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Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity.

Gutierrez DA, Kennedy A, Orr JS, Anderson EK, Webb CD, Gerrald WK, Hasty AH - Diabetes (2011)

Bottom Line: Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed.Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population.The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Objective: Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue-specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance.

Research design and methods: Mice with global or hematopoietic CCR2 deficiency (CCR2(-/-) and BM-CCR2(-/-), respectively) and wild-type controls (CCR2(+/+) and BM-CCR2(+/+), respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed.

Results: Flow cytometry analysis showed that two different populations of F4/80(+) myeloid cells (CD11b(lo)F4/80(lo) and CD11b(hi)F4/80(hi)) accumulated in the adipose tissue of CCR2(-/-) and BM-CCR2(-/-) mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b(hi)F4/80(hi) population was detected in the CCR2(+/+) and BM-CCR2(+/+) controls. After 20 weeks of HFD feeding, the CD11b(lo)F4/80(lo) cells were no longer present in the adipose tissue of CCR2(-/-) mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b(lo)F4/80(lo) cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue.

Conclusions: The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

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BM-CCR2−/− mice have decreased immune infiltration and macrophage marker expression after extended periods of HFD feeding. Adipose tissue was collected from BM-CCR2+/+ and BM-CCR2−/− mice fed an HFD for 6, 12, or 20 weeks and used to assess macrophage infiltration by histological and gene expression analysis. A–F: Representative TBO-stained images from mice as indicated. G: CLS in TBO-stained adipose tissue sections were quantified from eight different fields per mouse and presented as number of CLS per field (mean ± SEM; n = 5–7 mice per group). H–J: RNA was isolated from adipose tissue and used for gene expression analysis by real-time RT-PCR. The expression of the macrophage markers Emr1, Cd68, Itgam, and Itgax was assessed after 6, 12, and 20 weeks of HFD feeding and plotted as expression relative to the BM-CCR2+/+ group of each time point (mean ± SEM; n = 7–10 mice per group). *P < 0.05 compared with the 20-week BM-CCR2+/+ group. (A high-quality color representation of this figure is available in the online issue.)
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Figure 2: BM-CCR2−/− mice have decreased immune infiltration and macrophage marker expression after extended periods of HFD feeding. Adipose tissue was collected from BM-CCR2+/+ and BM-CCR2−/− mice fed an HFD for 6, 12, or 20 weeks and used to assess macrophage infiltration by histological and gene expression analysis. A–F: Representative TBO-stained images from mice as indicated. G: CLS in TBO-stained adipose tissue sections were quantified from eight different fields per mouse and presented as number of CLS per field (mean ± SEM; n = 5–7 mice per group). H–J: RNA was isolated from adipose tissue and used for gene expression analysis by real-time RT-PCR. The expression of the macrophage markers Emr1, Cd68, Itgam, and Itgax was assessed after 6, 12, and 20 weeks of HFD feeding and plotted as expression relative to the BM-CCR2+/+ group of each time point (mean ± SEM; n = 7–10 mice per group). *P < 0.05 compared with the 20-week BM-CCR2+/+ group. (A high-quality color representation of this figure is available in the online issue.)

Mentions: CLS, which are an accumulation of immune cells around presumably dead adipocytes, were used to quantify immune cell infiltration in TBO-stained sections of adipose tissue (Fig. 2A–F). There were no differences in numbers of CLS between BM-CCR2−/− and BM-CCR2+/+ mice after 6 or 12 weeks of HFD feeding (Fig. 2G). However, there was a significant decrease (P < 0.05) in CLS in the BM-CCR2−/− mice after 20 weeks of HFD feeding (Fig. 2E–F). Further analysis of TBO-stained adipose tissue from mice fed an HFD for 20 weeks showed that the number of CLS is dependent on the body weight of the mouse. There were no differences between weight-matched BM-CCR2−/− and BM-CCR2+/+ mice when they weighed <40 g; conversely, there was a decrease in CLS in the BM-CCR2−/− mice compared with weight-matched BM-CCR2+/+ controls at >45 g body weight (Supplementary Fig. 7). In accordance with our histological data, there were no differences in the expression of CD68 (Cd68), CD11b (Itgam), or CD11c (Itgax) in adipose tissue of BM-CCR2−/− compared with BM-CCR2+/+ mice after 6 or 12 weeks of HFD feeding (Fig. 2H and I); however, the expression of these genes was significantly lower (P < 0.05) for the BM-CCR2−/− mice by the 20-week time point compared with the control group (Fig. 2J). The expression of F4/80 (Emr1) in adipose tissue of BM-CCR2−/− mice was significantly (P < 0.05) increased after 6 weeks and unchanged after 12 weeks, and there was a trend (P < 0.07) toward a decrease after 20 weeks of an HFD in comparison with the BM-CCR2+/+ controls (Fig. 2H–J).


Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity.

