Background-Adipose tissue macrophages (ATMs) have become a focus of attention recently because they have been shown to accumulate with an increase in fat mass and to be involved in the genesis of insulin resistance in obese mice. However, the phenotype and functions of human ATMs are still to be defined. Methods and Results-The present study, performed on human subcutaneous AT, showed that ATMs from lean to overweight individuals are composed of distinct macrophage subsets based on the expression of several cell surface markers: CD45, CD14, CD31, CD44, HLA-DR, CD206, and CD16, as assessed by flow cytometry. ATMs isolated by an immunoselection protocol showed a mixed expression of proinflammatory (tumor necrosis factor-␣, interleukin-6 [IL-6], IL-23, monocyte chemoattractant protein-1, IL-8, cyclooxygenase-2) and antiinflammatory (IL-10, transforming growth factor-␤, alternative macrophage activation-associated cc chemokine-1, cyclooxygenase-1) factors. Fat mass enlargement is associated with accumulation of the CD206 ϩ /CD16 Ϫ macrophage subset that exhibits an M2 remodeling phenotype characterized by decreased expression of proinflammatory IL-8 and cyclooxygenase-2 and increased expression of lymphatic vessel endothelial hyaluronan receptor-1. ATMs specifically produced and released matrix metalloproteinase-9 compared with adipocytes and capillary endothelial cells, and secretion of matrix metalloproteinase-9 from human AT in vivo, assessed by arteriovenous difference measurement, was correlated with body mass index. Finally, ATMs exerted a marked proangiogenic effect on AT-derived endothelial and progenitor cells. Conclusions-The present results showed that the ATMs that accumulate with fat mass development exhibit a particular M2 remodeling phenotype. ATMs may be active players in the process of AT development through the extension of the capillary network and in the genesis of obesity-associated cardiovascular pathologies.

The stroma-vascular fraction (SVF) of human adipose tissue has recently been described to be composed of endothelial cells identified as CD34+/CD31+ cells, infiltrated/resident macrophages defined as CD14+/CD31+ cells, and a new cell population characterized as CD34+/CD31- cells. To elucidate the cell identity of the adipocyte precursor cells, fluorescent activating cell sorter (FACS) analyses were performed on crude SVF cultured under adipogenic conditions, i.e., serum-deprived medium containing insulin, cortisol, triiodothyronine, and supplemented with a PPARgamma agonist for the first 3 days. The progressive accumulation of lipid droplets was associated with a selective enrichment of the CD34+/CD31- cell population whereas control experiments performed in medium supplemented with 10% serum showed an overall downregulation of the three cell markers without adipogenesis. Among the different cell subsets, the CD34+/CD31- subset was the unique cell fraction able to answer to adipogenic culture conditions. Indeed, a time-dependent expression of adipocyte markers as well as acquisition of adipocyte-typical metabolic activities were observed. In parallel, the gene expression of lipogenic and lipolytic enzymes increased. The ability to differentiate into adipocytes was restricted to cells that did not express the mesenchymal stem cell marker CD105. Furthermore, the CD34+/CD31- cells did not respond to culture conditions used for hematopoietic colony assays. Taken together, the present study demonstrates that adipocyte progenitor cells, i.e., the preadipocytes, are included in the CD34+/CD31- cell fraction, which displays distinct features from the adult mesenchymal and hematopoietic stem cells.

Pattern recognition receptors link metabolite and bacteria-derived inflammation to insulin resistance during obesity. We demonstrate that NOD2 detection of bacterial cell wall peptidoglycan (PGN) regulates metabolic inflammation and insulin sensitivity. An obesity-promoting high-fat diet (HFD) increased NOD2 in hepatocytes and adipocytes, and NOD2−/− mice have increased adipose tissue and liver inflammation and exacerbated insulin resistance during a HFD. This effect is independent of altered adiposity or NOD2 in hematopoietic-derived immune cells. Instead, increased metabolic inflammation and insulin resistance in NOD2−/− mice is associated with increased commensal bacterial translocation from the gut into adipose tissue and liver. An intact PGN-NOD2 sensing system regulated gut mucosal bacterial colonization and a metabolic tissue dysbiosis that is a potential trigger for increased metabolic inflammation and insulin resistance. Gut dysbiosis in HFD-fed NOD2−/− mice is an independent and transmissible factor that contributes to metabolic inflammation and insulin resistance when transferred to WT, germ-free mice. These findings warrant scrutiny of bacterial component detection, dysbiosis, and protective immune responses in the links between inflammatory gut and metabolic diseases, including diabetes.

