Introduction

Obesity remains an issue in today’s society, with a recent epidemiological study projecting that 41% of the US population will be obese by 2015 [1]. Obesity is a critical component of the metabolic syndrome, along with hypertension, dyslipidaemia, impaired glucose tolerance and insulin resistance [2–4]. Obese individuals have a two- to threefold increased risk of death from all causes [5], with the majority of risk due to cardiovascular disease [6]. Because obesity is characterised by adipose tissue expansion, much effort is being placed into researching the contribution of adipose tissue to development of metabolic diseases associated with obesity.

One key cell type that may link obesity with metabolic disorders is the macrophage. Macrophages are cells of the innate immune system that have been implicated in development of diseases such as cancer, atherosclerosis and, most recently, insulin resistance [7–11]. Although it has long been known that macrophages infiltrate the artery wall and are integral to the formation of atherosclerotic lesions [12, 13], it was recently demonstrated that macrophages infiltrate white adipose tissue (WAT) and may contribute to inflammation and insulin resistance in obesity [8, 14]. Studies have shown that these adipose tissue macrophages (ATMs) contribute the majority of pro-inflammatory cytokine production in adipose tissue and that the presence of ATMs precedes hyperinsulinaemia [8, 14]. Importantly for our studies, ATMs have been shown to be derived from bone marrow [14].

Because of the close link between obesity, macrophages and inflammation, the activation of macrophages via toll-like receptor 4 (TLR4) has become an important area of research. TLR4 is a pathogen-associated molecular pattern recognition receptor that is activated by lipopolysaccharide (LPS). The lipid A moiety of LPS contains acylated hydroxyl saturated fatty acids (SFAs). Removal of these or their replacement with polyunsaturated fatty acids (PUFAs) results in a loss of LPS-induced inflammation [15]. This attribute of LPS led to the hypothesis that SFAs could be natural ligands for TLR4, a theory that has been confirmed by several different groups who have shown that the activation of TLR4 by SFAs leads to the induction of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphoinositide-3 kinase pathways, culminating in inflammatory gene expression [16–19]. TLR4 is able to sense and respond to different types of fatty acids, as SFAs result in activation, while PUFAs block the activation of TLR4 [17–22].

The responsiveness of TLR4 to fatty acids makes TLR4 an appealing intermediary between obesity and recruitment of macrophages to WAT and the artery wall. Recently, global TLR4 deletion studies have delivered conflicting results regarding the role of TLR4 in body composition and macrophage infiltration of WAT in mice challenged with high-fat diets. However, all of the studies shared the common finding that Tlr4 −/− mice were protected against inflammation and insulin resistance [18, 23–25]. Our current studies expand upon these findings by investigating the effect of macrophage Tlr4 on insulin resistance, the influx of macrophages to WAT, local inflammation and atherosclerosis.

Methods

Mice

All animal care and experimental procedures were performed with approval from the Institutional Animal Care and Usage Committee of Vanderbilt University. Tlr4 −/− mice on a C57BL/6 background were kindly provided by S. Uematsu and S. Akira (Department of Host Defense, University of Osaka, Japan). The Tlr4 −/− mice were subsequently crossed with C57BL/6 mice from our colony. The heterozygous offspring were intercrossed to produce the Tlr4 −/− and Tlr4 +/+ littermates used as bone marrow donors. The recipient, agouti (Ay/a), LDL-receptor (LDLR)-deficient (Ay/a;Ldlr −/−) mice were produced by intercrossing Ay/a mice with Ldlr −/− mice, both of which were on a C57BL/6 background [26, 27]. These studies were performed in male and female mice; however, data shown are from female mice only.

Bone marrow transplantation

For bone marrow transplantation (BMT), bone marrow cells collected from Tlr4 −/− and Tlr4 +/+ donors were injected into the retro-orbital venous plexus of lethally irradiated recipient Ay/a;Ldlr −/− mice. Reconstitution was confirmed by performing PCR for TLR4 genotype of DNA isolated from blood of recipient mice (data not shown).

