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538 Volume 70, February 2021

Multinucleated Giant Cells in Are Specialized in 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 (ATMs) and (AT) dysfunction. linked to . AT inflammation is histologi- is associated with chronic low-grade inflamma- cally indicated by the formation of so-called crown-like tion of visceral AT closely linked to 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 uptake in , 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- 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 -like in vivo. Finally, we showed that interleukin-4 treatment cells, which presumably form through -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 ) are important in bone (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, , , 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 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 ; there is an urgent need for new therapies. anti-inflammatory properties, uptake of from dying

1Institute of 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 , 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 (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 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). – 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 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. For imaging flow cytometry (ImageStream), AT. Interestingly, when comparing the characteristics of cells were stained using anti-CD80-AlexaFluor647, patients with MGCs with those of patients without detect- anti-CD86-AlexaFluor647, anti-MHC-II-AlexaFluor647, anti- able MGCs, blood leukocytes were significantly elevated in CD11c-AlexFluor647 (all 1:100; eBioscience), and anti- the MGC group. Furthermore, there was a trend toward CD206-AlexaFluor647 (ER-MP23; 1:100; AbD Serotec, higher values for plasma insulin, fasting plasma Kidlington, U.K.) for 20 min on ice, and Hoechst dye , and HOMA indices in the MGC group, suggesting 33342 (1:10,000) was applied for 30 min on ice for nuclear pathophysiological relevance (Table 1). However, these counterstaining followed by fixation. MGCs were preferentially observed near dead adipocytes For analysis of flow cytometric data, single cells were in CLSs. Expression of the pan-macrophage marker F4/80 1 1 1 gated for 7-AAD cells. Subsequently, CD45 and F4/80 and GFP-labeled macrophages (MacGreen mouse line) in cells were defined as ATMs (Supplementary Fig. 1). Anal- obese animals further validated their macrophage origin ysis was performed using an LSR II (BD Pharmingen) (Fig. 1A). Additionally, by long-term live imaging of AT equipped with FACS Diva software 8.0. Quantification explants, we could detect MGCs, which exhibited a remark- was performed using FlowJo software 10.0.5 (Tree Star, able motility (Fig. 1D and Supplementary Movie 1). There- Ashland, OR). For imaging flow cytometry, the Amnis fore, the aim of this study was to characterize MGCs and ImageStreamX Mark II Imaging Flow Cytometer (Luminex, their function in AT in more detail. Austin, TX) equipped with INSPIRE software (Luminex) was used. Data analysis was performed using IDEAS Formation of MGCs Occurs Late in AT Inflammation software (Luminex). Here, we established a model of AT inflammation in MacGreen mice using an HFD. After 12 weeks of HFD 3T3-L1 Adipocyte Feeding feeding, mice exhibited a significantly higher body weight 3T3-L1 adipocytes were differentiated and cultured as (Fig. 2A), and the weight of the epididymal AT also in- previously described (16,17). Afterward, adipocyte death was creased significantly compared with AT in their lean lit- induced using 25 nmol/L -a (TNFa)for termate controls (data not shown). Furthermore, the 1 24 h following established protocols (18). After incubation, frequency of GFP cells among SVF cells, corresponding adipocytes were cocultured with freshly isolated SVF cells of to ATMs in MacGreen (15), increased after 12 and 24 weeks epididymal AT from HFD-fed MacGreen mice for 24 h. of an HFD compared with that in their lean littermates (Fig. 2B), and the ratio of pro- (M1) to anti-inflammatory Statistical Analysis (M2) ATMs shifted toward a proinflammatory (M1) phe- Data are presented as means 6 SD or as box plots notype (Fig. 2C and D). In line with these findings, CLS (whiskers represent minima and maxima) for at least three density increased starting from 12 weeks of an HFD, animals evaluated by the Student t, Mann-Whitney U,or indicating adipocyte death (Fig. 2E and F). Furthermore, 1 Wilcoxon test, according to data distribution. Identifica- semiautomatic quantification of ATM size by GFP area tion of outliers (Grubbs test) and assessment of normality revealed a significantly enlarged cell size of ATMs after of data distribution (Shapiro-Wilk test) were performed 12 and 24 weeks of an HFD compared with that in lean using GraphPad Prism 8.0 (GraphPad Software, Inc., La littermates (Fig. 2G). MGCs in vivo were not detectable in Jolla, CA). The Pearson correlation coefficient was calcu- mice after 4 or 12 weeks of an HFD. However, after lated using GraphPad Prism (GraphPad). A P value ,0.05 24 weeks of an HFD, MGCs appeared in the SVF cells, was considered statistically significant. representing ;3% of all ATMs (Fig. 2H and I). Because MGCs were defined as ATMs with at least three nuclei, Data and Resource Availability they represented up to ;10% of ATM-associated nuclei in All data generated or analyzed during this study are in- obese AT. cluded in the published article (and its Supplementary Material). The mouse models analyzed during the current Adipocyte Death Prompts MGC Formation study are available from the corresponding author upon Formation of MGCs in obese AT occurred late in the course reasonable request. of AT inflammation, and their appearance was linked to inflammatory signs, such as CLS occurrence (Fig. 1A–C), RESULTS a rising number of ATMs (Fig. 2B), and a phenotypic switch Presence of MGCs in AT of Obese Mice and toward proinflammatory M1 macrophages (Fig. 2C and D). Previous studies focused on the role of ATMs in obesity Therefore, we next tested whether adipocyte death per se and AT inflammation in several mouse models. Extraor- could stimulate MGC formation ex vivo (Fig. 3). First, we dinarily large cells with multiple nuclei, often appearing in performed long-term cultivation of AT explants from lean a circular orientation, are regularly found in obese mice mice with almost no endogenous CLSs (,0.1% of adipo- and humans (Fig. 1A–C). Of note, MGCs were defined as cytes; Fig. 2E) or MGCs (Fig. 2H) and studied the potential macrophages with at least three nuclei according to pre- formation of CLSs and MGCs as a result of adipocyte death vious reports (12). In obese humans undergoing bariatric ex vivo. Interestingly, CLSs and MGCs both formed in surgery, 28% (7 of 25) exhibited MGCs within subcutaneous parallel starting from 7 days of long-term cultivation (Fig. diabetes.diabetesjournals.org Braune and Associates 541

