PPAR gamma is highly expressed in F4/80(hi) adipose tissue macrophages and dampens adipose-tissue inflammation

Abstract

Macrophage infiltration into adipose tissue is a hallmark of obesity. We recently reported two phenotypically distinct subsets of adipose tissue macrophages (ATM) based on the surface expression of the glycoprotein F4/80 and responsiveness to treatment with a peroxisome proliferator-activated receptor (PPAR) gamma agonist. Hence, we hypothesized that F4/80(hi) and F4/80(lo) ATM differentially express PPAR gamma. This study phenotypically and functionally characterizes F4/80(hi) and F4/80(lo) ATM subsets during obesity. Changes in gene expression were also examined on sorted F4/80(lo) and F4/80(hi) ATM by quantitative real-time RT-PCR. We show that while F4/80(lo) macrophages predominate in adipose tissue of lean mice, obesity causes accumulation of both F4/80(lo) and F4/80(hi) ATM. Moreover, accumulation of F4/80(hi) ATM in adipose tissue is associated with impaired glucose tolerance. Phenotypically, F4/80(hi) ATM express greater amounts of CD11c, MHC II, CD49b, and CX3CR1 and produce more TNF-alpha, MCP-1, and IL-10 than F4/80(lo) ATM. Gene expression analyses of the sorted populations revealed that only the F4/80(lo) population produced IL-4, whereas the F4/80(hi) ATM expressed greater amounts of PPAR gamma, delta, CD36 and toll-like receptor-4. In addition, the deficiency of PPAR gamma in immune cells favors expression of M1 and impairs M2 macrophage marker expression in adipose tissue. Thus, PPAR gamma is differentially expressed in F4/80(hi) versus F4/80(low) ATM subsets and its deficiency favors a predominance of M1 markers in WAT.

Fig. 1

F4/80^hi and F4/80^lo populations in lean and obese mice. White adipose tissue (WAT) was extracted from 10-week old lean (n=7) and obese mice (n=6) and flow cytometry was run on the stromal vascular fraction (SVF) obtained by digestion with collagenase and fractionation with percoll. (A) Representative density plots, (B) macrophage infiltration and (C) body weights. Data points with different asterisks indicate a statistically significant difference (P<0.05).

Fig. 2

Time course of adipose tissue macrophage (ATM) infiltration. C57BL6/J mice were fed either high-fat or low-fat diets (HFD vs LFD) for 0, 70, or 140 days. (A) Body weights were recorder for each group and (B) average adipocyte areas from intra-abdominal white adipose tissue were calculated from hemotoxylin and eosin (H&E) stained slides using Image Pro software. (C, D) Intraperitoneal glucose tolerance tests (IPGTT, 2 g glucose/kg body weight) were conducted at all timepoints for an assessment of glucose homeostasis. (E, F) Accumulation of F4/80^hi and F4/80^lo adipose tissue macrophages in the stromal vascular fraction (SVF) for each group is depicted. Data points with an asterisk are statistically different from group (P<0.05).

Fig. 3

Phenotype of F4/80^hi and F4/80^lo adipose tissue macrophages (ATMs) in lean and obese mice. Cells were stained with primary antibodies for surface proteins F4/80, CD11b, CX3CR1, CCR2, CD11c, Ly6C, CD49b and MHC II. (A-F) Representative histograms for F4/80^lo (blue) and F4/80^hi (green) ATMs from db/db mice are provided, and (G, H) the median fluorescence intensities (MFIs) for both lean and obese mice were statistically analyzed. Data points with different asterisks indicate a statistically significant difference (P<0.05).

Fig. 4

Heterogeneity in the F4/80^lo population. Ly6C^hiF4/80^lo adipose tissue macrophages (ATM) in lean and obese db/db mice were analyzed for alongside F4/80^loLy6C^lo/neg ATMs. (A) The percent of Ly6C^hi ATM and (B) the average median fluorescence intensities (MFIs) for chemokine receptors CCR2, CX3CR1, and CCR7 for both subsets were assessed. Data points with different asterisks indicate a statistically significant difference (P<0.05).

Fig. 5

Cytokine production in F4/80^lo and F4/80^hi adipose tissue macrophages (ATMs). The average median fluorescence intensity (MFI) for (A) interleukin-6 (IL-6), (B) tumor necrosis factor α (TNF-α) (C) monocyte chemoattractant protein-1 (MCP-1), and (D) interleukin-10 (IL-10) for F4/80^lo and F4/80^hi ATMs in lean and obese mice was determined. Data points with different asterisks indicate a statistically significant difference (P<0.05).

Fig. 6

Effect of lipopolysaccharide (LPS) stimulation on intracellular cytokine production by F4/80^lo and F4/80^hi adipose tissue macrophages (ATMs). The average median fluorescence intensity (MFI) for (A) interleukin-6 (IL-6), (B) tumor necrosis factor α (TNF-α) (C) monocyte chemoattractant protein-1 (MCP-1), and (D) interleukin-10 (IL-10) for F4/80^lo and F4/80^hi ATMs in lean and obese mice was determined. Data points with different asterisks indicate a statistically significant difference (P<0.05).

Fig. 7

Sorting of F4/80^lo and F4/80^hi adipose tissue macrophages. Stromal vascular cells (SVCs) from 10 db/db mice were enriched for CD11b using magnetic beads, stained with F4/80 PE-Cy7 and CD45 APC-Cy7, and then sorted into F4/80^lo and F4/80^hi fractions (over 98% purity). (A) Representative dot plots for all cells in the stromal vascular fraction (SVF) of white adipose tissue, presorted through CD11b enrichment by magnetic sorting, FACS Aria sorted F4/80^lo, and FACS Aria sorted F4/80^hi ATM are depicted. Gene expression analyses on sorted population was performed for (B) toll-like receptor 4 (TLR-4), (C) tumor necrosis factor-α (TNF-α), (D) peroxisome proliferator-activated receptor γ (PPAR γ), (E) CD36, (F) PPAR δ, and (G) interleukin-4 (IL-4). Data were analyzed as a ratio to the housekeeping gene β-actin. Each bar represents gene expression analyses with the pooled RNA of F4/80^lo and F4/80^hi macrophages sorted from the visceral adipose tissue of 10 db/db mice.

Fig. 8

Effect of deleting PPAR γ in immune cells on the expression of chemokine (C-C motif) ligand 1 and 17 (M1 markers), Arginase 1 (M2 marker) and IL-13 in the white adipose tissue of mice with diet-induced obesity. PPAR γ fl/fl; MMTV-Cre⁺ (tissue-specific PPAR γ null mice) and PPAR γ fl/fl; MMTV-Cre^- (floxed) littermate mice were fed high-fat diets for 28 weeks. Data are presented as least square means ± standard error of 10 observations. Data points with an asterisk are significantly different (P<0.05).