Diets

Mice had free access to food and water throughout the study. They were placed on experimental diets 4 weeks post-BMT and maintained on the respective diets for 12 weeks. All diets (Table 1) were purchased from Research Diets (New Brunswick, NJ, USA). Cocoa butter was selected as the source for high-fat diet with SFA source (HFSFA) to allow for the highest concentration of SFAs in the diet while still providing essential fatty acids.

Table 1 Composition of experimental diets
Full size table

Total body fat

Total body fat was determined by NMR using the Bruker Minispec (Woodlands, TX, USA) in the Mouse Metabolic Phenotyping Centre at Vanderbilt University.

Euglycaemic–hyperinsulinaemic clamps

Mice were catheterised at least 5 days before experimentation and euglycaemic–hyperinsulinaemic clamps were performed on conscious mice after a 5 h fast as previously described [28]. Briefly, a 185 kBq bolus of [3–3H]glucose was given, followed by a constant 1.85 kBq/min infusion for 90 min (t = –90 to 0 min), which served as the tracer equilibration period. At 0 min the insulin clamp began with a continuous infusion of human insulin at 4 mU kg−1 min−1 (Humulin R; Eli Lilly, Indianapolis, IN, USA). The [3–3H]glucose infusion was increased to 0.2 7.4 kBq/min for the clamp and euglycaemia (~8.33–8.89 mmol/l) was maintained by measuring arterial blood glucose every 10 min and infusing 50% dextrose as necessary. Following the 120 min insulin clamp, a 592 kBq bolus of 2-deoxy[14C]glucose was given to determine the tissue-specific glucose metabolic index (R g) as previously described [28]. At t = 150 min mice were killed with an overdose of sodium pentobarbital and the soleus, gastrocnemius, superficial vastus lateralis, WAT, liver, heart and brain were excised.

Blood collection and plasma variables

After 12 weeks of feeding on their respective diets, mice were fasted overnight before blood was collected. Plasma total cholesterol and triacylglycerol levels were measured using kits (Raichem, San Diego, CA, USA). NEFA measurements were performed using a kit (NEFA-C, Wako, Neuss, Germany). Glucose levels were determined on whole blood using a glucometer (Lifescan OneTouch; Johnson & Johnson, Northridge, CA, USA). Insulin and leptin measurements were performed by the Vanderbilt Hormone Assay and Analytical Services Core using RIAs.

F4/80 staining of adipose tissue

Perigonadal fat pads were extracted upon killing, fixed in 10% formalin (vol./vol.) and embedded in paraffin. Sections were stained for EGF-like module containing, mucin-like, hormone receptor-like sequence 1 (F4/80) using an antibody from Serotec (Raleigh, NC, USA) at a 1:100 dilution. Secondary antibody (Dako, Glostrup, Denmark) was used at a 1:100 dilution.

Gene expression

RNA was isolated from perigonadal WAT using the a kit (RNeasy Mini; Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Real-time RT-PCR was performed using Applied Biosystem 7700 sequence detection system (Foster City, CA, USA) or the iQ5 cycler from BioRad (Hercules, CA, USA). Primer-probe sets were purchased from Applied Biosystem’s Assays-on-demand. Gene expression was normalised to 18S and the ΔΔCt method was used to calculate relative gene expression [29].

Fatty acid composition of adipose tissue

Gas chromatography was used to analyse fatty acid composition of the triacylglycerol portion of perigonadal fat pads as described previously [27, 30].

Atherosclerotic lesion area

Hearts were excised upon killing, frozen and sectioned at the aortic root. Neutral lipids were stained using Oil-Red-O. Lesion area was quantified using Histometrix version 6 imaging and analysis software (Kinetic Imaging, Durham, NC, USA).

Statistics

Statistical analyses were performed using SPSS (SPSS, Chicago, IL, USA) for two-way ANOVA to detect main effects of diet and MθTLR4 genotype on specific phenotypic measures as well as the interactions between these effects. The Bonferroni procedure was used for post-hoc analyses. Prism 4 software (GraphPad, La Jolla, CA, USA) was used to perform Student’s t test (comparing mice with Tlr4 +/+ versus Tlr4 −/− bone marrow for each diet). Data are expressed as mean ± SEM and were considered significant at p < 0.05.

