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. 2009 Feb;52(2):318-28.
doi: 10.1007/s00125-008-1221-7. Epub 2008 Dec 4.

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice

K R Coenen  1 , M L Gruen, R S Lee-Young, M J Puglisi, D H Wasserman, A H Hasty
Affiliations

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice

K R Coenen et al. Diabetologia. 2009 Feb.

Abstract

Aims/hypothesis: Toll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders.

Methods: Bone marrow transplantation of agouti, LDL-receptor deficient (A(y)/a; Ldlr (-/-)) mice with marrow from either C57BL/6 or Tlr4 (-/-) mice was performed. Recipient mice with Tlr4 (+/+) marrow (MthetaTLR4(+/+)) or with Tlr4 (-/-) marrow (MthetaTLR4(-/-)) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HF(SFA)); and (4) HF(SFA) supplemented with fish oil.

Results: There were no differences in body composition or plasma lipids between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MthetaTLR4(-/-) mice compared with MthetaTLR4(+/+) mice. MthetaTLR4(-/-) mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed the HF(SFA) diet.

Conclusions/interpretation: These data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

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Conflict of interest statement

Duality of Interest The authors have no conflicts to disclose.

Figures

Figure 1
Figure 1. Total body weight and fat over 12 wks of diet
Body weight was measured every week and total body fat was measured every other week using NMR in mice that had received either TLR4+/+ (black bars) or TLR4-/- (white bars) marrow and had been fed LF diet (A & E), HF (B & F), HFSFA diet (C & G), and HFSFA+FO diet (D & H). Data are expressed as the mean ± SEM from 5-11 mice per group. *P<0.05.
Figure 2
Figure 2. Hyperinsulinemic-euglycaemic clamps in HFSFA fed mice
Blood glucose levels (A), average glucose infusion rate during the steady state period (80 to 120 min) (B), and tissue specific glucose metabolic index (Rg; C) during a 120min hyperinsulinemic-euglycaemic clamp in chronically catheterized, conscious MθTLR4+/+ (black) and MθTLR4-/- (white) mice fed HFSFA diet. Mice were fasted for 5h before the experiment. Data are mean ± SEM for 4-5 mice per group. Gastroc = gastrocnemius muscle; SVL = superficial vastus lateralis; WAT = white adipose tissue
Figure 3
Figure 3. F4/80 staining and Mθ expression in adipose tissue
WAT sections from mice fed LF (A & E), HF (B & F), HFSFA (C & G), and HFSFA+FO diets were immunostained with an antibody for F4/80, a macrophage surface marker. Images are shown at 10x magnification for mice receiving TLR4+/+ (A-D) and TLR4-/- (E-H) marrow. RNA was isolated from the perigondal fat pads and real-time RT-PCR was used to determine F4/80 expression (I-L) and CD68 expression (M-P) from mice on their respective diets. Mice receiving TLR4+/+ marrow are shown in black bars and those receiving TLR4-/- marrow are shown in white bars. Data are expressed as mean ± SEM from 5-11 mice per group. Two-way ANOVA revealed a main effect for diets for F4/80 (P<0.001) and CD68 (P<0.005). There were no significant main effects for macrophage TLR4 genotype. Significant findings of post-hoc tests are as indicated. *P<0.05.
Figure 4
Figure 4. Inflammation in WAT
Local inflammation was determined in WAT by analyzing gene expression using realtime RT-PCR. MIP-1α (A-D), SAA3 (E-H), and TLR4 (I-L) mRNA expression were measured in mice fed their respective diets. Data are expressed as mean ± SEM from 5-11 mice per group. Macrophage genotypes are indicated below graphs. Two-way ANOVA revealed a main effect for diets for MIP-1α (P<0.05) and SAA3 (P<0.00001). Main effects for macrophage TLR4 genotype reached near significance for TLR4 expression (P=0.08). Significant findings of post-hoc tests are as indicated. *P<0.05, **P<0.01.
Figure 5
Figure 5. M1 and M2 classification of ATMs
Markers of M1 and M2 macrophages were determined in WAT by analyzing gene expression for TNF-α, IL-12, MCP-1, iNOS, arginase, mannose receptor, Mgl1, and Mgl2 (M1 versus M2 as indicated on figure). Mice receiving TLR4+/+ marrow are shown in black bars and those receiving TLR4-/-marrow are shown in white bars. Data are shown only from mice fed the LF diet and are expressed as mean ± SEM from 5-11 mice per group.
Figure 6
Figure 6. Quantification of lesion area
Hearts were sectioned at the aortic root and atherosclerotic lesions were stained using ORO. Images are shown at 10x magnification (A-H). Lesion areas were quantified using Kinetic Histometrix software in 5-11 mice per group (I-L). Data are presented as mean ± SEM and macrophage genotypes are listed below graphs. *P<0.05.
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