538 Diabetes Volume 70, February 2021 Multinucleated Giant Cells in Adipose Tissue Are Specialized in Adipocyte Degradation Julia Braune,1 Andreas Lindhorst,1,2 Janine Fröba,1,2 Constance Hobusch,2 Peter Kovacs,3 Matthias Blüher,3 Jens Eilers,4 Ingo Bechmann,2 and Martin Gericke1 Diabetes 2021;70:538–548 | https://doi.org/10.2337/db20-0293 Obesity is associated with chronic low-grade inflamma- Therefore, it is essential to broaden our limited understand- tion of visceral adipose tissue (AT) characterized by an ing of the cellular mechanisms underlying adipose tissue increasing number of AT macrophages (ATMs) and (AT) dysfunction. linked to type 2 diabetes. AT inflammation is histologi- Obesity is associated with chronic low-grade inflamma- cally indicated by the formation of so-called crown-like tion of visceral AT closely linked to insulin resistance (3). structures, as ATMs accumulate around dying adipo- In obese individuals, a high caloric diet leads to massive cytes, and the occurrence of multinucleated giant cells lipid uptake in adipocytes, resulting in adipocyte hyper- (MGCs). However, to date, the function of MGCs in trophy. In addition, adipocyte death attracts macrophages, obesity is unknown. Therefore, the aim of this study which is indicative of AT inflammation, resulting in an was to characterize MGCs in AT and unravel the function accumulation of AT macrophages (ATMs), so-called crown- of these cells. We demonstrated that MGCs occurred in like structures (CLSs), surrounding dying adipocytes (4). obese patients and after 24 weeks of a high-fat diet in fl Additionally, there is a switch in ATM immune phenotype mice, accompanying signs of AT in ammation and then fl ∼ from an anti-in ammatory (M2) population in lean indi- representing 3% of ATMs in mice. Mechanistically, we fl found evidence that adipocyte death triggered MGC viduals to a more proin ammatory (M1) population in formation. Most importantly, MGCs in obese AT had obesity (5,6). PATHOPHYSIOLOGY a higher capacity to phagocytize oversized particles, Interestingly, in histological sections of obese AT, mul- such as adipocytes, as shown by live imaging of AT, tinucleated giant cells (MGCs) are frequently observed (4,7). 45-mm bead uptake ex vivo, and higher lipid content Before now, little was known about these macrophage-like in vivo. Finally, we showed that interleukin-4 treatment cells, which presumably form through cell-to-cell fusion. In was sufficient to increase the number of MGCs in AT, general, the function of MGCs has been debated and is whereas other factors may be more important for en- probably context specific. In tuberculosis, schistosomiasis, dogenous MGC formation in vivo. Most importantly, our and other granulomatous diseases, MGCs are disease hall- data suggest that MGCs are specialized for clearance of marks; foreign-body giant cells attack foreign material, and dead adipocytes in obesity. osteoclasts (endogenous MGCs of the bone) are important in bone homeostasis (8). In tuberculosis, MGCs seem to restrict the spread of mycobacteria, whereas in HIV infec- Obesity is a growing epidemic worldwide, leading to di- tion, virus particles seem to spread using cell-to-cell fusion, abetes type 2, stroke, cardiovascular disease, and cancer including MGCs (9,10). (1). For this reason, obesity is one of the main threats to Importantly, MGCs in obese AT have never been stud- global human health and life expectancy (2). To date, the ied in detail before. Therefore, the aim of this study was to only effective long-term treatment for obesity is invasive characterize MGCs with regard to the following: pro- and bariatric surgery; there is an urgent need for new therapies. anti-inflammatory properties, uptake of lipids from dying 1Institute of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, This article contains supplementary material online at https://doi.org/10.2337/ Halle, Germany figshare.13168862. 2 Institute of Anatomy, Leipzig University, Leipzig, Germany © 2020 by the American Diabetes Association. Readers may use this article as 3 Medical Department III, Leipzig University, Leipzig, Germany long as the work is properly cited, the use is educational and not for profit, and the 4 Carl-Ludwig Institute of Physiology, Leipzig University, Leipzig, Germany work is not altered. More information is available at https://www.diabetesjournals Corresponding author: Martin Gericke, [email protected] .org/content/license. Received 24 March 2020 and accepted 30 October 2020 diabetes.diabetesjournals.org Braune and Associates 539 adipocytes, mechanisms involved in MGC formation, and some experiments, AT explants were treated with indi- growth in MGC number. We tested the effect of different cated cytokines (50 ng/mL; PeproTech, Hamburg, Ger- cytokines on MGC formation in living AT ex vivo using an many, and Sigma-Aldrich). established AT explant model and found that interleukin (IL)-4 treatment was sufficient to enhance the generation Imaging of Whole Mounted or Living AT of MGCs in AT. Moreover, MGCs expressed not only Staining of whole mounted AT was performed as pre- pro- but also