Results

Use of BMT to develop a model with macrophage-specific deletion of Tlr4

Ay/a;Ldrl −/− mice were used as bone marrow recipients because of their susceptibility to hyperlipidaemia, insulin resistance, obesity and atherosclerosis [26, 27]. These mice were lethally irradiated followed by transplantation with marrow from C57BL/6 or Tlr4 −/− donors (Electronic supplementary material [ESM] Fig. 1) to create recipient mice with Tlr4 +/+ marrow (MθTLR4+/+) and recipient mice with Tlr4 −/− marrow (MθTLR4−/−) respectively. At 4 weeks post-BMT, mice were placed on one of four experimental diets (Table 1) for 12 weeks. All diets were high in cholesterol (0.15%) and sucrose (341 g/kg diet). The low-fat diet was low in total fat (12%) and energy (16.5 kJ/g) and used olive oil as the fat source, providing 14.2% of fat from SFAs, 72.1% from monounsaturated fatty acids (MUFAs) and 13.6% of fat from PUFAs. The other three diets were high in total fat (41%) and energy (19.6 kJ/g). The high-fat diet also used olive oil as the fat source; thus the fatty acid composition was the same as the low-fat diet, but the total fat content was higher. The HFSFA contained cocoa butter as the fat source resulting in 62.5% SFAs, 34.4% MUFAs and 3% PUFAs. Finally, the HFSFA with added fish oil (HFSFA+FO) diet also used cocoa butter as the fat source, but was supplemented with menhaden oil, providing 56% SFAs, 30.4% MUFAS and 13.6% PUFAs (Table 1).

Body composition of recipient mice

Body weights were measured weekly and total body fat was analysed biweekly (Fig. 1). We observed diet effects on weight gain and adiposity, such that the HFSFA+FO mice gained the most weight and had the greatest total adipose tissue, as well as the largest perigonadal, perirenal and subscapular fat pads (p < 0.005) (Table 2). MθTLR4−/− mice fed low-fat diets tended to weigh less and have less total fat than MθTLR4+/+ mice; however, at death there were no significant differences between recipients of Tlr4 +/+ and Tlr4 −/− marrow (Fig. 1a,e). In contrast, the MθTLR4−/− mice fed any of the three high-fat diets demonstrated modest but non-significant increases in body weight (Fig. 1b–d) and total body fat (Fig. 1f–h) compared with MθTLR4+/+ mice. Upon killing, no significant differences were seen in total body weight, total fat mass or perigonadal, perirenal or subscapular fat pad weight between MθTLR4+/+ and MθTLR4−/− mice on any of the diets (Table 2).

Fig. 1
figure 1

Total body weight and fat over 12 weeks 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 circles) or Tlr4 −/− (white symbols) marrow and had been fed low-fat diet (a, e), high-fat diet (b, f), HFSFA diet (c, g) and HFSFA+FO diet (d, h). Data are expressed as the mean ± SEM (n = 5–10 mice per group). *p < 0.05

Full size image
Table 2 Body composition of recipient mice after 12 weeks on respective diets
Full size table

Insulin sensitivity in HFSFA fed mice

Euglycaemic–hyperinsulinaemic clamps were performed on mice with or without macrophage TLR4 that were fed the HFSFA diet. Arterial glucose levels were clamped at similar levels in both groups (Fig. 2a). Glucose infusion rates required to maintain euglycaemia were not significantly different between the two groups, suggesting similar levels of insulin sensitivity (Fig. 2b). A bolus infusion of 2-deoxy[14C]glucose was given during the clamp to determine R g, a measure of tissue glucose uptake. We saw no significant differences in the uptake of glucose in skeletal muscle from mice in the presence or absence of macrophage Tlr4 (Fig. 2c). R g was also not different in adipose tissue or brain retrieved from MθTLR4+/+ or MθTLR4−/− mice fed HFSFA diet (Fig. 2c).

