Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes

Abstract

Objective: To establish the mechanism of the phenotypic switch of adipose tissue macrophages (ATMs) from an alternatively activated (M2a) to a classically activated (M1) phenotype with obesity.

Research design and methods: ATMs from lean and obese (high-fat diet-fed) C57Bl/6 mice were analyzed by a combination of flow cytometry, immunofluorescence, and expression analysis for M2a and M1 genes. Pulse labeling of ATMs with PKH26 assessed the recruitment rate of ATMs to spatially distinct regions.

Results: Resident ATMs in lean mice express the M2a marker macrophage galactose N-acetyl-galactosamine specific lectin 1 (MGL1) and localize to interstitial spaces between adipocytes independent of CCR2 and CCL2. With diet-induced obesity, MGL1(+) ATMs remain in interstitial spaces, whereas a population of MGL1(-)CCR2(+) ATMs with high M1 and low M2a gene expression is recruited to clusters surrounding necrotic adipocytes. Pulse labeling showed that the rate of recruitment of new macrophages to MGL1(-) ATM clusters is significantly faster than that of interstitial MGL1(+) ATMs. This recruitment is attenuated in Ccr2(-/-) mice. M2a- and M1-polarized macrophages produced different effects on adipogenesis and adipocyte insulin sensitivity in vitro.

Conclusions: The shift in the M2a/M1 ATM balance is generated by spatial and temporal differences in the recruitment of distinct ATM subtypes. The obesity-induced switch in ATM activation state is coupled to the localized recruitment of an inflammatory ATM subtype to macrophage clusters from the circulation and not to the conversion of resident M2a macrophages to M1 ATMs in situ.

FIG. 1.

Resident M2a ATMs in lean mice are MGL1⁺ and are recruited independent of CCR2/CCL2. Immunofluorescence localization of ATMs in adipose tissue using confocal microscopy. Samples from male C57Bl/6 mice fed chow diet. Similar results were obtained from three to five independent mice. Bar = 50 μm. A: MGL1⁺ ATMs are present in interstitial spaces between adipocytes in multiple adipose tissue depots. Caveolin staining (red) delineates adipocytes. B: MGL1⁺ cells (red) are macrophages based on costaining with F4/80 (left panels) and CD11b (right panel). Images from epididymal fat pads. C: Perivascular localization of a portion of MGL1⁺ ATMs. Isolectin (red) labels endothelial cells to highlight vasculature in fat. MGL1⁺ ATMs express IL-10 (D) and CCR2 (E). F: MGL1⁺ ATMs are retained in epididymal adipose tissue from Ccl2^−/− and Ccr2^−/− mice. (Please see http://dx.doi.org/10.2337/db08-0872 for a high-quality digital representation of this figure.)

FIG. 2.

ATMs in clusters in obese mice are MGL1⁻CCR2⁺ and MGL1⁺ ATMs are retained. Characterization of ATMs in high-fat diet (HFD)-fed C57Bl/6 mice. Caveolin staining (blue) demonstrates loss of membrane integrity in lipid droplets. Staining patterns were observed in at least four independent mice. Bar = 50 μm. A and B: Interstitial ATMs are retained with obesity and are uniformly MGL1⁺F4/80⁺CD11b⁺. CD11c (C) and TLR4 (D) are strongly expressed in MGL1⁻ ATMs in clusters. Isolectin binding (E) and CCR2 expression (F) are increased in ATM clusters and do not overlap with MGL1 expression. G: IL-10 expression is retained in interstitial ATMs and is downregulated in ATM clusters. H: Partial loss of caveolin staining in a dying adipocyte surrounded by a thin wall of MGL1⁻CD11c⁺ ATMs. I: Reduced numbers of ATM clusters in epididymal fat in high-fat diet Ccr2^−/− mice. ATM clusters in Ccr2^−/− mice express CD11c similar to obese Ccr2^+/+ mice. J: ATM clusters in Ccr2^−/− mice downregulate MGL1 expression. (Please see http://dx.doi.org/10.2337/db08-0872 for a high-quality digital representation of this figure.)

FIG. 3.

