Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation

Abstract

Adipose tissue macrophages are associated with insulin resistance and are linked to changes in the extracellular matrix. To better characterize adipose macrophages, the extracellular matrix, and adipocyte-macrophage interactions, gene expression from adipose tissue and the stromal vascular fraction was assessed for markers of inflammation and fibrosis, and macrophages from obese and lean subjects were counted and characterized immunohistochemically. Coculture experiments examined the effects of adipocyte-macrophage interaction. Collagen VI gene expression was associated with insulin sensitivity and CD68 (r = -0.56 and 0.60, P < 0.0001) and with other markers of inflammation and fibrosis. Compared with adipose tissue from lean subjects, adipose tissue from obese subjects contained increased areas of fibrosis, which correlated inversely with insulin sensitivity (r = -0.58, P < 0.02) and positively with macrophage number (r = 0.70, P < 0.01). Although macrophages in crownlike structures (CLS) were more abundant in obese adipose tissue, the majority of macrophages were associated with fibrosis and were not organized in CLS. Macrophages in CLS were predominantly M1, but most other macrophages, particularly those in fibrotic areas, were M2 and also expressed CD150, a marker of M2c macrophages. Coculture of THP-1 macrophages with adipocytes promoted the M2 phenotype, with a lower level of IL-1 expression and a higher ratio of IL-10 to IL-12. Transforming growth factor-β (TGF-β) was more abundant in M2 macrophages and was further increased by coculture with adipocytes. Downstream effectors of TGF-β, such as plasminogen activator inhibitor-1, collagen VI, and phosphorylated Smad, were increased in macrophages and adipocytes. Thus adipose tissue of insulin-resistant humans demonstrated increased fibrosis, M2 macrophage abundance, and TGF-β activity.

Fig. 1.

Changes in gene expression with insulin resistance and inflammation. Adipose tissue gene expression was assessed in 86 subjects covering a range of obesity and insulin sensitivity. A: relationship between collagen VI mRNA level and insulin sensitivity (S_I; r = −0.56, n = 86, P < 0.000001). B: relationship between collagen VI mRNA level and CD68 mRNA level (r = 0.60, n = 86, P < 0.000001). C: relationship between collagen VI mRNA level and expression of connective tissue growth factor (CTGF; r = 0.77, n = 86, P < 0.000001).

Fig. 2.

Characterization of collagen VI and macrophages in human adipose tissue. Human adipose tissue was double-stained with antibodies to CD68 (brown) and collagen VI (blue). A: adipocytes were surrounded by intense collagen staining in some areas (bottom left), but not others. Magnification ×200. Arrows, CD68-stained macrophages. B: a crownlike structure (CLS) in an area with intense collagen VI staining. Magnification ×400. C: area of fibrosis (F) in adipose tissue, with isolated interstitial macrophages (arrows). Magnification ×200. D: area of fibrosis (F) in adipose tissue with collagen staining extending into surrounding adipocytes, with isolated macrophages. Magnification ×200. E: Masson's trichrome stain of adipose tissue demonstrating layers of fibrosis (blue) surrounding adipocytes. Magnification ×100. Scale bar, 50 μm.

Fig. 3.

Relationship between fibrosis, macrophages, and insulin sensitivity. Areas covered by fibrosis and collagen VI and number of macrophages in adipose tissue sections were quantitated in 16 subjects and expressed in terms of S_I. A: percent fibrosis in relation to S_I. B: percent collagen VI staining in relation to S_I. C: macrophage number (Macs) in relation to S_I.

Fig. 4.

Immunofluorescent analysis of macrophage phenotype. Adipose tissue samples were immunoreacted with antibodies to identify M1 and M2 macrophages. Photomicrographs are representative of a field that included a CLS, as well as interstitial macrophages. A: bright-field overlay showing triple-immunofluorescence reactivity to antibodies recognizing a pan-monocyte/macrophage marker (CD68, green), a marker preferentially expressed on M1 macrophages (CD86, blue), and a marker for M2 macrophages (CD206, red). B, C, and D: individual reactivity to CD68, CD86, and CD206, respectively. Scale bar, 50 μm.

Fig. 5.

Identification of CD150 in adipose tissue macrophages. A–C: photomicrographs representative of a fibrotic area with interstitial macrophages. A: bright-field overlay of CD68 (green) and CD150 (red) double-immunofluorescence with background Sudan Black B staining. B: CD68 immunofluorescence. C: CD150 immunofluorescence. D: immunohistochemical double-staining of a relatively nonfibrotic area of adipose tissue with CD150 (brown) and collagen VI (blue). All CD150-positive cells also stained positively with anti-CD206, indicating that they were M2 macrophages (data not shown). Arrows, CD150-positive macrophages. Scale bars, 50 μm.

Fig. 6.

Assessment of M1 and M2 macrophages in adipose tissue. A: adipose tissue from lean and obese subjects was stained for CD86 and CD206, and number of macrophages that stained positively for each antigen were counted and expressed as percentage of total macrophages (CD68-positive cells). For each macrophage, CD86-, CD206-, or CD86- and CD206- (double) positive staining was determined. B: mRNA levels of TGF-β, plasminogen activator inhibitor-1 (PAI-1), IL-10, and IL-1 from stromal vascular fraction of lean and obese subjects. Data were normalized to 18S RNA. *P < 0.05 vs. lean.

Fig. 7.

Coculture of macrophages and adipocytes: effects on gene expression. THP-1 gene expression. THP-1 cells were differentiated into M1, M2a, or M2c macrophages, and IL-1, IL-12, IL-10, and PAI-1 gene expression was assessed in these cells when cultured alone or when cocultured with preadipocytes or adipocytes. Gene expression for each condition was normalized to expression of THP-1 monocytes that were not induced to differentiate. *P < 0.05.

Fig. 8.

Effects of coculture on collagen VI expression. Macrophages were induced to differentiate and cultured alone or cocultured with adipocytes. After 72 h in culture, RNA was prepared from the cells, and collagen VI expression was measured. A: collagen VI expression in macrophages. THP-1, undifferentiated macrophages. B: collagen VI expression in adipocytes, cultured alone (adipocytes) or in the presence of differentiated macrophages for 72 h.

Fig. 9.

Secretion of transforming growth factor (TGF)-β1 precursor. A: TGF-β1 precursor identified by Western blotting in THP-1 macrophages, without differentiation, or following differentiation to M1, M2a, or M2c macrophages. B: TGF-β1 precursor measurement in the medium by ELISA of macrophages alone or following 24 h of coculture with adipocytes. *P < 0.05 vs. macrophages alone.

Fig. 10.

TGF-β signaling in adipocytes. Conditioned medium (CM) from M1, M2a, or M2c macrophages was added to adipocytes for 24 h in the presence or absence of the TGF-β receptor kinase inhibitor SB-431542. PAI-1 mRNA and phosphorylated Smad2/3 (p-Smad2/3) were measured as markers of TGF-β activity. A: SB-431542 (0.1 and 5 μM) was added to cultures, and adipocyte PAI-1 mRNA levels were measured. Data are expressed relative to adipocytes that were not cultured with conditioned medium. B: adipocytes treated with conditioned medium with or without 5 μM SB-431542 were blotted for p-Smad2/3 and then for total Smad2/3.