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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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CD11bloF4/80lo cells have an inflammatory expression profile and a monocytic morphology. CD11bloF4/80lo and CD11bhiF4/80hi cells were isolated by fluorescence-activated cell sorting from CCR2−/− mice fed an HFD for 10 weeks. A: Gating strategy used for cell sorting; CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2). RNA was isolated from the sorted cells and used for gene expression analysis by real-time RT-PCR. B: Gene expression profiles in CD11bloF4/80lo (P2) cells relative to CD11bhiF4/80hi (P1) cells in CCR2−/− mice were assessed (mean ± SEM; n = 4 biological replicates). C: Representative images of H-E–stained CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2) cells. *P < 0.05 compared with CD11bhiF4/80hi. **P < 0.01 compared with CD11bhiF4/80hi. ***P < 0.001 compared with CD11bhiF4/80hi. (A high-quality color representation of this figure is available in the online issue.)
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Figure 5: CD11bloF4/80lo cells have an inflammatory expression profile and a monocytic morphology. CD11bloF4/80lo and CD11bhiF4/80hi cells were isolated by fluorescence-activated cell sorting from CCR2−/− mice fed an HFD for 10 weeks. A: Gating strategy used for cell sorting; CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2). RNA was isolated from the sorted cells and used for gene expression analysis by real-time RT-PCR. B: Gene expression profiles in CD11bloF4/80lo (P2) cells relative to CD11bhiF4/80hi (P1) cells in CCR2−/− mice were assessed (mean ± SEM; n = 4 biological replicates). C: Representative images of H-E–stained CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2) cells. *P < 0.05 compared with CD11bhiF4/80hi. **P < 0.01 compared with CD11bhiF4/80hi. ***P < 0.001 compared with CD11bhiF4/80hi. (A high-quality color representation of this figure is available in the online issue.)

Mentions: CD11bhiF4/80hi and CD11bloF4/80lo cells were isolated from the adipose tissue of CCR2−/− mice 10 weeks post–HFD feeding (Fig. 5A). Gene expression analysis was performed for both populations and is presented as expression relative to the CD11bhiF4/80hi cells (Fig. 5B). Expression of Emr1 (P < 0.001) was significantly lower for CD11bloF4/80lo cells, as expected based on our gating strategy. The expression of Arg1 (P < 0.05), Cd163 (P < 0.001), Il10 (P < 0.01), Il6 (P < 0.01), Ccr5 (P < 0.05), Ccl2 (P < 0.01), and Csfr1 (P < 0.001) was significantly lower in the CD11bloF4/80lo cells in comparison with the CD11bhiF4/80hi macrophages. Expression of Itgax (P < 0.01), Nos2 (P < 0.05), Ccl5 (P < 0.01), Il8rb (P < 0.05), and Csf1 (P < 0.05) was significantly higher for the CD11bloF4/80lo cells. H-E staining of the sorted CD11bloF4/80lo cells showed that these cells have a monocytic morphology and are smaller in size than the CD11bhiF4/80hi macrophages (Fig. 5C). Despite several reports of neutrophilia at sites of inflammation in CCR2−/− mice (25,26), flow cytometry analysis of the CD11bloF4/80lo population in the global CCR2−/− mice showed that these cells are Ly6G negative and therefore unlikely to be neutrophils (Supplementary Fig. 9).


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)

CD11bloF4/80lo cells have an inflammatory expression profile and a monocytic morphology. CD11bloF4/80lo and CD11bhiF4/80hi cells were isolated by fluorescence-activated cell sorting from CCR2−/− mice fed an HFD for 10 weeks. A: Gating strategy used for cell sorting; CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2). RNA was isolated from the sorted cells and used for gene expression analysis by real-time RT-PCR. B: Gene expression profiles in CD11bloF4/80lo (P2) cells relative to CD11bhiF4/80hi (P1) cells in CCR2−/− mice were assessed (mean ± SEM; n = 4 biological replicates). C: Representative images of H-E–stained CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2) cells. *P < 0.05 compared with CD11bhiF4/80hi. **P < 0.01 compared with CD11bhiF4/80hi. ***P < 0.001 compared with CD11bhiF4/80hi. (A high-quality color representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 5: CD11bloF4/80lo cells have an inflammatory expression profile and a monocytic morphology. CD11bloF4/80lo and CD11bhiF4/80hi cells were isolated by fluorescence-activated cell sorting from CCR2−/− mice fed an HFD for 10 weeks. A: Gating strategy used for cell sorting; CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2). RNA was isolated from the sorted cells and used for gene expression analysis by real-time RT-PCR. B: Gene expression profiles in CD11bloF4/80lo (P2) cells relative to CD11bhiF4/80hi (P1) cells in CCR2−/− mice were assessed (mean ± SEM; n = 4 biological replicates). C: Representative images of H-E–stained CD11bhiF4/80hi (P1) and CD11bloF4/80lo (P2) cells. *P < 0.05 compared with CD11bhiF4/80hi. **P < 0.01 compared with CD11bhiF4/80hi. ***P < 0.001 compared with CD11bhiF4/80hi. (A high-quality color representation of this figure is available in the online issue.)
Mentions: CD11bhiF4/80hi and CD11bloF4/80lo cells were isolated from the adipose tissue of CCR2−/− mice 10 weeks post–HFD feeding (Fig. 5A). Gene expression analysis was performed for both populations and is presented as expression relative to the CD11bhiF4/80hi cells (Fig. 5B). Expression of Emr1 (P < 0.001) was significantly lower for CD11bloF4/80lo cells, as expected based on our gating strategy. The expression of Arg1 (P < 0.05), Cd163 (P < 0.001), Il10 (P < 0.01), Il6 (P < 0.01), Ccr5 (P < 0.05), Ccl2 (P < 0.01), and Csfr1 (P < 0.001) was significantly lower in the CD11bloF4/80lo cells in comparison with the CD11bhiF4/80hi macrophages. Expression of Itgax (P < 0.01), Nos2 (P < 0.05), Ccl5 (P < 0.01), Il8rb (P < 0.05), and Csf1 (P < 0.05) was significantly higher for the CD11bloF4/80lo cells. H-E staining of the sorted CD11bloF4/80lo cells showed that these cells have a monocytic morphology and are smaller in size than the CD11bhiF4/80hi macrophages (Fig. 5C). Despite several reports of neutrophilia at sites of inflammation in CCR2−/− mice (25,26), flow cytometry analysis of the CD11bloF4/80lo population in the global CCR2−/− mice showed that these cells are Ly6G negative and therefore unlikely to be neutrophils (Supplementary Fig. 9).

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