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

Obesity is a disease of epidemic proportions and it is closely linked to an increased risk of developing cardiovascular diseases, stroke, cancer, and type 2 diabetes (1). As a result, obesity is one of the main threats to global human health and life expectancy (2). Therefore, it is important to understand the mechanisms underlying obesity and associated diseases, such as type 2 diabetes.

During obesity development significant metabolic and immunological changes occur, especially in adipose tissue (AT). The first evidence of a positive correlation between inflammatory changes and insulin resistance appeared by the observation that TNF-α secretion is chronically elevated in obese rodent AT compared with lean animals (3). Since this initial finding, numerous studies have demonstrated that obesity is associated with chronic low-grade inflammation in AT, reflected by increased levels of inflammatory cytokines (4). Furthermore, the obesity-associated inflammatory state is linked to accumulation of AT macrophages (ATMs) and other immune cells (4).

Macrophages are generally classified as classically activated (M1) or alternatively activated (M2). This activation state can be affected by either a pro- or anti-inflammatory micromilieu. Polarization of M1 macrophages is induced by proinflammatory cytokines, such as TNF-α, INF-γ, or microbial products, such as LPS (5). M1 macrophages are characterized by the expression of the surface marker CD11c and production of proinflammatory cytokines. Additionally, they produce reactive oxygen and nitrogen intermediates (6). In contrast, M2-polarized macrophages induce the production of anti-inflammatory cytokines, express high levels of arginase 1, and can be identified by the expression of CD163, CD206, and CD301 on their cell surface (6, 7). They are thought to participate in tissue homeostasis, remodeling, and wound healing. Alternative activation of macrophages is stimulated by Th2 cytokines, such as IL-4, IL-13, and partially IL-10 (5). Interestingly, also IL-6, a classical proinflammatory cytokine, which is upregulated during several kinds of inflammation, including obesity, promotes M2 polarization (8). Additionally, IL-6 has been shown to sustain systemic glucose tolerance and insulin sensitivity (8–11). Importantly, a growing body of evidence suggests that the established M1/M2 paradigm is an oversimplification of the macrophage biology found in vivo. In obesity, ATMs exhibit an activated phenotype that is distinct from classical activation (M1) or alternative activation (M2). Therefore, the term “metabolic activation” has been introduced by Kratz et al. (7), which is characterized by an increased machinery of lipid catabolism in ATMs (12).

In lean individuals, visceral fat depots contain a relatively low amount of tissue-resident macrophages (10–15% of stroma cells), and these cells exhibit an alternatively activated phenotype. They are distributed in an even manner (13) and have been shown to promote insulin sensitivity (14). The insulin-sensitive state of AT in lean mice is thought to be preserved by eosinophils due to local IL-4 secretion and maintenance of an anti-inflammatory milieu (15). During weight gain, a chronic low-grade inflammation is induced and the number of macrophages in AT rises. Therefore, the percentage of ATMs in obese AT can be as high as 50% of stroma cells (4). Additionally, a phenotypic switch from an M2 toward a more classically activated ATM phenotype occurs (13). Diet-induced obesity also alters the distribution of macrophages within the tissue, which leads to the formation of crown-like structures (CLS) around dying adipocytes (16). Notably, 90% of ATMs in obesity are localized in CLS (17, 18).

Considering the dramatic gain of AT mass in obesity, the absolute number of both M1- and M2-polarized ATMs increases (6). However, mechanisms leading to increased macrophage numbers seem to differ between these two macrophage subpopulations. Recently, we and others have shown that ATMs have the ability to proliferate within AT, especially within CLS (17, 19, 20). Notably, proliferating ATMs expressed CD206 and CD301 at a high level, suggesting that these cells exhibit an alternatively activated state (17).

M2 macrophages in AT can positively alter the outcome of obesity due to their beneficial function in apoptotic cell clearance, tissue repair, and remodeling (21). Therefore, the aim of this study was to identify the microenvironmental factors that lead to obesity-induced local proliferation of M2 ATMs. We tested the effect of various cytokines and neutralizing Abs in an organotypic AT model (AT explants) for their ability to influence local ATM proliferation. The gene expression of identified cytokines and their putative receptors was further analyzed in AT from lean and obese mice to define microenvironmental cues stimulating ATM proliferation in obesity.