Obesity induces the recruitment of M1 MGL1⁻ ATMs to adipose tissue. A: Accumulation of MGL1⁻ ATMs with high-fat diet (HFD) feeding. SVF was isolated from epididymal fat pads and stained to identify MGL1⁺ and MGL1⁻ F4/80⁺ ATMs. Percentages of total SVF provided from a representative experiment. Similar results were observed for six animals per group. B: Induction of MGL1⁻F4/80⁺ ATMs with obesity is more pronounced in visceral compared with subcutaneous fat. ATM MGL1 subtypes were quantitated by flow cytometry and expressed as a percentage of SVF, total cells per fat pad, and total cells per gram fat. Visceral fat from epididymal fat pads (Epi) were compared with subcutaneous fat (SQ) from the inguinal fat pad. n = 6 per group. Values are expressed as means ± SE. *P < 0.05. **P < 0.005. C: Differential inflammatory gene expression between ATM subtypes shows M2a polarization of MGL1⁺ ATMs. MGL1⁺ and MGL1⁻ ATMs were isolated by fluorescence-activated cell sorting, and RNAs were prepared for real-time RT-PCR analysis. Gene expression is expressed as relative to MGL1⁺ ATMs. M2a genes (left panel) and M1 genes (right panels) were analyzed. n = 4 per group. Values are expressed as means ± SE. *P < 0.05. A.U., arbitrary units; ND, normal diet.

FIG. 4.

MGL1⁻ ATMs are preferentially recruited to clusters with high-fat diet feeding. A and B: Pulse labeling of ATMs demonstrates recruitment of ATMs to clusters and not interstitial sites with high-fat diet feeding. A: High-fat diet–fed C57Bl/6 mice were injected with PKH26 to label ATMs in visceral fat and analyzed by confocal microscopy at the indicated time after injection. Imaging focused either on ATM clusters (top) or interstitial areas (bottom). PKH26⁻ ATMs accumulate in clusters while the interstitial ATMs retain PKH26⁺ as long as 28 days after labeling. B: Quantitation of PKH26⁻F4/80⁺ ATMs during time course. From two to three mice per time point, 25–30 clusters or high-power fields were analyzed and scored for the percentage of PKH26⁻ ATMs. **P < 0.005 vs. interstitial. C and D: MGL1⁺ ATMs preferentially retain PKH26. C: Immunofluorescence on mice 28 days after PKH26 injection, demonstrating the retention of dye in MGL1⁺ ATMs. PKH26⁻ ATMs are primarily MGL1⁻CD11c⁺ cells in clusters. D: Flow cytometry of SVF from mice 28 days after PKH26 injection. PKH26⁺ gates (W0) were defined by comparing ATMs from uninjected (white) versus PKH26 injected (yellow) mice 3 days after injection. PKH26⁺ (R1) and PKH26⁻ (R2) ATMs (F4/80⁺) were gated and examined for MGL1 expression. PKH26⁺ ATMs have high MGL1 expression compared with low MGL1 expression in PKH26⁻ ATMs. Representative experiment is shown from one of three independent experiments. E: Decreased rate of recruitment of ATMs to clusters in obese Ccr2^−/− mice. Ccr2^−/− mice were analyzed 28 days after injection by microscopy and scored for the percentage of PKH26⁻ ATMs in clusters or interstitial areas. *P < 0.05 vs. Ccr2^+/+ mice. (Please see http://dx.doi.org/10.2337/db08-0872 for a high-quality digital representation of this figure.)

FIG. 5.

Differential effects of M1 and M2a macrophages on adipocyte insulin sensitivity and adipogenesis. A: Conditioned medium (CM) from M1 but not M2a macrophages blocks insulin-stimulated glucose uptake. Differentiated 3T3-L1 adipocytes were treated with conditioned media from macrophages treated with vehicle, LPS (10 ng/ml), or IL-4 (10 ng/ml) for 12 h and assayed for insulin-stimulated 2-DG uptake, demonstrating selective inhibition with LPS conditioned medium. No effects on glucose uptake were seen with LPS or IL-4 treatment alone. n = 3 per group. Values are expressed as means ± SE. *P < 0.05. B: Conditioned medium from M1 but not M2a macrophages blocks adipogenesis. Conditioned medium from treated macrophages was added during differentiation of 3T3-L1 adipocytes at different percentages by volume, and cells were stained with Oil-Red-O, demonstrating that M2a macrophages are permissive toward adipogenesis in contrast to M1 macrophages. Similar results were from three experiments, and a representative experiment is shown. C: Downregulation of GLUT4 and insulin receptor (IR) expression with M1 conditioned medium but not M2a or unstimulated macrophage conditioned medium during adipogenesis. Immunoblots of lysates from 3T3-L1 cells treated with conditioned media during differentiation. (Please see http://dx.doi.org/10.2337/db08-0872 for a high-quality digital representation of this figure.)