CX3CR1 deficiency does not influence trafficking of adipose tissue macrophages in mice with diet-induced obesity

Abstract

Adipose tissue macrophages (ATMs) accumulate in fat during obesity and resemble foam cells in atherosclerotic lesions, suggesting that common mechanisms underlie both inflammatory conditions. CX(3)CR1 and its ligand fractalkine/CX(3)CL1 contribute to macrophage recruitment and inflammation in atherosclerosis, but their role in obesity-induced adipose tissue inflammation is unknown. Therefore, we tested the hypothesis that CX(3)CR1 regulates ATM trafficking to epididymal fat and contributes to the development of adipose tissue inflammation during diet-induced obesity. Cx(3)cl1 and Cx(3)cr1 expression was induced specifically in epididymal fat from mice fed a high-fat diet (HFD). CX(3)CR1 was detected on multiple myeloid cells within epididymal fat from obese mice. To test the requirement of CX(3)CR1 for ATM trafficking and obesity-induced inflammation, Cx(3)cr1(+/GFP) and Cx(3)cr1(GFP/GFP) mice were fed a HFD. Ly-6c(Low) monocytes were reduced in lean Cx(3)cr1(GFP/GFP) mice; however, HFD-induced monocytosis was comparable between strains. Total ATM content, the ratio of type 1 (CD11c(+)) to type 2 (CD206(+)) ATMs, expression of inflammatory markers, and T-cell content were similar in epididymal fat from obese Cx(3)cr1(+/GFP) and Cx(3)cr1(GFP/GFP) mice. Cx(3)cr1 deficiency did not prevent the development of obesity-induced insulin resistance or hepatic steatosis. In summary, our data indicate that CX(3)CR1 is not required for the recruitment or retention of ATMs in epididymal adipose tissue of mice with HFD-induced obesity even though CX(3)CR1 promotes foam cell formation. This highlights an important point of divergence between the mechanisms regulating monocyte trafficking to fat with obesity and those that contribute to foam cell formation in atherogenesis.

Figure 1

Cx₃cl1 and Cx₃cr1 expression is increased in epididymal fat during high-fat diet (HFD)-induced obesity in mice. (a) Chemokine/receptor gene expression in epididymal (visceral) and inguinal (subcutaneous) adipose tissue from male C57BL/6J mice fed a normal diet (ND; 4.5% fat; n = 4) or HFD (60% fat; n = 4) for 20 weeks. (b) Cx₃cl1 expression in isolated adipocytes (Adipo) and stromal vascular cells (SVCs) within epididymal fat pads from ND and HFD mice (n = 4 per group). (c) Cx₃cl1 expression in differentiated 3T3-L1 adipocytes grown in 5 mmol/l and 25 mmol/l glucose and stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 24 h (n = 3–4 replicates per treatment). (d) Cx₃cl1 and TNF-α expression in bone marrow-derived macrophages (BMMϕ) and cultured SVCs stimulated with LPS (100 ng/ml) for 24 h (n = 3 replicates per treatment). *P < 0.05; **P < 0.01; ***P < 0.001. TNF-α, tumor necrosis factor-α.

Figure 2

CX₃CR1 is expressed on adipose tissue macrophages (ATMs) in epididymal adipose tissue. (a) Identification of GFP⁺ (green) and F4/80⁺ (red) ATMs in epididymal fat from obese Cx₃cr1^+/GFP reporter mice by confocal microscopy. Isolectin (blue) labels endothelial cells and identifies vasculature. (b) GFP⁺ (green) and CD11c⁺ (red) type 1 ATMs in epididymal fat. (c) GFP⁺ (green) and MGL1⁺ (red) type 2 ATMs in epididymal fat. (d) Representative image of unique GFP⁺ (green) cell clusters in hypervascularized regions devoid of adipocytes and F4/80⁺ (red) ATMs. Caveolin (blue) identifies vasculature. Bar = 100 μm; original magnification = ×60. GFP, green fluorescent protein.

Figure 3

Characterization of CX₃CR1/GFP⁺ stromal vascular cells (SVCs) in epididymal adipose tissue of obese Cx₃cr1^+/GFP mice by flow cytometry. Cx₃cr1^+/GFP were fed a high-fat diet (HFD; 60% fat) for 12 weeks before SVCs were isolated from epididymal adipose tissue and phenotyped by flow cytometry. (a) Identification of GFP⁺ SVCs in obese Cx₃cr1^+/GFP (open) and C57BL/6 (shaded) mice. (b) Identification of CD11b^HighF4/80^High (adipose tissue macrophages; ATMs) and CD11b^HighF4/80^Low (immature myeloid cells; iMCs) populations in SVCs from obese mice. (c) Representative histogram showing Ly-6c expression in ATMs (shaded) and iMCs (open). Scatter plots (SSC vs. FSC) of gated ATMs (middle) and iMCs (right) demonstrate the difference in cell size and granularity between the two populations. (d) Distribution of GFP⁺ SVCs from a representative obese Cx₃cr1^+/GFP mouse. GFP expression in ATMs and iMCS is shown. (e) Representative scatter plots showing CD11b⁺GFP⁻ and CD11b⁺GFP⁺ SVCs (left) and F4/80⁺GFP⁻ and F4/80⁺GFP⁺ SVCs (right) within visceral fat from obese Cx₃cr1^+/GFP mice. (f) CD11c⁺GFP⁻ and CD11c⁺GFP⁺ ATMs (left) and CD206⁺GFP⁻ and CD206⁺GFP⁺ ATMs (right) within visceral fat from obese Cx₃cr1^+/GFP mice. (g) CD11c⁺GFP⁻ and CD11c⁺GFP⁺ iMCs (left) and CD206⁺GFP⁻ and CD206⁺GFP⁺ iMCs (right) within visceral fat from from obese Cx₃cr1^+/GFP mice. In e–g, percentage of gated cells is indicated in each quadrant. APC, allophycocyanin; FSC, forward scatter; GFP, green fluorescent protein; PE, R-phycoerythrin; SSC, side scatter.