anti-inflammatory marker proteins. Most im- viously described (14). Images were taken using an Olym- portantly, MGCs exhibited significantly higher lipid content pus FV1000 confocal laser scanning microscope (Olympus in vivo and were able to phagocytize larger particles compared Deutschland GmbH, Hamburg, Germany). Live imaging with regular, mononucleated ATMs in vitro. Therefore, our was performed as described previously with a special focus data suggest that MGCs are specialized for clearance of dead on putative MGCs using an Olympus FV300 confocal laser adipocytes in obesity. scanning microscope (Olympus) (15). RESEARCH DESIGN AND METHODS Isolation of Stroma Vascular Fraction, Quantification of MGCs, and Phagocytosis Experimental Animals To determine the formation of MGCs, the stroma vascular Mice strains were maintained in the local animal facility fraction (SVF) of 100 mg freshly dissected epididymal AT in 12-h light/dark cycles and with free access to food or 10 cultured AT explants was isolated using collagenase and water. For diet-induced obesity, male C57BL/6J, 1/2 flx/2 Dmyel type II (Worthington, Lakewood, NJ). Subsequently, the LysMCre 3 IL-4Ra (IL-4Ra ), or MacGreen 1 2 fi m (CSF1R-eGFP / ) mice on a C57BL/6 background were fed SVF cell suspension was ltered through 75- m mesh and seeded onto a poly-L-lysine–coated (Sigma-Aldrich) cover- a high-fat diet (HFD; 60% kcal fat; Ssniff-Spezialdiäten slip in RPMI medium containing 10% FCS (Gibco) and GmbH, Soest, Germany) starting at 6–8 weeks of age. antibiotics (1% penicillin/streptomycin; Gibco). After 2 h Control littermates were kept on a regular chow diet of attachment, cells were counterstained with Hoechst dye (chow; 9% kcal fat; Ssniff-Spezialdiäten). Leptin receptor– 33342 (1:10,000; Life Technologies) and subsequently deficient mice (The Jackson Laboratory, Bar Harbor, ME) fixed for 5 min using zinc formalin (Polysciences, Hirsch- were analyzed at 12 weeks of age. All experiments were berg, Germany). performed in accordance with the National Institutes of fi Health guidelines and approved by the local authorities For quanti cation, 10 randomly chosen images were taken per condition with a 103 objective using an Olym- (Regierungspräsidium Leipzig). pus BX51 epifluorescence microscope (Olympus), and 1 1 Human Samples GFP cells in MacGreen mice or Mac-2 cells in nonrep- Twenty-five obese patients who underwent laparoscopic orter mice were semiautomatically counted using cellSens fi Roux-en-Y gastric bypass surgery were recruited at the software (Olympus). MGCs were de ned as macrophages Leipzig University Medical Center (Leipzig, Germany). The with at least three nuclei according to previous reports human study was approved by the ethics committee of the (12). University of Leipzig (approval number: 017–12–23012012), For the phagocytosis assay, SVF cells were incubated for m and all participants provided written informed consent 48 h with serum-opsonized and BODIPY-stained 45- m before taking part in the study. Exclusion criteria included polystyrene beads (30,000 beads per well; Polysciences). chronic or acute inflammatory disease as defined by white cell Representative images were acquired using a confocal counts and/or hs-CRP along with clinical symptoms, antibi- FV1000 microscope (Olympus) to ensure full engulfment otic treatment within 2 months before surgery, pregnancy, of the phagocytized beads. and/or nursing. All individuals underwent routine clinical phenotyping as listed in Table 1. Abdominal subcutaneous AT Flow Cytometry and Imaging Flow Cytometry samples were acquired during surgery, paraffin embedded, To perform flow cytometric analysis, freshly dissected AT sectioned, and stained as described previously (11). After or cultured AT explants were digested as described above. staining, two sections per individual were evaluated with After digestion, the cell suspension was filtered through regard to occurrence of MGCs within the total sections in 75-mm mesh, and Fc receptors were blocked by using anti- 1 ablindedfashion.MGCsweredefined as Mac-2 cells CD16/32 (1:100; eBioscience, Frankfurt, Germany) treat- (1:1,000; Cedarlane, Burlington, ON, Canada) with at least ment for 10 min on ice. Subsequently, cells were stained three nuclei according to previous reports (12). Finally, for 20 min on ice with anti-CD45-FITC (30-F11), anti-F/ patients with detectable MGCs and without detectable 480-PE-Cy7 (BM8), anti-CD11b (M1/70), anti-CD11c-PE MGCs were statistically analyzed. (N418, all 1:100; all from eBioscience), and anti-CD36-PE (HM36, 1:100; Biolegend, San Diego, CA) for standard Cultivation of Organotypic AT (AT Explants) flow cytometry. After extracellular live staining, cells were Cultivation of AT explants from chow-fed C57BL6 and fixed using the Fixation/Permeabilization Solution Kit and 2 2 D IL-4Ra / mice or HFD-fed MacGreen or IL-4Ra myel stained with 7-AAD (both BD Pharmingen, Heidelberg, mice was performed as described previously (13). In Germany) for 5 min at room temperature for nuclear 540 Giant Cells in Adipose Tissue Diabetes Volume 70, February 2021 counterstain.