We demonstrate that Th2 cytokines that drive M2 polarization can also increase local ATM proliferation. Furthermore, we identify IL-6 as a main driver of ATM proliferation in obesity, presumably due to upregulation of IL-4Rα. Hence, our data indicate a distinct M2 population of ATMs in obesity, characterized by enhanced Il4ra expression and a high proliferation rate.

Mouse strains were maintained in the local animal facility at a 12-h light/dark cycle with free access to food and water. For diet-induced obesity, male wild-type C57BL/6 mice or MacGreen (CSF1R-eGFP^+/−) mice on a C57BL/6 background were fed a high-fat diet (60% kcal fat; Ssniff Spezialdiäten, Soest, Germany) for 24 wk, starting at 6 wk of age. Control littermates were kept on a regular chow diet (9% kcal fat; Ssniff Spezialdiäten). All experiments were approved by the local authorities (Landesdirektion Leipzig).

Epididymal AT of male MacGreen mice after 24 wk of high-fat diet was used to generate organotypic AT cultures (AT explants). MacGreen mice develop AT inflammation similar to wild-type C57BL/6 mice (22) with the additional opportunity to observe ATMs during the experiment ex vivo. Mice were killed and the rostral part of the epididymal fat pad was dissected sterilely. Then, the fat pad was placed in a PBS-filled culture dish and was further cut into 1 mm³ pieces using a sterile razor blade. Explants were cultured for 48 or 96 h in RPMI 1640 medium (Sigma-Aldrich, Deisenhofen, Germany) supplemented with 1% insulin/transferrin/selenium mixture and antibiotics (100 U/ml penicillin and streptomycin; all reagents from Sigma-Aldrich) at 5% CO₂/21% O₂ and 37°C. For analysis of cell proliferation, 10 μM BrdU was applied to the culture medium (BD Pharmingen, Heidelberg, Germany). Explants were stimulated by several cytokines (1–250 ng/ml as indicated; PeproTech, Hamburg, Germany; Sigma-Aldrich). Depletion of cytokines was performed by using anti–IL-4 (11B11), anti–IL-13 (eBio1316H), or anti–IL-6 (MP5-20F3) Abs. Rat IgG1 control was performed in parallel in every experiment (all 10 μg/ml; eBioscience, Frankfurt, Germany). After 24 h of incubation with neutralizing Abs, BrdU was applied to the culture medium for the last 24 h. Detection of BrdU uptake of ATMs was performed as described below. For Th2 susceptibility test, AT explants were generated as described above and directly stimulated with IL-13 or IL-4 (both 50 ng/ml) at 37°C. After 30 min, AT explants were snap frozen in liquid nitrogen and processed for Western blot analysis.

For flow cytometry analyses, freshly dissected AT or cultured AT explants were digested using collagenase type II (Worthington Biochemical, Lakewood, NJ). Subsequently, the cell suspension was filtered through a 75 μm mesh, followed by blocking Fc receptors by anti-CD16/32 (1:100; eBioscience) for 15 min on ice. Next, cells were stained by anti–CD45-FITC (30-F11; including 99.9% of GFP cells, data not shown), anti–F4/80-PE-Cy7 (BM8), anti–CD11c-Brilliant Violet 421 (N418), anti–CD11c-PE (N418; all 1:100; eBioscience), anti–CD206-Alexa Fluor 647 (MR5D3; 1:50; AbD Serotec, Kidlington, U.K.), and/or anti–CD301-Alexa Fluor 647 (ER-MP23; 1:200; AbD Serotec) for 20 min on ice. Fixation and permeabilization for cell proliferation assays were performed using the allophycocyanin BrdU flow kit (BD Pharmingen) according to the manufacturer’s protocol. For detection of BrdU, cells were further treated with DNase IV (Sigma-Aldrich), followed by anti–BrdU-Alexa Fluor 647 (PRB-1; 1:50; Abcam, Cambridge, U.K.). For detection of intracellular proteins, DNase treatment was omitted and appropriate Abs, such as anti-Ki67 (SP6; 1:100; DCS Immunoline, Hamburg, Germany), anti–IL-13-PE (eBio13A; 1:100; eBioscience), or anti–IL-6-PE (MP5-20F3; 1:100; eBioscience), were applied. For visualizing Ki67 staining we used goat anti-rabbit Alexa Fluor 647 (1:200; Invitrogen, Darmstadt, Germany). Finally, 7-aminoactinomycin D (BD Pharmingen) was used for DNA staining. Additionally, fluorescence minus one and isotype controls were carried out for all experiments. Isotype controls of the CD11c/CD206/CD301 Abs were used for gating ATM subsets, and secondary Ab controls were used for defining Ki67⁻/Ki67⁺ cells (Supplemental Fig. 1). For analysis of flow cytometry data, cells were gated first for 7-aminoactinomycin D⁺ cells. Subsequently, CD45⁺ and F4/80⁺ cells were defined as ATMs, which further could be differentiated into M1 (CD11c⁺; CD206⁻/CD301⁻) and M2 (CD11c⁻; CD206⁺/CD301⁺) macrophages. Analysis was performed using an LSR II (BD Pharmingen) equipped with FACSDiva software 8.0. Quantification was performed using FlowJo software 10.0.5 (Tree Star, Ashland, OR).