3A–D), indicating a strong association between both pro- intensity (Fig. 4G). Of note, ;90% of all MGCs expressed cesses. For a more controlled experimental condition, GFP and F4/80 as well as the most common proinflam- TNFa overstimulation of adipocytes was successfully matory (M1) marker, CD11c (Fig. 4A–E), which has been established as model to kill 3T3-L1 adipocytes in vitro validated by whole mount staining of obese AT in situ (Fig. as described previously (18) (data not shown). Interest- 4F). Most interestingly, more anti-inflammatory markers, ingly, cocultivation of dead adipocytes with SVF cells of such as CD206 and CD80, were expressed on ;50% of obese AT led to a significantly higher number of MGCs all MGCs, indicating a possible fusion of pro- and anti- compared with the number in the PBS-treated control (Fig. inflammatory ATMs or a de novo expression of M2 markers 3E and F), indicating that adipocyte death increases MGC by MGCs (Fig. 4A–E and Supplementary Fig. 2). Further- formation. more, expression of MHC-II as well as T-cell costimulatory markers CD80 and CD86 indicated a role in antigen pre- MGCs Express Both Pro- and Anti-Inflammatory sentation of MGCs (Fig. 4A–E). In comparison, MGCs Markers In Vivo expressed higher levels of GFP and F4/80 as mononucleated fl To further characterize MGCs in AT, we established ow ATMs (Fig. 4G). cytometric imaging of SVF cells from epididymal AT of obese MacGreen mice (Fig. 4A). As expected, MGCs were MGCs Have a Higher Phagocytic Capacity Ex Vivo and threefold bigger in size than mononucleated ATMs (Fig. 4B). In Vivo Moreover, MGCs did show a significantly higher fluores- Next, we specifically focused on MGCs in living AT by long- cence intensity for nuclear counterstaining (Hoechst re- term live imaging. Putative MGCs were identified by an lated to the cell size) compared with regular ATMs as increased cell size, which was several-fold larger than the asignofpolyploidy(Fig.4D). Furthermore, the intensity size of regular ATMs also expressing GFP (Fig. 5A). In- of the nuclear staining was significantly correlated with terestingly, regular ATMs struggled with the uptake of cell area (Fig. 4C), validating our approach of detecting large lipid remnants (;25 mm), resulting in several failed MGCs by imaging flow cytometry. More importantly, MGCs attempts at phagocytosis by regular ATMs (t 5 3–6h). expressed both pro- and anti-inflammatory marker pro- After 13 h of observation, an MGC formed and rapidly teins, such as CD11c, CD206, CD80, CD86, and MHC-II phagocytized the respective lipid remnant, with subse- (Fig. 4A–E), with a substantial variety of expression quent intracellular degradation (13–30 h; Fig. 5A and

Figure 1—Occurrence and dynamics of MGCs in obese AT. A: Whole mount staining of epididymal AT of MacGreen mice after 24 weeks of an HFD. GFP and pan-macrophage marker F4/80 indicate macrophage origin. B: Hematoxylin-eosin (H&E)–stained section of epididymal AT of leptin receptor–deficient mice at 12 weeks of age. Note multiple MGCs within CLSs. C: Section of human subcutaneous AT of an obese individual stained against the macrophage marker Mac-2 (red), the adipocyte marker Perilipin (green), and the nuclear counterstain DAPI (blue). D: Representative images of a long-term live-imaging experiment on AT explants of obese MacGreen mice after 24 weeks of an HFD. Macrophages are visible because of GFP expression (red), and adipocytes were stained using the neutral lipid stain BODIPY (green). Corresponding movie is provided as Supplementary Movie 1. Arrows indicate regular ATMs; double arrows indicate MGCs. Scale bar, 50 mm. 542 Giant Cells in Adipose Tissue Diabetes Volume 70, February 2021