Fig. 2
figure 2

Euglycaemic–hyperinsulinaemic 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 (R g) (c) during a 120-min euglycaemic–hyperinsulinaemic clamp in chronically catheterised, conscious MθTLR4+/+ (black bars) and MθTLR4−/− (white bars) mice fed a HFSFA diet. Mice were fasted for 5 h before the experiment. Data are mean ± SEM for (n = 4–5 mice per group). Gast gastrocnemius muscle, SVL superficial vastus lateralis

Full size image

Fatty acid composition of WAT

Gas chromatography analyses of WAT revealed that the fatty acid composition of triacylglycerol reflected the dietary fat sources, but was not influenced by the absence of macrophage Tlr4 (ESM Fig. 2). The total triacylglycerol portion of WAT from mice fed the low-fat diet consisted of 21% SFAs (14:0, 16:0 and 18:0 fatty acids), 73% MUFAs (16:1 and 18:1 fatty acids) and 6% PUFAs (18:2 fatty acids). Similarly, WAT from mice fed the high-fat diet contained 16% SFAs, 77% MUFAs and 7% PUFAs. WAT from the HFSFA fed mice had a slight increase in SFAs and decrease in PUFAs (25% SFAs, 72% MUFAs, 3% PUFAs). As expected, WAT from mice fed HFSFA+FO diet were the only ones to contain the long-chain PUFAs eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6), while displaying 33% SFAs, 60% MUFAs and 7% PUFAs (18:2, 20:4, 20:5, 22:5 and 22:6 fatty acids).

Plasma variables of recipient mice

After 12 weeks on their respective diets, plasma was collected from overnight fasted mice and used to analyse total cholesterol, total triacylglycerol, NEFA, glucose, insulin and leptin levels (Table 3). Because of the high cholesterol content of all the diets and the Ldrl deficiency of recipient mice, plasma lipids were elevated by all four diets. Dietary effects were observed for the degree of hyperlipidaemia (p < 0.001), with the HFSFA+FO mice having the lowest total cholesterol and NEFA levels. NEFA levels were also significantly lower in the HFSFA group than in the high-fat and low-fat groups. Blood glucose levels were highest in HFSFA and HFSFA+FO fed mice. Likewise, plasma insulin levels were significantly higher in these groups than in the groups fed low-fat and high-fat diets (p < 0.01). Finally, in agreement with the increased body weight and adiposity, leptin levels were highest in the HFSFA+FO groups (p < 0.00001). Despite these main effects of the different diets, we saw no significant differences in any of the plasma variables between MθTLR4+/+ and MθTLR4−/− mice on any of the diets.

Table 3 Plasma variables of recipient mice at death
Full size table

Macrophage accrual in adipose tissue of recipient mice

Paraffin sections of perigonadal WAT from MθTLR4+/+ and MθTLR4−/− mice stained with an antibody for the macrophage surface marker F4/80 demonstrated no overt differences between MθTLR4+/+ and MθTLR4−/− mice (Fig. 3a–h). However, quantitative analysis of mRNA expression of F4/80 (also known as Emr1) in adipose tissue revealed that when on the low-fat diet, MθTLR4−/− mice had significantly less F4/80 gene expression than MθTLR4+/+ mice (Fig. 3i; p < 0.05). The MθTLR4−/− mice also demonstrated a trend toward a reduction in Cd68 expression (Fig. 3m). This difference was not seen in mice challenged with any of the three high-fat diets (Fig. 3j–l, n–p).

Fig. 3
figure 3

F4/80 staining and macrophage expression in adipose tissue. WAT sections from mice fed low fat (a, e), high fat (b, f), HFSFA (c, g) and HFSFA+FO (d, h) diets were immunostained with an antibody for F4/80, a macrophage surface marker. Images are shown at ×10 magnification for mice receiving Tlr4 +/+ (a–d) and Tlr4 −/− (e–h) marrow. RNA was isolated from the perigonadal 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. Black bars, mice receiving Tlr4 +/+ marrow; white bars, mice receiving Tlr4 −/− marrow. Data are expressed as mean ± SEM (n = 5–10 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: *p < 0.05

