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

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^loF4/80^lo and CD11b^hiF4/80^hi) accumulated in the adipose tissue of CCR2^−/− and BM-CCR2^−/− mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b^hiF4/80^hi population was detected in the CCR2^+/+ and BM-CCR2^+/+ controls. After 20 weeks of HFD feeding, the CD11b^loF4/80^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^loF4/80^lo cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b^loF4/80^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.Obesity is an independent risk factor for type 2 diabetes, cardiovascular disease, fatty liver disease, atherosclerosis, and several cancers. Chronic adipose tissue inflammation is a cardinal feature of obesity that leads to other health complications such as insulin resistance (1–3). Recruitment of macrophages is an important factor in adipose tissue inflammation (4) and has been shown to be temporally associated with insulin resistance (5,6).CCR2 regulates monocyte chemotaxis through direct interactions with its ligands, monocyte chemoattractant protein (MCP)-1 and -3 (7). CCR2^−/− mice have an immune deficiency in Th1 responses characterized by low levels of interferon-γ (IFN-γ) production and delayed macrophage recruitment to sites of inflammation (8). A study by Tsou et al. (9) found that CCR2^−/− mice have a defect in the egress of inflammatory monocytes from the bone marrow, resulting in a dramatic reduction of these cells in the circulation (9,10). Thus, CCR2^−/− mice have a decreased pool of circulating inflammatory monocytes and reduced numbers of differentiated myeloid cells recruited to sites of inflammation.The role of CCR2 and MCP-1 in macrophage recruitment to adipose tissue and the liver and their contribution to insulin resistance have been evaluated by various groups (11–16). One study showed that obese CCR2^−/− mice have a modest decrease in macrophage marker expression and macrophage number in adipose tissue after 24 weeks on a 60% fat diet compared with wild-type controls (11). Furthermore, a bone marrow transplant (BMT) study showed that hematopoietic CCR2 deficiency leads to decreased F4/80 expression in the adipose tissue of obese ob/ob mice (13). These experiments indicate that substantial obesity (i.e., long periods of high-fat diet [HFD] feeding and/or a genetically morbidly obese model) is required before differences in macrophage recruitment and improvements in insulin sensitivity are observed in CCR2^−/− mice, despite their striking reduction in circulating inflammatory monocytes. We set out to investigate the mechanism responsible for the delayed protection in adipose tissue inflammation and macrophage recruitment in global and hematopoietic models of CCR2 deficiency (CCR2^−/− and BM-CCR2^−/−, respectively).In this study, we analyzed the changes in myeloid populations that take place in the adipose tissue of CCR2^−/− and BM-CCR2^−/− mice during HFD-induced obesity and contrasted them with changes in CCR2^+/+ and BM-CCR2^+/+ controls. Our data showed that global and hematopoietic CCR2 deficiency leads to the accumulation of CD11b^loF4/80^lo myeloid cells during early periods of HFD feeding. Further analysis of the CD11b^loF4/80^lo cells showed that they have a monocytic and inflammatory gene expression profile characterized by significantly elevated expression of Itgax, Il8rb, Ccl5, Nos2, and Csf1. In addition, no differences in inflammation or glucose tolerance were observed between BM-CCR2^+/+ and BM-CCR2^−/− mice until after substantial obesity was reached during extended periods of HFD feeding—a condition that corresponds temporally with the absence of CD11b^loF4/80^lo cells.