IL-6 Regulates M2 Polarization and Local Proliferation of Adipose Tissue Macrophages in Obesity.

Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor (Il4ra) and its ligand IL-13 are elevated in AT of obese C57BL/6 mice. Most importantly, Il4ra expression and susceptibility to IL-4 or IL-13 treatment depend on IL-6 signaling, which seems to be the underlying mechanism of local ATM proliferation in obesity. We conclude that IL-6 acts as a Th2 cytokine in obesity by stimulating M2 polarization and local ATM proliferation, presumably due to upregulation of the IL-4 receptor α.

Hedgehog signalling in myeloid cells impacts on body weight, adipose tissue inflammation and glucose metabolism.

AIMS/HYPOTHESIS: Recently, hedgehog (Hh) was identified as a crucial player in adipose tissue development and energy expenditure. Therefore, we tested whether Hh ligands are regulated in obesity. Further, we aimed at identifying potential target cells of Hh signalling and studied the functional impact of Hh signalling on adipose tissue inflammation and glucose metabolism. METHODS: Hh ligands and receptors were analysed in adipose tissue or serum from lean and obese mice as well as in humans. To study the impact on adipose tissue inflammation and glucose metabolism, Hh signalling was specifically blocked in myeloid cells using a conditional knockout approach (Lys-Smo -/-). RESULTS: Desert Hh (DHH) and Indian Hh (IHH) are local Hh ligands, whereas Sonic Hh is not expressed in adipose tissue from mice or humans. In mice, obesity leads to a preferential upregulation of Hh ligands (Dhh) and signalling components (Ptch1, Smo and Gli1) in subcutaneous adipose tissue. Further, adipose tissue macrophages are Hh target cells owing to the expression of Hh receptors, such as Patched1 and 2. Conditional knockout of Smo (which encodes Smoothened, a mandatory Hh signalling component) in myeloid cells increases body weight and adipose tissue inflammation and attenuates glucose tolerance, suggesting an anti-inflammatory effect of Hh signalling. In humans, adipose tissue expression of DHH and serum IHH decrease with obesity and type 2 diabetes, which might be explained by the intake of metformin. Interestingly, metformin reduced Dhh and Ihh expression in mouse adipose tissue explants. CONCLUSIONS/INTERPRETATION: Hh signalling in myeloid cells affects adipose tissue inflammation and glucose metabolism and may be a potential target to treat type 2 diabetes.

A method for long-term live imaging of tissue macrophages in adipose tissue explants.

Obesity is frequently associated with a chronic low-grade inflammation within adipose tissue (AT). Although classical signs of inflammation are missing in AT inflammation, there is a significant increase in macrophages and, to a lesser extent, other immune cells, such as T cells, B cells, mast cells, and neutrophils. The spatial and temporal activation of these cells as well as their accumulation in the AT seem to be tightly linked to so-called crown-like structures (CLS). CLS are accumulations of adipose tissue macrophages (ATMs) around dead adipocytes and are thought to reflect a scavenger response. At present, data on the life cycle of CLS are missing. To better understand the cellular events underlying AT inflammation, we developed an approach that allows long-term imaging of ATMs, adipocytes, and CLS within live AT explants. We tested three putative reporter mouse lines for myeloid cells in regard to their suitability for live imaging. Thereby, we identified ATMs from CSF1R-eGFP mice to exhibit the most robust expression of eGFP. AT explants from these mice allowed stable live imaging for more than 7 days without significant phototoxicity. Long-term imaging thus revealed the accumulation of ATMs around dying adipocytes, migration of ATMs within AT, and also the degradation of the lipid remnants of perishing adipocytes. The observed behavior of ATMs in the context of AT inflammation is in line with previous studies but for the first time provides data on the specific behavior of individual ATMs and on the life cycle of CLS with unprecedented spatiotemporal resolution.

Local proliferation of macrophages in adipose tissue during obesity-induced inflammation.

AIMS/HYPOTHESIS: Obesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans. METHODS: We studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n = 239). The cell fate of dividing macrophages was assessed by live imaging of AT explants. RESULTS: We show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68(+) macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients. CONCLUSIONS/INTERPRETATION: Local proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.