Full size image

Adipose tissue inflammation of recipient mice

To determine the inflammatory status of adipose tissue in response to the presence or absence of macrophage Tlr4, real-time RT-PCR was used to analyse gene expression of several inflammatory genes. Dietary effects were seen for both Mip-1α (also known as Ccl3) and Saa, whereby the HFSFA+FO mice had significantly greater expression of these inflammatory genes than the low-fat and HFSFA groups (p < 0.05). Comparisons between MθTLR4−/− mice and MθTLR4+/+ mice demonstrated that Mip-1α mRNA levels were significantly lower in MθTLR4−/− than in MθTLR4+/+ mice when fed a low-fat diet (p < 0.05) (Fig. 4a). Similarly, Saa3 and Tlr4 expression levels were reduced in MθTLR4−/− mice (p < 0.01) (Fig. 4e,i). When mice were challenged with any of the three high-fat diets, there were no significant differences in Mip-1α (Fig. 4b–d), Saa3 (Fig. 4f–h) or Tlr4 (Fig. 4j–l) mRNA levels among MθTLR4−/− mice and MθTLR4+/+ mice.

Fig. 4
figure 4

Inflammation in WAT. Local inflammation was determined in WAT by analysing gene expression using real-time RT-PCR. Mip-1α (a–d), Saa3 (e–h) and Tlr4 (i–l) mRNA expression were measured in mice fed their respective diets (low fat a, e, i; high fat b, f, j; HFSFA c, g, k; HFSFA+FO d, h, l) Data are expressed as mean ± SEM (n = 5–10 mice per group). 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: *p < 0.05, **p < 0.01

Full size image

Classical versus alternative macrophage marker phenotype of adipose tissue from mice fed low-fat diet

To further characterise the macrophages present in adipose tissue, the gene expression of classical macrophage markers (M1) versus alternative activation macrophage markers (M2) was assessed by real-time RT-PCR in perigonadal fat pads. The HFSFA+FO mice demonstrated a significant dietary effect by displaying elevated levels of both M1 and M2 compared with mice fed the other diets (data not shown). There were no significant differences in expression of M1 markers such as Tnf-α (also known as Tnf), Il-12 (also known as Il12), monocyte chemoattractant protein-1 and inducible nitric oxide synthase or M2 markers such as arginase, mannose receptor, macrophage galactose N-acetyl-galactosamine specific lectin 1 (Mgl1) and Mgl2 in mice with or without MθTLR4 that were on a low-fat diet (Fig. 5).

Fig. 5
figure 5

M1 and M2 classification of ATMs. Markers of M1 and M2 macrophages were determined in WAT by analysing gene expression. M1: a Tnf-α, c Il-12, e Mcp-1 and g iNos, and for M2 macrophages. M2: b arginase, d mannose receptor, f Mgl1 and h Mgl2. Black bars, mice receiving Tlr4 +/+ marrow; white bars, mice receiving Tlr4 −/− marrow. Data are shown only from mice fed the low-fat diet and are expressed as mean±SEM (n = 5–10 mice per group)

Full size image

Atherosclerotic lesion area of recipient mice

To quantify atherosclerotic lesion area, hearts were sectioned at the aortic root and neutral lipids were stained with Oil-Red-O (Fig. 6a–h). The mice fed the low-fat diet displayed the largest lesion areas (Fig. 6i) compared with mice fed the high-fat diets (Fig. 6j–l). In accordance with their reduced plasma lipid levels, mice fed the HFSFA+FO diet were protected from atherosclerotic lesion development (Fig. 6l). In mice on low-fat diet, MθTLR4−/− mice demonstrated smaller lesions (130,998 ± 15,719 μm2) than MθTLR4+/+ mice (176,856 ± 11,671 μm2; p < 0.05) (Fig. 6i). There were no significant differences in lesion areas between mice with or without macrophage Tlr4 that were fed the high-fat diets (Fig. 6j–l).