Figure 2—Late AT inflammation implicates the formation of MGCs. A: Body on normal chow diet (NCD) (dots) and HFD (squares) for a period of 24 weeks starting at 6–8 weeks of age (n 5 4–7). B: Frequency of GFP-expressing ATMs in the SVF cells of MacGreen mice (n 5 4–7). C and D: Ratio of proinflammatory (M1; CD11c1/CD2062) to anti-inflammatory (M2; CD11c2/CD2061) ATMs (n 5 3–6) (D) and representative flow cytometric images (C). E: Frequency of CLSs per area in epididymal murine AT. F: Representative image of an epididymal AT section after 24 weeks of an HFD stained for the adipocyte marker perilipin (green) and the macrophage marker Mac-2. Arrows indicate CLSs. G and H: Area of ATMs in SVF cells (G) and frequency of MGCs (H) related to all ATMs in the AT of chow-fed (white) or HFD-fed (gray) mice. I: Representative image of MGCs after isolation from mice after 24 weeks of an HFD. Arrows indicate regular ATMs; double arrows indicate MGCs. Scale bars, 250 (F)or25mm(I). *P , 0.05, **P , 0.01, ****P , 0.0001.

Supplementary Movie 2). Thus, live imaging indicated that an intermediate phenotype between mono- and multinu- MGCs could take up lipid remnants, which are indigestible cleated ATMs with regard to cell area, Hoechst intensity, for regular ATMs. To quantify the phagocytosis of large and lipid content (Supplementary Fig. 3). Next, MGCs particles, acutely isolated SVF cells of obese MacGreen showed a significantly higher side scatter signal related mice were incubated with 45-mm beads, which are too big to cell area, indicating greater cell granularity (Fig. 5G and for phagocytosis by regular ATMs (cell size ;20 mm) (19). H), as a result of more intracellular vesicles, which was also Interestingly, MGCs were responsible for all observed positively correlated with respective cell size (Fig. 5I). Of events of successful bead uptake (14 of 14); none were note, granules did not colocalize with intracellular lipid attributable to mononucleated ATMs (Fig. 5B and C). droplets (Supplementary Fig. 4). Next, we aimed at estimating the phagocytic capacity of MGCs in vivo. Therefore, we used flow cytometric imaging Th2 Cytokines Induce MGC Formation in AT to compare the lipid content and cell granularity of ATMs Finally, we tested the effect of different cytokines on their and MGCs (Fig. 5D–I). Interestingly, MGCs exhibited ability to force MGC formation ex vivo. Therefore, we used a significantly higher lipid content (related to cell area) an obese AT explant model treated for 48 h with several than mononucleated ATMs measured by BODIPY staining cytokines. Interestingly, stimulation with granulocyte- (Fig. 5D and E). In addition, BODIPY fluorescence was macrophage colony-stimulating factor (GM-CSF), IL-4, strongly correlated with cell size (Fig. 5F), indicating and IL-13 led to a significantly enhanced frequency of higher lipid uptake. Of note, binucleated ATMs exhibited MGCs in the SVF by approximately twofold (Fig. 6A and diabetes.diabetesjournals.org Braune and Associates 543

Figure 3—Adipocyte death provokes MGC formation. A: Representative images of AT explants ex vivo after days 1 and 10 of cultivation. Arrows indicate CLSs. B: Frequency of CLSs per adipocyte of AT explants ex vivo. d, day. C: Representative images of isolated stroma cells of AT explants at distinct time points of cultivation. Arrows indicate MGCs. D: Frequency of MGCs per GFP1 cells (ATMs) of AT explants ex vivo. d, day. E: Representative images of 3T3-L1 feeding experiments of GFP1 ATMs (green) and BODIPY-stained 3T3-L1 adipocytes (red), either untreated or killed by TNFa overstimulation (25 nmol/L). Double arrows indicate MGCs. F: Number of MGCs per high-power field (HPF; 203) in coculture with dead 3T3-L1 adipocytes as a result of TNFa overstimulation (gray) or PBS-treated control (white) (n 5 5). *P , 0.05, **P , 0.01, ***P , 0.001.

