Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice

Abstract

Objective: To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity.

Research design and methods: The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment.

Results: Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-alpha and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue.

Conclusions: M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.

FIG. 1.

Effect of HFD and pioglitazone on gene expressions of epididymal fat tissue. Total RNA was isolated from the whole epididymal fat tissue, and real-time RT-PCR for the indicated genes was conducted as described in the research design and methods section. Data were normalized according to 18S mRNA level and presented as a value relative to that for normal diet–fed mice. The results are shown as means ± SE for five to eight animals per group. *P < 0.05; **P < 0.01.

FIG. 2.

Gene profiles of macrophages positive or negative for CD11c and CD206 in epididymal fat tissue. Cells in the SVF of epididymal fat tissue from mice fed a HFD for 12 weeks were analyzed using flow cytometry. F4/80-positive cells were further analyzed with anti-CD11c and anti-CD206 antibodies. A: Representative flow cytometry results. Blue dots show isotype control. Total RNA was isolated from F4/80-positive/CD11c-positive/CD206-negative cells or F4/80-positive/CD11c-negative/CD206-positive cells. B: Real-time RT-PCR for the indicated genes was conducted as described in the research design and methods section. Data were normalized according to the 18S mRNA level. The results are shown as the means ± SE for four to seven animals per group. *P < 0.05; **P < 0.01. The correlation between CD206 expression and MglI expression was examined. C: Representative results.

FIG. 3.

Effect of HFD and pioglitazone treatment on the number of M1/M2 macrophages in epididymal fat tissue. Cells in the SVF of epididymal fat tissue were analyzed using flow cytometry as described in Fig. 2. A: Representative results of flow cytometry are shown. B: The cell numbers in the F4/80-positive fraction. C and D: F4/80-positive/CD11c-positive/CD206-negative fraction (■) or F4/80-positive/CD11c-negative/CD206-positive fraction (□) were counted as described in the research design and methods section. C: The cell numbers in the CD11c-positive/CD206-negative and CD11c-negative/CD206-positive fractions are shown according to the percentage of each fraction per total F4/80-positive cells. D: The cell number in each fraction from 1 g of epididymal fat tissue. Epididymal fat tissues were stained with anti-F4/80 (E) or anti-CD11c antibody (F and G). E and F: The number of CLSs was counted as described in the research design and methods section. The results are shown as the means ± SE for five to eight animals per group. *P < 0.05; **P < 0.01. G: Representative images with anti-CD11c antibody. Bar = 200 μm.

FIG. 4.

Effect of pioglitazone on gene expressions in CD11c-positive and -negative fractions. Macrophages in the F4/80-positive/CD11c-positive/CD206-negative fraction or the F4/80-positive/CD11c-negative/CD206-positive fraction of epididymal fat tissue were sorted using flow cytometry as described in Fig. 2. Total RNA was isolated, and real-time RT-PCR for the indicated genes was conducted as described in the research design and methods section. Data were normalized according to the 18S mRNA level. The results are shown as the means ± SE for six animals per group. *P < 0.05.

FIG. 5.

Effect of HFD and pioglitazone on gene expressions of adipocyte fraction in epididymal fat tissue. Total RNA from the adipocyte fraction was isolated, and real-time RT-PCR for the indicated genes was conducted as described in the research design and methods section. Data were normalized according to the 18S mRNA level. The results are shown as the means ± SE for six animals per group. *P < 0.05.

FIG. 6.

Effect of IL-10 overexpression on macrophage genes in epididymal fat tissue. Mice were fed with a normal chow (NC) or a HFD and were subsequently injected with either Ad-hIL-10 (IL-10) or a control vector (R2), then killed 4 days after injection. Purification of the mRNA and real-time RT-PCR for the indicated genes were performed as described in the research design and methods section. F: Epididymal fat tissues were stained with anti-F4/80 antibody. Then the number of CLSs was counted, as described in the research design and methods section. Results are shown as the mean ± SE of at least four mice per group. *P < 0.05; **P < 0.01.