mice show increased body weight gain but decreased sub-cutaneous and perirenal fat index.In contrast,both 12-months and 18-months TLR4knockout mice show increased body weight gain,subcutaneous fat index,and perire-nal fat index,but only 18-months TLR4^-/-mice show increased Lee`s index.HE staining and Oil red O staining results showed that 18-months TLR4^-/-mice promote lipid accumulation in liver and adipose tissue.2.18-months TLR4^-/-mice show significantly upregulated the mRNA expression levels of Ch REBP and Leptin,and also decreased the mRNA expression level of Ad-ipoq in liver,subcutaneous and perirenal fat;18-months TLR4^-/-show upregulated the protein expression level of the Ch REBP,and downregulated the protein expression level of Adipoq in liver.3.18-months TLR4^-/-mice show significantly upregulated the mRNA expression levels of IL1βand CD86,and decreased the mRNA expression levels of IL10 and CD163 in the liver;18-months TLR4^-/-mice increased the number of CD86-positive macrophages and decreased the number of CD163-positive macrophages in the liver4.18-months TLR4^-/-mice show significantly upregulated TRIF mRNA expres-sion,TRIF,p IRF3 protein expression levels,and increased p IRF3 immunoreactivity in liver;whereas 18-months TLR4^-/-mice did not affect the mRNA expression levels of S100A8,S100A9,and My D88 in liver.5.18-months TLR4^-/-mice show significantly upregulated the mRNA expression levels of Tbet,Rorγt,IL2,and IL17a,and downregulated the mRNA expression lev-els of GATA3,Foxp3,and IL4 in spleen;18-months TLR4^-/-mice also show signifi-cantly increased the proteins expression levels of Tbet and Rorγt and decreased the expression levels of Gata3 and Foxp3 proteins in the spleen;Besides that,18-months TLR4^-/-mice show increased T-bet and IL17a immunoreactivities,and reduced Gata3and Foxp3 immunoreactivities in the spleen.Elisa result showed that 18-months TLR4^-/-mice upregulated the protein expression levels of IL6 and Leptin in serum.Conclusion:1.TLR4 knockout can induce spontaneous obesity in aged mice.2.TLR4 knockout increased the expression of the adipogenic gene Ch REBP and facilitated lipid metabolism in liver and adipose tissue of aged mice.3.TLR4 knockout induces inflammation in the liver of aged mice,and the pos-sible mechanism may promote the polarization of M2 to M1 by activating the TRIF signal transduction pathway.4.TLR4 knockout increased the spleen index and induce the imbalances of Th1/Th2 and Th17/Treg cells which indicated the occurrence of chronic low-grade inflammation in aged mice.