Gutierrez DA, Kennedy A, Orr JS, Anderson EK, Webb CD, Gerrald WK, Hasty AH - Diabetes (2011)

BM-CCR2−/− mice have decreased immune infiltration and macrophage marker expression after extended periods of HFD feeding. Adipose tissue was collected from BM-CCR2+/+ and BM-CCR2−/− mice fed an HFD for 6, 12, or 20 weeks and used to assess macrophage infiltration by histological and gene expression analysis. A–F: Representative TBO-stained images from mice as indicated. G: CLS in TBO-stained adipose tissue sections were quantified from eight different fields per mouse and presented as number of CLS per field (mean ± SEM; n = 5–7 mice per group). H–J: RNA was isolated from adipose tissue and used for gene expression analysis by real-time RT-PCR. The expression of the macrophage markers Emr1, Cd68, Itgam, and Itgax was assessed after 6, 12, and 20 weeks of HFD feeding and plotted as expression relative to the BM-CCR2+/+ group of each time point (mean ± SEM; n = 7–10 mice per group). *P < 0.05 compared with the 20-week BM-CCR2+/+ group. (A high-quality color representation of this figure is available in the online issue.)
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Figure 2: BM-CCR2−/− mice have decreased immune infiltration and macrophage marker expression after extended periods of HFD feeding. Adipose tissue was collected from BM-CCR2+/+ and BM-CCR2−/− mice fed an HFD for 6, 12, or 20 weeks and used to assess macrophage infiltration by histological and gene expression analysis. A–F: Representative TBO-stained images from mice as indicated. G: CLS in TBO-stained adipose tissue sections were quantified from eight different fields per mouse and presented as number of CLS per field (mean ± SEM; n = 5–7 mice per group). H–J: RNA was isolated from adipose tissue and used for gene expression analysis by real-time RT-PCR. The expression of the macrophage markers Emr1, Cd68, Itgam, and Itgax was assessed after 6, 12, and 20 weeks of HFD feeding and plotted as expression relative to the BM-CCR2+/+ group of each time point (mean ± SEM; n = 7–10 mice per group). *P < 0.05 compared with the 20-week BM-CCR2+/+ group. (A high-quality color representation of this figure is available in the online issue.)
Mentions: CLS, which are an accumulation of immune cells around presumably dead adipocytes, were used to quantify immune cell infiltration in TBO-stained sections of adipose tissue (Fig. 2A–F). There were no differences in numbers of CLS between BM-CCR2−/− and BM-CCR2+/+ mice after 6 or 12 weeks of HFD feeding (Fig. 2G). However, there was a significant decrease (P < 0.05) in CLS in the BM-CCR2−/− mice after 20 weeks of HFD feeding (Fig. 2E–F). Further analysis of TBO-stained adipose tissue from mice fed an HFD for 20 weeks showed that the number of CLS is dependent on the body weight of the mouse. There were no differences between weight-matched BM-CCR2−/− and BM-CCR2+/+ mice when they weighed <40 g; conversely, there was a decrease in CLS in the BM-CCR2−/− mice compared with weight-matched BM-CCR2+/+ controls at >45 g body weight (Supplementary Fig. 7). In accordance with our histological data, there were no differences in the expression of CD68 (Cd68), CD11b (Itgam), or CD11c (Itgax) in adipose tissue of BM-CCR2−/− compared with BM-CCR2+/+ mice after 6 or 12 weeks of HFD feeding (Fig. 2H and I); however, the expression of these genes was significantly lower (P < 0.05) for the BM-CCR2−/− mice by the 20-week time point compared with the control group (Fig. 2J). The expression of F4/80 (Emr1) in adipose tissue of BM-CCR2−/− mice was significantly (P < 0.05) increased after 6 weeks and unchanged after 12 weeks, and there was a trend (P < 0.07) toward a decrease after 20 weeks of an HFD in comparison with the BM-CCR2+/+ controls (Fig. 2H–J).

Bottom Line: Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed.Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population.The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Objective: Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue-specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance.

Research design and methods: Mice with global or hematopoietic CCR2 deficiency (CCR2(-/-) and BM-CCR2(-/-), respectively) and wild-type controls (CCR2(+/+) and BM-CCR2(+/+), respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed.

Results: Flow cytometry analysis showed that two different populations of F4/80(+) myeloid cells (CD11b(lo)F4/80(lo) and CD11b(hi)F4/80(hi)) accumulated in the adipose tissue of CCR2(-/-) and BM-CCR2(-/-) mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b(hi)F4/80(hi) population was detected in the CCR2(+/+) and BM-CCR2(+/+) controls. After 20 weeks of HFD feeding, the CD11b(lo)F4/80(lo) cells were no longer present in the adipose tissue of CCR2(-/-) mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b(lo)F4/80(lo) cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue.

Conclusions: The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

Show MeSH
Related in: MedlinePlus