Fig. 6
figure 6

Quantification of lesion area. Hearts were sectioned at the aortic root and atherosclerotic lesions were stained using Oil-Red-O for low fat (a, e), high fat (b, f), HFSFA (c, g) and HFSFA+FO (d, h) diets. Images are shown at ×10 magnification for mice receiving Tlr4 +/+ (a–d) and Tlr4 −/− (e–h) marrow. Lesion areas were quantified using Kinetic Histometrix software (n = 5–10 mice per group): low-fat (i), high-fat (j), HFSFA (k) and HFSFA+FO (l). Data are presented as mean ± SEM. *p < 0.05

Full size image

Discussion

Our results support the possibility of a role for macrophage Tlr4 in regulating ATM content and inflammation in the presence of low-fat diet feeding. Despite an absence of differences in body weight, total fat or perigonadal fat pad weight, MθTLR4−/− mice fed a diet low in total fat displayed decreased ATM accumulation as evidenced by F4/80 mRNA expression (Fig. 3). MθTLR4−/− mice also exhibited decreased local inflammation with significantly lower Mip-1α and Saa3 expression levels in WAT (Fig. 4), although significant differences were not detected for other inflammatory markers tested (Fig. 5). These data support a capacity for macrophage Tlr4 in regulating macrophage accumulation and inflammation in WAT, even in the absence of differences in body weight and adipose tissue mass. In addition, our studies are the first to demonstrate that deficiency of macrophage Tlr4 can protect against atherosclerotic lesion formation.

Several studies have investigated the effects of global TLR4 deficiency on body composition, adipose tissue physiology, inflammation and insulin resistance in vivo [18, 23–25, 31]. These investigators found that the absence of whole-body TLR4 results in a significant decrease in inflammation and insulin resistance; however, conflicting results with regard to body weight and fat gain, as well as ATM accumulation, have been reported [18, 23–25, 31]. Similar to studies involving global Tlr4 deletions [18], our results involving macrophage Tlr4 deletion were specific to female mice, suggesting a sexual dimorphism in the effects of macrophage Tlr4. Our studies also demonstrate that dietary fat can modulate metabolic responses to macrophage Tlr4 deficiency.

Previous reports have established that in the absence of whole-body TLR4, mice fed high-fat diets or subjected to lipid infusion are protected against insulin resistance as evidenced by glucose tolerance tests, insulin signalling and euglycaemic–hyperinsulinaemic clamps [18, 23–25]. Because SFAs are the key ligands for TLR4, our current studies investigated the more specific question of the effect of macrophage Tlr4 on insulin sensitivity in the presence of a high-fat diet rich in SFAs. However, using highly sensitive euglycaemic–hyperinsulinaemic clamp techniques, we demonstrated that macrophage Tlr4 exerts little to no effect on insulin sensitivity when this mouse model is challenged with a HFSFA diet (Fig. 2).

While our results provide evidence supporting a role for macrophage Tlr4 in ATM content and inflammation, these effects were apparent only in the context of low-fat diet feeding and were only moderate. There are several reasons why the impact of high-fat diet feeding may not have been affected by macrophage Tlr4 deficiency in this model system. First, it is possible that in the presence of a high-fat diet, the system is challenged to a point that subtle differences in ATM content are no longer detected when only macrophage Tlr4 expression is different between groups. Furthermore, dietary cholesterol itself can contribute to macrophage accumulation in WAT [32], while the interaction between dietary cholesterol and fatty acids is unexplored. Second, our studies used bone marrow transplantation, which results in suppression of body weight gain and fat expansion and could have influenced the results in our high-fat diet groups. Third, the source and type of SFAs are also important. In our studies, we used cocoa butter as a fat source rich in SFAs. Cocoa butter is almost solely composed of SFAs; however, they are medium-chain SFAs that are absorbed and metabolised differently than long-chain SFAs, which are more prominent in animal fat sources. Fourth, because we were also interested in atherosclerotic lesion development, we performed these studies in Ldlr −/− mice. It is possible that the LDLR is also important for dietary fatty acid interactions and may influence macrophage recruitment. Fifth and lastly, it should also be noted that Ay/a mice develop adult-onset obesity due to hyperphagia resulting from ectopic expression of the agouti protein that acts as an antagonist of the melanocortin 4 receptor [33, 34]. Thus, it cannot be ruled out that the effects of ectopic expression of the agouti protein might influence results seen in this mouse model. Although more work is required to understand the role of macrophage Tlr4 in macrophage recruitment to WAT and the artery wall, our data demonstrate that the absence of macrophage Tlr4 exerts effects on macrophage accrual under certain dietary conditions.