Figure 4

Effects of high-fat diet (HFD)-induced obesity on blood monocyte populations in Cx₃cr1^GFP/GFP and control mice. Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice were fed a normal diet (ND) or HFD (60% fat) for 12 or 40 weeks before blood leukocytes were examined by flow cytometry. (a) Total circulating CD115⁺ Ly-6g⁻ monocytes in Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice. (b) Representative scatter plots showing the distribution of blood monocyte subsets (Ly-6c^High and Ly-6c^Low) from lean (ND) and obese (12- and 40-weeks HFD) mice. (c,d) Quantification of blood monocyte subsets after gating Ly-6c^High and Ly-6c^Low populations as indicated in b. (e) Relative change in blood monocytes in Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice with HFD-induced obesity. Data are expressed as percentage of total blood leukocytes from lean mice. n = 5–8 per group; *P < 0.05; ***P < 0.001; ^†P < 0.05 vs. ND. GFP, green fluorescent protein.

Figure 5

Cx₃cr1 deficiency does not alter adipose tissue macrophage (ATM) content in visceral fat from lean or obese mice. Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice were fed a normal diet (ND) or high-fat diet (HFD) for 12, 16, 30 or 40 weeks before stromal vascular cells (SVCs) were isolated from epididymal adipose tissue and examined by flow cytometry. (a) ATM content in epididymal fat shown as percentage of total SVCs and normalized to visceral fat mass. (b) Immature myeloid cell (iMC) content in epididymal fat shown as percentage of total SVCs and normalized to visceral fat mass. (c) Distribution of GFP⁺ and GFP⁻ SVCs in visceral fat after 12 weeks of HFD. Data are shown as percentage of total SVCs. (d) ATM content in visceral fat from C57BL/6 and Cx₃cr1^GFP/GFP mice after 20 weeks of HFD. Data are shown as percentage of total SVCs and normalized to visceral fat mass. ND: n = 4 per group; HFD: n = 4–5 per group; ^†P < 0.05 vs. ND. GFP, green fluorescent protein.

Figure 6

Cx₃cr1 deficiency does not alter the distribution of type 1 and type 2 adipose tissue macrophages (ATMs) in epididymal adipose tissue from lean or obese mice. Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice were fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks before stromal vascular cells (SVCs) were isolated from epididymal fat and examined by flow cytometry. (a) Distribution of ATM subsets in visceral fat from Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice shown as percentage of total ATMs. (b) Ratio of type 1 (CD11c⁺) to type 2 (CD206⁺) ATMs in visceral fat after 12- and 40-weeks of HFD. (c) ATM infiltration assessed by immunofluorescence microscopy. Caveolin (green) and Mac2 (red) staining in epididymal fat from obese (12 weeks HFD) mice. Bar = 100 μm. (d) Relative expression of M1 and M2 macrophage markers in visceral fat from Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice after 12 weeks of HFD. (e) Relative expression of a subset of cytokines and chemokines in epididymal fat after 12 weeks of HFD. n = 4–5 mice per group. ^†P < 0.05 vs. ND. CLS, crown-like structures; GFP, green fluorescent protein.

Figure 7

Cx₃cr1 deficiency does not prevent diet-induced metabolic disease. (a) Fasting (6 h) glucose and insulin levels lean Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice and after 23 weeks of high-fat diet (HFD)-induced obesity (n = 8–10 per group). (b) Glucose tolerance tests (GTT) performed in obese Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice after 23 weeks of HFD (n = 12–13 per group). GTT curves for lean (ND) mice (n = 3–4 per group) are shown (circles) for comparison. (c) Insulin tolerance tests (ITT) were performed on Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice (n = 12–13 per group) after 29 weeks of HFD. Data is expressed as % of initial blood glucose levels. (d) Representative hematoxylin and eosin-stained sections of liver biopsies from lean and obese (30 weeks HFD) Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice. Bar = 200 μm. (e) Liver triglyceride content in lean and obese Cx₃cr1^+/GFP and Cx₃cr1^GFP/GFP mice (n = 4 per group). ^†P < 0.05 vs. ND. GFP, green fluorescent protein; ND, normal diet.