Supplementary Fig. 5A). In contrast, other factors well signaling was also a prerequisite for MGCs in obese Δ known to induce formation of MGCs in vitro by fusion of animals in vivo. Surprisingly, IL-4Ra myel did exhibit –derived macrophages, such as Rankl, had no a trend toward more MGCs within the AT of obese mice significant effect on MGC generation in AT (Fig. 6A). after 24 weeks of an HFD, which could have been related to Additionally, quantification of the cell size of ATMs did an altered immune phenotype, body composition, or CLS verify a general increase in mean ATM size after GM-CSF density in these animals (data not shown). Therefore, we and IL-4 stimulation (Fig. 6B), as seen for ATMs after used our established model of MGC induction produced by 12 and 24 weeks of an HFD in vivo. Furthermore, we long-term cultivation of lean AT explants to ensure similar studied whether IL-4 directly stimulated MGC generation conditions (Fig. 3A–D). However, the number of MGCs was or functioned through other cell types in AT. Therefore, not affected by knockout of IL-4Ra after 10 days of long- the effect of a myeloid cell–specific knockout of IL-4Ra, term cultivation, suggesting that although IL-4 can be used the mandatory signaling component of the IL-4 receptor, to increase MGCs in AT, other signals dominate physio- 2 fl was tested ex vivo. In control mice (IL-4Ra / ), the logical MGC formation in vivo. accumulation of MGCs as a result of IL-4 stimulation was unaffected, whereas in myeloid-specific knockout DISCUSSION Δ mice (IL-4Ra myel), IL-4 was unable to generate MGCs The link between obesity and associated diseases is AT (Fig. 6D). Next, we aimed at testing whether IL-4Ra dysfunction caused by chronic low-grade inflammation (1). 544 Giant Cells in Adipose Tissue Diabetes Volume 70, February 2021

Figure 4—MGCs express both pro- and anti-inflammatory markers in vivo. A: Representative images of flow cytometric imaging of pro- and anti-inflammatory markers (red) of ATMs and MGCs from MacGreen mice after 24 weeks of an HFD. MGCs were defined as GFP1 (green), F4/ 801 (magenta), and polyploid (nuclei .3; Hoechst; gray) cells. Scale bar, 10 mm. B and D: Area (B) and Hoechst fluorescence (D) related to cell area of MGCs (gray) compared with ATMs (white) (n 5 6). C: Linear regression of Hoechst signal to cell area of all GFP1 cells of SVF cells from obese MacGreen mice (n . 1,000). E: Frequency of marker expression by MGCs (n 5 4–6). F: Representative image of a CD11c1 MGC in epididymal AT of obese MacGreen mice after 24 weeks of an HFD. Scale bar, 50 mm. G: Mean fluorescence intensity (FI) of different markers (related to cell area) comparing ATMs (white) and MGCs (gray) (n 5 4–6). *P , 0.05, **P , 0.01, ****P , 0.0001.

Accordingly, AT inflammation can be observed before extraordinarily long clearing periods of adipocyte rem- obesity-associated diseases manifest, and immune cell in- nants. For instance, after global induction of adipocyte filtration is the strongest predictor of in death, the formation of CLSs occurs several days after obese patients (20). AT inflammation is characterized by induced cell death (26). In contrast, apoptotic cells in an increase of immune cells, mostly resulting from an other tissues are cleaved in ,24 h (27–29). More impor- increase in ATMs, an increasing number of CLS, and a shift tantly, after switching back from a CLS-inducing HFD to in ATM polarization toward a more proinflammatory (M1) a chow diet in mice, increased CLS density was detectable phenotype (5,21). Mechanistically, this proinflammatory for up to 6 months, indicating that adipocyte degradation phenotype of ATMs is closely linked to adipocyte death takes several months (30). This implies that local ATM and the occurrence of CLS (22). Of note, up to 90% of proliferation and activation toward a M1 phenotype ATMs are found in CLS in AT of obese mice (4,23). continue for this prolonged period. In line with these Although susceptibility of AT depots to obesity-induced findings, several studies have shown that induction of AT adipocyte death is different, adipocyte death is increased inflammation as a result of HFD feeding is not (or only by 10- to 100-fold as a result of obesity (24). However, the partially) reversible after switching back to regular chow clearing of dead adipocytes seems challenging for resident (30,31). ATMs, because hypertrophic adipocytes are fivefold larger In general, the ineffective clearing of dead cells increases than regular phagocytes (25). This seems to result in the release of proinflammatory cytokines (32–34), leading diabetes.diabetesjournals.org Braune and Associates 545