Not only is macrophage content in WAT important, the classification of macrophages provides an indication of their activation status. M1 polarised macrophages are pro-inflammatory, while M2 polarised macrophages are anti-inflammatory. We had anticipated that absence of macrophage Tlr4 might cause WAT from MθTLR4−/− mice to have lower expression of M1 markers and higher expression of M2 markers; however, our results revealed no differences between MθTLR4+/+ and MθTLR4−/− mice (Fig. 5). Thus, in this mouse model, expression of Tlr4 by ATMs does not appear to influence the recruitment of one type of macrophage over another (M1 vs M2), although inflammatory markers such as Mip-1α and Saa were reduced in WAT of MθTLR4−/− mice. While our data do not suggest a role for macrophage Tlr4 in M1 versus M2 phenotype in WAT in this model, a role of Tlr4 in general in M1/M2 responses cannot be ruled out considering the importance of both Tlr4 and M1/M2 classification in the initiation of an inflammatory response.

We have previously demonstrated that fish oil supplementation of an olive oil-based diet increases body fat mass and decreases plasma lipids, atherosclerosis, ATMs and inflammation in Ldlr −/− mice [35]. In the current study, feeding of the BMT recipient Ay/a;Ldlr −/− mice with the HFSFA+FO diet resulted in many of the same outcomes, with the exception of beneficial effects in WAT. In fact, HFSFA+FO feeding appeared to induce the greatest amount of inflammatory macrophage accumulation in WAT (Fig. 4). The differences between these two studies could be explained by the base fat source of the diet, the effect of BMT or the extent of obesity between the two models. Taken together our studies demonstrate that fish oil supplementation has beneficial effects on plasma lipids and atherogenesis regardless of the other components of the diet, but that it cannot ameliorate metabolic disorders related to adipose tissue when the diet is also rich in SFAs. This is an important distinction when considering supplementation with fish oil for the treatment of metabolic diseases.

Several studies have suggested a role for TLR4 in atherosclerotic lesion area development in mice [36–39]. In addition, patients heterozygous or homozygous for two single nucleotide polymorphisms (Asp299Gly and Thr399Ile) are hyporesponsive to TLR4 activation and as a result are less susceptible to atherosclerosis [40–42]. Our studies show the novel finding that Ay/a;Ldlr −/− mice fed a diet low in total fat supplemented with cholesterol develop smaller lesions in the absence of macrophage Tlr4. These effects were lost when the mice were challenged with any of the three high-fat diets. Previous work from our laboratory has shown that while hyperlipidaemia was critical in macrophage influx into the artery wall, obesity was a better predictor of macrophage entrenchment in WAT [26, 27]. The current studies suggest that macrophage Tlr4 plays a definitive role in macrophage entry into the artery wall and WAT, providing a commonality in mechanisms of macrophage recruitment to these two tissues.

The low-fat diet was low in total fat, yet mice fed this diet displayed the highest plasma lipid levels (Table 3) and greatest atherosclerotic lesion areas (Fig. 6). While the low-fat diet was low in total fat, all of the diets contained equal amounts of added cholesterol and sucrose. The added cholesterol and high carbohydrates are likely to have contributed to the resulting hyperlipidaemia and large atherosclerotic lesions seen in animals fed the low-fat diet. A similar effect was recently noted by Hartvigsen and colleagues, who showed that a diet high in cholesterol but low in fat caused hyperlipidaemia and atherosclerotic lesion formation in Ldlr −/− mice [43]. Together, our data suggest that complex interactions exist between dietary levels of total fat, fatty acid composition, cholesterol and sucrose, resulting in differences in plasma lipids and atherosclerosis (Table 3, Fig. 6).

Our current studies establish a potential role for macrophage TLR4 in metabolic diseases such as obesity and cardiovascular disease. Macrophage Tlr4 deletion protects against macrophage accumulation in WAT and the resulting pro-inflammatory response that follows, as well as macrophage accumulation in the artery wall and the resulting formation of atherosclerotic lesions. These results suggest a specific capacity for macrophage Tlr4 to influence macrophage accrual and inflammation in pathological conditions related to obesity.