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, 98 (4), 467-77

CCR2 Deficiency Leads to Increased Eosinophils, Alternative Macrophage Activation, and Type 2 Cytokine Expression in Adipose Tissue

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CCR2 Deficiency Leads to Increased Eosinophils, Alternative Macrophage Activation, and Type 2 Cytokine Expression in Adipose Tissue

W Reid Bolus et al. J Leukoc Biol.

Abstract

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2(-/-) AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2(-/-) mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2(-/-) bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2(-/-) mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2(-/-) mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation.

Keywords: inflammation; interleukin 5; obesity.

Figures

Figure 1.
Figure 1.. Identification of eosinophils by flow cytometry, Fast Green/Neutral Red, and major basic protein.
eAT from lean WT and CCR2−/− mice was collected, the SVF isolated, and cells sorted by FACS. FACS (A and C) eosinophils (CD11blo, F4/80lo, SiglecF+) and (B and D) macrophages (CD11bhi, F4/80hi, SiglecF) from the SVF of AT of WT (left) and CCR2−/− (right) mice. SSC, Side-scatter. (E–H) Fast Green/Neutral Red staining and (I–L) MBP detection by HRP were performed on eosinophils (E, I, G, and K) and macrophages (F, J, H, and L). Images are representative of n = 2–3 mice/group. The only modification to images was removal of background discoloration and neutralizing to a white background for even comparison.
Figure 2.
Figure 2.. HFD-induced obesity in CCR2−/− mice leads to increased eosinophils in white AT.
White AT from lean and obese WT and CCR2−/− mice was collected, the SVF isolated, and the percentage of eosinophils quantified by flow cytometry. (A) eAT, (B) pAT, (C) mesenteric AT, and (D) subcutaneous AT. Data are presented as means ± sem representing the difference in percent eosinophils between lean and obese for each genotype; n = 5–10 mice/group. *P < 0.05, difference between genotypes of mice on the same diet; **P < 0.005, difference between genotypes of mice on the same diet; ****P < 0.0001, difference between genotypes of mice on the same diet.
Figure 3.
Figure 3.. Localization of eosinophils and macrophages to interstitial spaces or CLSs in eAT.
eAT was collected from lean and obese WT and CCR2−/− mice. AT was stained with SiglecF (green) for eosinophils, F4/80 (red) for macrophages, and DAPI (blue) for nuclei and imaged by confocal immunofluorescence microscopy, visualized as a computer-generated 3D rendering. Interstitially spaced macrophages and eosinophils from lean (A) WT and (D) CCR2−/− mice and from obese (B) WT and (E) CCR2−/− mice. CLS-localized macrophages and eosinophils from obese (C) WT and (F) CCR2−/− mice. (G) Magnified image of juxtaposed macrophage and eosinophil for comparison. (H and I) High magnification images of CCR2−/− AT eosinophils exhibiting prototypical multilobular or doughnut-shaped nuclei. Arrowheads demonstrate some of the eosinophils visible in each image. Images are representative of 3 images/mouse, with n = 4 mice/group.
Figure 4.
Figure 4.. Quantification of macrophages and eosinophils in CLS or interstitially spaced regions of eAT.
Immune cell localization from images in Fig. 3 was quantified by percent of DAPI+ cells (white bars) and absolute cell number per high-power field (HPF; gray bars). (A) Macrophages in eAT of lean mice. (B) Macrophages in eAT of obese mice.; (C) Eosinophils in eAT of lean mice. (D) Eosinophils in eAT of obese mice. Data are shown as the means ± sem, with n = 3–4 mice/group. *P < 0.05, difference between locations within the same genotype; **P < 0.005, difference between locations within the same genotype; ***P < 0.0005, difference between locations within the same genotype; ^P < 0.05, difference between genotypes within the same location; ^^P < 0.005, difference between genotypes within the same location; ^^^P < 0.0001, difference between genotypes within the same location.
Figure 5.
Figure 5.. Bone marrow-derived CCR2−/− eosinophils display increased expression of key eosinophil genes during differentiation in vitro.
Bone marrow cells were collected from lean WT and CCR2−/− mice and differentiated into eosinophils in vitro, according to Materials and Methods. In brief, cells were cultured with SCF and FLT3 ligand for 4 days and subsequently cultured with IL-5 for 8 days. Expression of eosinophil-specific genes (A) Epx, (B) Prg2, (C) Il5ra, (D) Ccr3, (E) Gata1; eosinophil-secreted cytokines (F) Il4 and (G) Il6; and noneosinophil specific gene (H) Mpo throughout differentiation. Cytospin Hemacolor images of bone marrow-derived eosinophils at 10 days of differentiation from (I) WT and (K) CCR2−/− mice. Percent purity of eosinophils from (J) WT and (L) CCR2−/− at 10 days of differentiation. (M) Total cell growth rate throughout differentiation expressed as change in cell number over time. Data are normalized to day 0 for each genotype and are presented as means ± sem, with n = 2 for day 0, and n = 4 for days 4–12. *P < 0.05, difference between genotypes; **P < 0.01, difference between genotypes; ***P < 0.0005, difference between genotypes; ****P < 0.0001, difference between genotypes.
Figure 6.
Figure 6.. Eosinophil-chemoattractant expression in AT.
Total RNA was isolated from eAT of lean and obese WT and CCR2−/− mice and used for real-time RT-PCR analyses. Relative expression of (A) Il5, (B) Ccr3, (C) Ccl11, (D) Ccl24, (E) Ccl3, and (F) Ccl5. Data are normalized to lean WT control and presented as means ± sem, with n = 5–7 mice/group. *P < 0.05, difference between genotypes of mice on the same diet; ^P < 0.05, difference between diets in mice of the same genotype.
Figure 7.
Figure 7.. AT eosinophil accumulation is associated with type 2 cytokine expression.
Total RNA was isolated from eAT of lean and obese WT and CCR2−/− mice and used for real-time RT-PCR analysis. Relative expression of (A) Il4, (B) Il13, (C) Foxp3, and (D) Arg1. Data are presented as means ± sem, with n = 5–7 mice/group. *P < 0.05, difference between genotypes of mice on the same diet; **P < 0.01, difference between genotypes of mice on the same diet; ^^P < 0.01, difference between diets in mice of the same genotype; ^^^P < 0.001, difference between diets in mice of the same genotype.
Figure 8.
Figure 8.. CCR2 deficiency counteracts typical AT M1 macrophage polarization associated with obesity.
F4/80hi;CD11bhi;SiglecF macrophages were sorted from the eAT SVF of lean and obese WT and CCR2−/− mice and used for real-time RT-PCR analysis. Relative expression of (A) Itgax (CD11c), (B) Retnla (Fizz1), (C) Arg1 (arginase), and (D) Chil3 (Ym1). Data are normalized to lean WT control and presented as means ± sem, with n = 4–5 mice/group. *P < 0.05, difference between genotypes of mice on the same diet; **P < 0.01, difference between genotypes of mice on the same diet.

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