Figure 5—MGCs have a higher phagocytotic capacity than regular ATMs. A: Representative images of a long-term live-imaging experiment on AT explants of obese MacGreen mice after 24 weeks of an HFD. Macrophages are visible because of GFP expression (red), and adipocytes were stained using the neutral lipid stain BODIPY (green). Of note, after two failed attempts at phagocytosis by regular ATMs (upper row), an MGC successfully phagocytized and digested the lipid remnant (lower row). Corresponding movie is provided as Supplementary Movie 2. B: Representative image of a phagocytized 45-mm bead (magenta) by an MGC (green). A and B: Arrows indicate regular ATMs; double arrows indicate MGCs. C: Quantification of four independent experiments counting uptake of 45-mm beads (total of 14 events) by either mononucleated ATMs or MGCs. D and G: Representative images of MGCs (defined as GFP1 [green], F4/801 [magenta], and polyploid [nuclei .3; Hoechst; gray] cells) by flow cytometric imaging studied for lipid content by BODIPY staining (D) or granularity (G). E and H: Quantification of mean fluorescence intensity (FI) of the BODIPY signal (E) or granularity (H), measured by side scatter intensity (SSC) in either regular ATMs (white) or MGCs (gray). F and I: Linear regression of the BODIPY signal (F) or the granularity (I), measured by SSC, relative to the cell area of all GFP1 cells in the SVF cells of obese MacGreen mice. Scale bars, 10 (D and G), 25 (A), or 50 mm(B). **P , 0.01, ***P , 0.001.

to chronic inflammation and autoimmune diseases (33). Importantly, one way that ATMs digest large cells, such This failure may also involve the dysregulation of receptors as adipocytes, was recently described and includes the mediating contact between MGCs/macrophages and dying extracellular degradation of lipid remnants as a result of adipocytes or lipid remnants. This could include BAI-1 or lysosomal exocytosis. Here, CLS-associated ATMs secrete TIM-4, which are generally involved in efferocytosis (35), lysosomal enzymes to the adipocyte-macrophage interface or TREM-2, which was recently shown to mediate macro- and subsequently take up liberated lipids (18,25). In this phage-adipocyte interaction (36,37). report, we describe a potential second alternative of the 546 Giant Cells in Adipose Tissue Diabetes Volume 70, February 2021

Figure 6—IL-4 can stimulate ΜGC formation in AT via CD11c binding. A: Frequency of MCGs relative to the number of all ATMs after treatment of obese AT explants for 48 h compared with PBS control (50 ng/mL each; n 5 4–8). B: Mean ATM area after stimulation of obese AT explant with either IL-4 or GM-CSF compared with PBS (n 5 10). C: Representative image of GFP1 MGCs after cell isolation from MacGreen mice after 24 weeks of HFD. Arrow indicates a regular ATM; double arrows indicate MGCs. Scale bar, 25 mm. D: Frequency of MGCs as percentage of all ATMs after IL-4 stimulation for 48 h (black) of obese AT explants derived from either IL-4Ra2/fl (control) or IL-4RaΔmyel (conditional knockout [KO]) mice (n 5 5–7). PBS (white) served as control. E: Frequency of MGCs in epididymal AT from either IL-4Ra2/fl (control; white) or IL-4RaΔmyel (conditional KO; black) mice (n 5 9–11) after 24 weeks of HFD in vivo. F: Frequency of MGCs in AT explants from either IL-4Ra1/1 (control; white) or IL-4Ra2/2 (KO; black) mice (n 5 6) after 10 days of long-term cultivation ex vivo. *P , 0.05, **P , 0.01.

immune system to improve large-cell efferocytosis through estimate the phagocytic activity in adipocyte degradation the generation of MGCs, possibly as a result of macrophage in vivo, we measured the lipid content of MGCs, which was fusion. Direct macrophage fusion was recently confirmed threefold higher than that in mononucleated ATMs of the to be a mechanism for the maintenance of osteoclasts, respective cell size, indicating a higher density of lipid which are physiologically occurring MGCs of the bone (38). droplets as a result of increased lipid uptake. AT foam cells Of note, fusion of other myeloid cell types, such as (lipid laden macrophages) are associated with an unhealthy dendritic cells, cannot be excluded, because of the lack obese phenotype and AT inflammation (42–44), similar to of exclusive markers as discussed by others (39). However, MGCs, but they form earlier in obesity (45). However, the endoreplication (nucleus replication without cell division) influence of polyploidy on the lipid content of ATMs has as an alternative origin of MGCs may also play a role in AT not been studied before. Of note, binucleated ATMs ex- (40). IL-4 and GM-CSF have convincingly been demon- hibit an intermediate phenotype between mono- and strated to induce macrophage fusion in vitro (8,41), and multinucleated ATMs, and some of them may represent IL-4 signaling (IL-13–, IL-4Ra–, and IL-4–stimulated precursors of MGCs. However, uptake of liberated fatty STAT6 phosphorylation) is also increased in AT from acids after extracellular fat mobilization resulting from obese mice as shown previously (13). Although our data lysosomal exocytosis could also explain the higher lipid on obese IL-4Ra knockout mice suggest that signaling content of mononucleated ATMs and lead to a metaboli- other than IL-4Ra signaling dominates the formation of cally activated immune phenotype (25). Either way, our MGCs in vivo, the increase in the number of MGCs in AT data show for the first time that MGCs found in CLSs are via IL-4 stimulation seems possible and may have thera- specialized in adipocyte degradation. Our findings also peutic potential. show that provoked adipocyte death induces MGC forma- Importantly, we demonstrated that MGCs isolated tion in vitro. from the AT of obese mice had a higher phagocytic capacity Whether the formation of MGCs is protective of or ex vivo, because only these cells were able to take up large detrimental to AT inflammation requires further study. Of particles, such as 25-mm lipid remnants or 45-mm poly- note, patients with detectable MGCs within AT sections styrene beads, which are otherwise indigestible for regular, had significantly elevated blood leukocytes as an indicator mononucleated ATMs. Of note, the specialization of of a more proinflammatory AT phenotype. However, we in vitro–generated, bone barrow–derived MGCs for large also showed that ;50% of MGCs also expressed anti- particle uptake has been previously demonstrated (19). To inflammatory marker proteins, such as CD206. This could diabetes.diabetesjournals.org Braune and Associates 547