Relative gene expression was analyzed by quantitative real-time PCR (Maxima SYBR Green quantitative PCR master mix; Thermo Fisher Scientific, Schwerte, Germany) on a Bio-Rad CFX96 Manager system (Bio-Rad, Munich, Germany). After extraction of total RNA (TRI Reagent solution; Thermo Fisher Scientific), cDNA was synthesized by using oligo(dT) primers and a ProtoScript first-strand cDNA synthesis kit (New England Biolabs, Frankfurt am Main, Germany). Primer 3 software was used to design gene-specific primers (presented in Supplemental Table I). Gene of interest mRNA expression levels were measured in duplicate and normalized to Ipo8. Relative gene expression data were analyzed using the ∆∆Ct method by Pfaffl (23). Fractionation of AT to separate stroma cells (including macrophages) and adipocytes was performed as described previously (24).

Western blot analysis was performed as described earlier (25). Proteins were extracted using RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with 1% PMSF and 1% protease inhibitor mixture [Sigma-Aldrich]). Blots were incubated with polyclonal antisera against phospho-STAT6 (ab54461; 1:500; Abcam) or pan-STAT6 (9362; 1:500; Cell Signaling Technology, Boston, MA) at 4°C overnight. Immunoreactions were detected with the appropriate peroxidase-conjugated anti-rabbit IgG secondary Ab (1:10,000; Vector Laboratories, Peterborough, U.K.) at room temperature for 2 h. Peroxidase activity was visualized with an ECL kit (Amersham Pharmacia, Freiburg, Germany). Semiquantitative evaluation of arbitrary units was performed with the Gel analyzer software (Media Cypernetics, Bethesda, MD).

Data are presented as mean ± SD or as box plots (whiskers represent minima and maxima) of at least three animals evaluated by the Student–Newman–Keuls method for multiple comparisons, the Student t test, or the Mann–Whitney U test, respectively. The Pearson correlation coefficient was calculated using GraphPad Prism (GraphPad Software, La Jolla, CA). A p value < 0.05 was considered statistically significant.

First, we established an organotypic culture system of AT (AT explants) to study the impact of cytokine stimulation on local ATM proliferation without the confounding effect of monocyte recruitment from the circulation. After 48 h ex vivo, we analyzed the immune phenotype of ATMs by flow cytometry. AT explants from lean mice exhibited an almost exclusive M2 phenotype of ATMs (CD301⁺, CD11c⁻), whereas AT explants from obese mice displayed a decrease of M2 macrophage proportion and an increase of classically activated M1 ATMs (CD301⁻, CD11c⁺; Fig. 1A, 1C). Hence, the ratio of M1 to M2 macrophages was elevated in AT explants from obese mice, compared with AT explants from lean mice (Fig. 1C). Furthermore, we studied the ability of ATMs to proliferate locally within AT. We quantified proliferating (BrdU⁺) and nonproliferating (BrdU⁻) ATMs based on their BrdU incorporation after 48 h ex vivo (Fig. 1B, 1D). In AT explants from obese mice, we found significantly more BrdU⁺ ATMs than in AT explants from lean mice (Fig. 1D). Hence, the proinflammatory ATM phenotype and the increased proliferation rate of ATMs previously documented in vivo are sustained in AT explants for at least 48 h ex vivo.