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysisinduced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. rophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysisinduced AKI progression and advocate the utility of long-term follow-up for patients with this disease.

Objective-Adipose tissue (AT) plays a major role in the low-grade inflammatory state associated with obesity. The aim of the present study was to characterize the human AT lymphocytes (ATLs) and to analyze their interactions with adipocytes. Methods and Results-Human ATL subsets were characterized by flow cytometry in subcutaneous ATs from 92 individuals with body mass index (BMI) ranging from 19 to 43 kg/m 2 and in paired biopsies of subcutaneous and visceral AT from 45 class II/III obese patients. CD3ϩ ATLs were composed of effector and memory CD4 ϩ helper and CD8 ϩ cytotoxic T cells. The number of ATLs correlated positively with BMI and was higher in visceral than subcutaneous AT. Mature adipocytes stimulated the migration of ATLs and released the chemokine CCL20, the receptor of which (CCR6) was expressed in ATLs. The expression of adipocyte CCL20 was positively correlated with BMI and increased in visceral compared to subcutaneous adipocytes. ATLs expressed inflammatory markers and released interferon gamma (IFN␥). Progenitor and adipocyte treatment with ATL-conditioned media reduced the insulinmediated upregulation of lipogenic enzymes, an effect involving IFN␥. Conclusions-Therefore, crosstalk occurs between adipocytes and lymphocytes within human AT involving T cell chemoattraction by adipocytes and modulation of lipogenesis by ATLs.

We have recently demonstrated that natriuretic peptides (NPs), which are known for regulation of blood pressure via membrane guanylyl cyclase (GC) receptors, are lipolytic in human adipose tissue. In this study, we compared the NP control of lipolysis in adipocytes from humans, nonhuman primates (macaques), rodents (rats, mice, hamsters), and nonrodent mammals (rabbits, dogs). Isolated adipocytes from these species were exposed to increasing concentrations of atrial NP (ANP) or isoproterenol (beta-adrenergic agonist). Although isoproterenol was lipolytic in all of the species, ANP only enhanced lipolysis in human and macaque adipocytes. In primate fat cells, NP-induced lipolysis involved a cGMP-dependent pathway. Binding studies and real-time quantitative PCR assays revealed that rat adipocytes expressed a higher density of NP receptors compared with humans but with a different subtype pattern of expression; type-A GC receptors predominate in human fat cells. This was also confirmed by the weak GC-activity stimulation and the reduced cGMP formation under ANP exposure in rat adipocytes compared with human fat cells. In conclusion, NP-induced lipolysis is a primate specificity, and adipocytes from ANP-nonresponsive species present a predominance of "clearance" receptors and very low expression of "biologically active" receptors.

Adipose tissue fibrosis development blocks adipocyte hypertrophy and favors ectopic lipid accumulation. Here, we show that adipose tissue fibrosis is associated with obesity and insulin resistance in humans and mice. Kinetic studies in C3H mice fed a high-fat diet show activation of macrophages and progression of fibrosis along with adipocyte metabolic dysfunction and death. Adipose tissue fibrosis is attenuated by macrophage depletion. Impairment of Toll-like receptor 4 signaling protects mice from obesity-induced fibrosis. The presence of a functional Toll-like receptor 4 on adipose tissue hematopoietic cells is necessary for the initiation of adipose tissue fibrosis. Continuous low-dose infusion of the Toll-like receptor 4 ligand, lipopolysaccharide, promotes adipose tissue fibrosis. Ex vivo, lipopolysaccharide-mediated induction of fibrosis is prevented by antibodies against the profibrotic factor TGFβ1. Together, these results indicate that obesity and endotoxemia favor the development of adipose tissue fibrosis, a condition associated with insulin resistance, through immune cell Toll-like receptor 4.

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