fi fl Table 1—Participant demographic and clinical characteristics t into a model of resolution of in ammation after tissue clearing by MGCs. Although this is a tempting speculation, No MGCs MGCs more data on the function and extensive transcriptional pro- P Mean SD Mean SD filing of AT-derived MGCs are needed to prove this concept. Participants, In summary, for the first time, we characterized nat- n (%) 18 (72.0) 7 (28.0) urally occurring MGCs in AT of obese mice in detail. We Sex, n (%) showed that these unique cells represented a significant Female 13 (72.2) 5 (71.4) 0.97 Male 5 2 number of ATMs in obesity, making up to 3% of ATMs or ;10% ATM-associated nuclei. Of note, because the criteria Diagnosed with fi T2D, % 61.1 42.9 0.43 for detecting MGCs seemed to t with the exclusion criteria from other laboratories, this cell population may Age, years 49.0 11.8 46.4 13.2 0.63 be underrepresented in other studies. However, our data Body weight, kg 146.1 27.4 137.1 18.1 0.43 indicate that adipocyte death is a trigger for MGC forma- Height, m 1.7 0.1 1.7 0.1 0.38 tion. Most importantly, MGCs seem to be specialized for BMI, kg/m2 49.2 5.7 48.9 4.8 0.91 large cell destruction, such as adipocytes, and express pro- Body fat, % 47.4 8.7 51.3 10.0 0.34 and anti-inflammatory marker proteins. However, more Creatinine, data are needed to unravel the function of these unique mmol/L 131.2 193.0 80.8 27.5 0.50 cells in the course of AT inflammation as well as in the CRP, mg/L 8.9 12.4 7.1 6.4 0.73 resolution of obesity-associated tissue damage in AT. Albumin, g/dL 4.4 0.3 4.5 0.2 0.45 Fasting plasma Acknowledgments. The authors are grateful for the technical assistance glucose, of Christine Fröhlich and Michaela Kirstein (Institute of Anatomy and Cell Biology, mmol/L 6.2 1.3 7.3 3.5 0.26 Martin-Luther-University Halle-Wittenberg). The authors also thank Kathrin Jäger Fasting plasma and Andreas Lösche from the FACS core unit, University of Leipzig, as well as insulin, pmol/L 164.9 128.2 255.8 227.3 0.21 Alexander Navarrete-Santos from the Flow Cytometry core unit, Martin-Luther- HOMA index 6.8 7.5 14.3 19.3 0.18 University Halle-Wittenberg, and Claudia Müller from the Fraunhofer Institute Leipzig for providing flow imaging cytometers. Furthermore, the authors thank C- 2.2 1.0 1.8 0.8 0.42 Frank Brombacher from the Health Sciences Faculty, University of Cape Town, IL-6 24.0 53.5 4.8 2.2 0.56 Cape Town, South Africa, and David Hume from the Roslin Institute and Royal 2/2 HbA1c, mmol/L 7.4 1.3 7.0 1.6 0.59 School of Veterinary Studies, Roslin, U.K., for kindly providing IL-4Ra , 1/2 3 aflx/2 aDmyel 1/2 HbA1c, % 6.2 0.9 6.0 1.0 0.59 LysMCre IL-4R (IL-4R ), and MacGreen (CSF1R-eGFP ) Cholesterol, mice, respectively. mmol/L 4.3 1.0 4.1 1.0 0.62 Funding. This work was funded by Deutsche Forschungsgemeinschaft project number 209933838–SFB 1052 (project B09 to M.G. and I.B.). HDL cholesterol, Duality of Interest. No potential conflicts of interest relevant to this article mmol/L 1.0 0.3 1.0 0.4 0.84 were reported. LDL cholesterol, Author Contributions. J.B. designed, performed, and analyzed gene mmol/L 2.4 0.8 2.5 0.9 0.96 expression, flow cytometry, imaging flow cytometry, and microscopic imaging , data and wrote the manuscript. A.L. performed the beat assay. J.F. performed and mmol/L 2.3 2.7 1.5 0.6 0.46 analyzed murine explants experiments. C.H. performed murine Leukocytes, experiments. P.K. and M.B. analyzed human data. J.E. and I.B. performed live Gpt/L 8.6 2.0 10.5 2.0 0.04* imaging and edited the manuscript. M.G. designed the study and wrote the paper. Erythrocytes, M.G. is the guarantor of this work and, as such, had full access to all the data in the Tpt/L 4.7 0.5 5.0 0.5 0.26 study and takes responsibility for the integrity of the data and the accuracy of the Thrombocytes, data analysis. Gpt/L 268.9 73.3 261.9 36.4 0.81 References ALT, mkat/L 0.6 0.3 0.7 0.5 0.56 1. Blüher M. Obesity: global epidemiology and pathogenesis. Nat Rev Endo- m AST, kat/L 0.6 0.2 0.6 0.3 0.60 crinol 2019;15:288–298 GGT, mkat/L 0.7 0.5 0.5 0.2 0.35 2. Olshansky SJ, Passaro DJ, Hershow RC, et al. A potential decline in life ex- TSH, mU/L 1.5 0.9 1.3 1.1 0.69 pectancy in the United States in the 21st century. N Engl J Med 2005;352:1138–1145 3. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. T2D, type 2 diabetes; TSH, thyrotropin. *Statistically significant. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–1808 4. Cinti S, Mitchell G, Barbatelli G, et al. Adipocyte death defines macrophage have been due to endogenous de novo expression of M2 localization and function in adipose tissue of obese mice and humans. J Lipid Res fl markers by MGCs or due to fusion of proin ammatory 2005;46:2347–2355 (M1) with anti-inflammatory (M2) macrophages. Thus, 5. Lumeng CN, DelProposto JB, Westcott DJ, Saltiel AR. Phenotypic switching of a reprogramming of proinflammatory macrophages to- adipose tissue macrophages with obesity is generated by spatiotemporal dif- ward a tissue-remodeling M2 phenotype in MGCs would ferences in macrophage subtypes. Diabetes 2008;57:3239–3246 548 Giant Cells in Adipose Tissue Diabetes Volume 70, February 2021

6. Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch in 25. Coats BR, Schoenfelt KQ, Barbosa-Lorenzi VC, et al. Metabolically activated adipose tissue macrophage polarization. J Clin Invest 2007;117:175–184 adipose tissue macrophages perform detrimental and beneficial functions during 7. Strissel KJ, Stancheva Z, Miyoshi H, et al. Adipocyte death, adipose tissue diet-induced obesity. Cell Rep 2017;20:3149–3161 remodeling, and obesity complications. Diabetes 2007;56:2910–2918 26. Murano I, Rutkowski JM, Wang QA, Cho YR, Scherer PE, Cinti S. Time course 8. Helming L, Gordon S. Macrophage fusion induced by IL-4 alternative ac- of histomorphological changes in adipose tissue upon acute lipoatrophy. Nutr tivation is a multistage process involving multiple target molecules. Eur J Immunol Metab Cardiovasc Dis 2013;23:723–731 2007;37:33–42 27. Kourtzelis I, Li X, Mitroulis I, et al. DEL-1 promotes macrophage efferocytosis 9. Byrd TF. Multinucleated giant cell formation induced by IFN-gamma/IL-3 is and clearance of inflammation. Nat Immunol 2019;20:40–49 associated with restriction of virulent Mycobacterium tuberculosis cell to cell 28. Elliott MR, Chekeni FB, Trampont PC, et al. Nucleotides released by apoptotic invasion in human monocyte monolayers. Cell Immunol 1998;188:89–96 cells act as a find-me signal to promote phagocytic clearance. Nature 2009;461: 10. Bracq L, Xie M, Lambelé M, et al. T cell-macrophage fusion triggers multi- 282–286 nucleated giant cell formation for HIV-1 spreading. J Virol 2017;91:e01237-17 29. Elliott MR, Ravichandran KS. The dynamics of apoptotic cell clearance. Dev 11. Braune J, Weyer U, Matz-Soja M, et al. Hedgehog signalling in myeloid cells Cell 2016;38:147–160 impacts on body weight, adipose tissue inflammation and glucose . 30. Zamarron BF, Mergian TA, Cho KW, et al. Macrophage proliferation sustains Diabetologia 2017;60:889–899 adipose tissue inflammation in formerly obese mice. Diabetes 2017;66:392–406 12. Trout KL, Holian A. Factors influencing multinucleated giant cell formation 31. Vatarescu M, Bechor S, Haim Y, et al. Adipose tissue supports normalization in vitro. Immunobiology 2019;224:834–842 of macrophage and lipid handling in obesity reversal. J Endocrinol 2017;233: 13. Braune J, Weyer U, Hobusch C, et al. IL-6 regulates M2 polarization and local 293–305 proliferation of adipose tissue macrophages in obesity. J Immunol 2017;198: 32. Elliott MR, Ravichandran KS. ELMO1 signaling in apoptotic germ cell 2927–2934 clearance and spermatogenesis. Ann N Y Acad Sci 2010;1209:30–36 14. Haase J, Weyer U, Immig K, et al. Local proliferation of macrophages in 33. Elliott MR, Koster KM, Murphy PS. Efferocytosis signaling in the regulation of adipose tissue during obesity-induced inflammation. Diabetologia 2014;57:562– macrophage inflammatory responses. J Immunol 2017;198:1387–1394 571 34. Fullerton JN, Gilroy DW. Resolution of inflammation: a new therapeutic 15. Gericke M, Weyer U, Braune J, Bechmann I, Eilers J. A method for long-term frontier. Nat Rev Drug Discov 2016;15:551–567 live imaging of tissue macrophages in adipose tissue explants. Am J Physiol 35. Mazaheri F, Breus O, Durdu S, et al. Distinct roles for BAI1 and TIM-4 in the Endocrinol Metab 