Toll-like receptor 4 deficiency promotes the alternative activation of adipose tissue macrophages

Abstract

Obesity is characterized by adipose tissue (AT) macrophage (ATM) accumulation, which promotes AT inflammation and dysfunction. Toll-like receptor 4 (TLR4) deficiency attenuates AT inflammation in obesity but does not impede the accumulation of ATMs. The purpose of the current study was to determine whether TLR4 deficiency alters ATM polarization. TLR4(-/-) and wild-type mice were fed a low-fat, high-monounsaturated fat (HF(MUFA)), or a high-saturated fat (HF(SFA)) diet for 16 weeks. Further, we used a bone marrow transplant model to determine the influence of hematopoietic cell TLR4 signaling. The metabolic and inflammatory responses to high-fat feeding and ATM phenotype were assessed. Global and hematopoietic cell TLR4 deficiency, irrespective of recipient genotype, produced a shift in ATM phenotype toward an alternatively activated state, which was accompanied by reduced AT inflammation. Despite the observed shift in ATM phenotype, neither global nor hematopoietic cell TLR4 deficiency influenced systemic insulin sensitivity after high-fat feeding. Results of the current study suggest that TLR4 directly influences ATM polarization but question the relevance of TLR4 signaling to systemic glucose homeostasis in obesity.

FIG. 1.

TLR4 deficiency attenuates weight gain and hepatic TG accumulation after HF feeding. A: WT and TLR4-deficient mice display similar growth curves on a LF diet, whereas weight gain is attenuated in TLR4^−/− mice after HF^MUFA and HF^SFA feeding (n = 13–15 for all diet conditions). B: TLR4 deficiency reduces HF-feeding–induced hepatic TG accumulation (n = 13–16). C: Representative images of ORO-stained liver sections. Data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 for WT HF^SFA vs. TLR4^−/− HF^SFA; #P < 0.05 for WT HF^MUFA vs. TLR4^−/− HF^MUFA; ‡P < 0.01 for genotype effect; and P < 0.05 for groups not connected by the same letter. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

AT inflammation. Real time RT-PCR was used to assess AT mRNA expression of Tnfa (P = 0.07 for genotype effect; P < 0.10 for diet effect) (A), Ccl3 (B), Tlr2 (C), Ccl2 (D), Il6 (E), and Il10 (F). Data are presented as mean ± SEM (n = 5–16). *P < 0.05 for HF^SFA compared with LF in (B) and (C), *P < 0.05 for HF^SFA TLR4^−/− compared with HF^SFA WT in (D), and P < 0.05 for groups not connected by the same letter.

FIG. 3.

TLR4 deficiency promotes alternative activation of ATMs. Real-time RT-PCR was used to assess AT mRNA expression of Emr1 (A), Cd68 (B), Mrc1 (C), Clec10a (D), Mgl2 (E), Cd163 (F), Nos2 (G), and Arg1 (H) (n = 7–16). I: Representative FACS plot of F4/80⁺Mgl1⁺ ATMs from HF^SFA-fed WT and TLR4^−/− mice. J: Quantification of F4/80⁺ ATMs from HF^SFA-fed WT and TLR4^−/− mice (n = 6–9). K: Quantification of F4/80⁺Mgl1⁺ ATMs from HF^SFA-fed WT and TLR4^−/− mice (n = 4–5). Data are presented as mean ± SEM. *P < 0.05 for HF^SFA TLR4^−/− compared with HF^SFA WT; ‡P < 0.05 for genotype effect; and P < 0.05 for groups not connected by the same letter. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

TLR4 deficiency does not attenuate systemic IR after HF^SFA feeding. A: Time course of blood glucose throughout the hyperinsulinemic-euglycemic clamp is presented to illustrate clamp quality. B: The glucose infusion rate required to maintain blood glucose between 120 and 130 mg/dL did not differ between HF^SFA-fed TLR4^−/− and WT mice. WT and TLR4^−/− mice displayed similar rates of endogenous glucose production (EndoR_a) (C) and glucose disappearance (R_d) (D). E: Glucose uptake (R_g) was significantly reduced in the gastrocnemius (Gastroc), superficial vastus lateralis (SVL), and AT of TLR4^−/− mice compared with WT. Data are presented as mean ± SEM (n = 4–7). **P < 0.01 and ***P < 0.0001 for TLR4^−/− compared with WT.

FIG. 5.

Hematopoietic cell TLR4 deficiency attenuates AT inflammation in WT recipient mice. Real time RT-PCR was used to assess adipocyte fraction mRNA expression of Tnfa (A), Ccl3 (B), Ccl2 (C), and Il6 (D) as well as SVF mRNA expression of Tnfa (E), Ccl3 (F), Ccl2 (G), Il6 (H), and Tlr2 (I). Data are presented as mean ± SEM (n = 7–16). *P < 0.05 for WT^{TLR4−/−BM} compared with WT^WTBM , and #P < 0.01 for TLR4^{−/−TLR4−/−BM} compared with TLR4^−/−WTBM.

FIG. 6.

Hematopoietic cell TLR4 deficiency promotes the alternative activation of ATMs. Real-time RT-PCR was used to assess SVF mRNA expression of Emr1 (A), Cd68 (B), Clec10a (C), Mgl2 (D), Mrc1 (E), Nos2 (F), Arg1 (G), and Il10 (H). Data are presented as mean ± SEM (n = 7–16). *P < 0.05 for WT^{TLR4−/−BM} compared with WT^WTBM, and #P < 0.05 for TLR4^{−/−TLR4−/−BM} compared with TLR4^−/−WTBM.

FIG. 7.

A: Hematopoietic cell TLR4 deficiency does not influence systemic insulin sensitivity. WT and TLR4^−/− mice displayed similar glucose excursion curves before BMT (n = 5–9). After 20 weeks of an HF^SFA diet, TLR4^−/− recipient mice exhibited impaired glucose (n = 8–15) (B) and insulin (n = 6–8) (C) tolerance compared with WT recipients, regardless of hematopoietic cell TLR4 expression. D: Fasting blood glucose (n = 8–18). E: Fasting plasma insulin (n = 8–18). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 for recipient genotype effect, and P < 0.05 for groups not connected by the same letter.