2015;308:E1023–E1033 engulfment of dying neurons by microglia. Nat Commun 2014;5:4046 16. Gericke MT, Kosacka J, Koch D, et al. Receptors for NPY and PACAP differ in 36. Liu C, Li P, Li H, et al. TREM2 regulates obesity-induced insulin resistance via expression and activity during in the murine 3T3-L1 fibroblast cell adipose in mice of high-fat feeding. J Transl Med 2019;17:300 line. Br J Pharmacol 2009;157:620–632 37. Jaitin DA, Adlung L, Thaiss CA, et al. Lipid-associated macrophages control 17. Gericke MT, Schröder T, Kosacka J, Nowicki M, Klöting N, Spanel-Borowski metabolic homeostasis in a Trem2-dependent manner. Cell 2019;178:686– K. Neuropeptide Y impairs insulin-stimulated translocation of 4 in 698.e14 3T3-L1 adipocytes through the Y1 receptor. Mol Cell Endocrinol 2012;348:27–32 38. Jacome-Galarza CE, Percin GI, Muller JT, et al. Developmental origin, 18. Haka AS, Barbosa-Lorenzi VC, Lee HJ, et al. Exocytosis of macrophage functional maintenance and genetic rescue of osteoclasts. Nature 2019;568:541– lysosomes leads to digestion of apoptotic adipocytes and foam cell formation. J 545 Lipid Res 2016;57:980–992 39. Hume DA. Macrophages as APC and the dendritic cell myth. J Immunol 2008; 19. Milde R, Ritter J, Tennent GA, et al. Multinucleated giant cells are specialized 181:5829–5835 for complement-mediated phagocytosis and large target destruction. Cell Rep 40. Herrtwich L, Nanda I, Evangelou K, et al. DNA damage signaling instructs 2015;13:1937–1948 polyploid macrophage fate in granulomas. Cell 2016;167:1264–1280.e18 20. Klöting N, Fasshauer M, Dietrich A, et al. Insulin-sensitive obesity. Am J 41. Helming L, Winter J, Gordon S. The scavenger receptor CD36 plays a role in Physiol Endocrinol Metab 2010;299:E506–E515 cytokine-induced macrophage fusion. J Cell Sci 2009;122:453–459 21. Osborn O, Olefsky JM. The cellular and signaling networks linking the 42. Shapiro H, Pecht T, Shaco-Levy R, et al. Adipose tissue foam cells are present immune system and metabolism in disease. Nat Med 2012;18:363–374 in human obesity. J Clin Endocrinol Metab 2013;98:1173–1181 22. Lumeng CN, Deyoung SM, Bodzin JL, Saltiel AR. Increased inflammatory 43. Prieur X, Mok CY, Velagapudi VR, et al. Differential lipid partitioning between properties of adipose tissue macrophages recruited during diet-induced obesity. adipocytes and tissue macrophages modulates macrophage and M2/ Diabetes 2007;56:16–23 M1 polarization in obese mice. Diabetes 2011;60:797–809 23. Murano I, Barbatelli G, Parisani V, et al. Dead adipocytes, detected as crown- 44. Wentworth JM, Naselli G, Brown WA, et al. Pro-inflammatory CD11c1CD2061 like structures, are prevalent in visceral fat depots of genetically obese mice. J adipose tissue macrophages are associated with insulin resistance in human Lipid Res 2008;49:1562–1568 obesity. Diabetes 2010;59:1648–1656 24. Altintas MM, Azad A, Nayer B, et al. Mast cells, macrophages, and crown-like 45. Muir LA, Kiridena S, Griffin C, et al. Frontline science: rapid adipose tissue structures distinguish subcutaneous from visceral fat in mice. J Lipid Res 2011;52: expansion triggers unique proliferation and lipid accumulation profiles in adipose 480–488 tissue macrophages. J Leukoc Biol 2018;103:615–628