Next, BrdU incorporation and the immune phenotype of ATMs were studied by flow cytometry upon stimulation of obese AT explants with various cytokines for 48 h. ATMs in the control group treated with PBS showed an initial proliferation of ∼30% and a more classically activated immune phenotype. Stimulation with IL-4, IL-13, or GM-CSF led to an enhanced incorporation of BrdU reflecting more proliferating ATMs. Additionally, Th2 cytokines increased the number of M2-polarized ATMs. In general, Th2 cytokine stimulation is sufficient to shift the M1-to-M2 ratio toward a preferential M2 polarization (Fig. 2A, 2B) and also to enhance the local proliferation rate of ATMs, whereas LPS or TNF-α stimulation shifted ATMs toward M1 polarization and attenuated local ATM proliferation (Fig. 2A–C; individual frequencies of all ATMs and M1- and M2-polarized ATMs are presented in Supplemental Fig. 2). GM-CSF, IL-4, or IL-13 was also applied in increasing concentrations from 1 to 250 ng/ml, respectively. We found that GM-CSF is the most potent stimulus for ATM proliferation, whereas IL-13 seems to be marginally less effective (Fig. 2D–F). In summary, we performed correlation analyses of all experiments. We found that the number of M1-polarized ATMs negatively correlated with the number of proliferating ATMs (r = −0.47, p < 0.0001; Fig. 2G). In contrast, the number of M2-polarized ATMs positively correlated with the number of proliferating ATMs (r = 0.52, p < 0.0001; Fig. 2H).

Next, we determined the expression of Ki67, a marker protein of cell proliferation, by flow cytometry as an additional independent measure of ATM proliferation. After 48 h of IL-4 or GM-CSF stimulation, the number of Ki67⁺ ATMs increased. After 96 h of stimulation with IL-4, GM-CSF, or IL-13, the local proliferation rate of ATMs was also enhanced as measured by Ki67 expression (Fig. 3A, 3B). Importantly, after 96 h of stimulation using either IL-4 or IL-13, also the percentage of ATMs among all isolated stroma cells increased significantly within the treated AT explants (Fig. 3C). These results demonstrate that Th2 cytokines are sufficient to increase both cell proliferation rate and the number of ATMs.

To identify the local stimulus of M2 polarization in obese animals, we next analyzed the expression of putative Th1 and Th2 cytokines and their receptors in AT from lean and obese mice. Although obesity induced an increase of proinflammatory factors, such as MCP-1 and TNF-a, we also found elevated mRNA expression of cytokines that are well-documented inducers of M2 polarization, such as Il-6, Il-10, and Il-13 (5, 8) (Fig. 4A). Furthermore, expression of the respective cytokine receptors Il6ra, Il10ra, Il10rb, Il4ra, and Il13ra2 was also increased in obese AT, suggesting that signaling of these cytokines could be elevated in obesity (Fig. 4B). Of note, Il-4, Il-13, and Il-6 expression was preferentially found in stroma cells of AT, including macrophages and other immune cells, whereas their respective receptors (Il4ra, Il6ra, and Il13ra1) were equally expressed by stroma cells and adipocytes within AT. Interestingly, the IL-13 decoy receptor Il13ra2 was preferentially expressed in adipocytes, rather than in stroma cells (Fig. 4C).

We hypothesized that IL-13 could be a possible stimulus of local ATM proliferation in obesity. We performed flow cytometry and observed that 7.9% of ATMs produced IL-13 in obesity, which represents 96.4% of overall IL-13 protein expression in stroma cells. Furthermore, IL-13–producing ATMs were preferentially M2 polarized (Fig. 4D). Next, we focused on the IL-4Rα, which is altered in obesity and essential for IL-13 signaling. We stimulated acutely isolated AT explants with IL-13 and IL-4. Interestingly, IL-4 stimulation of obese AT explants led to a 10-fold enhanced STAT6 phosphorylation compared with AT from lean littermates, possibly reflecting the enhanced expression of Il4ra in AT from obese mice, as shown above on the mRNA level. In contrast, IL-13 stimulation of STAT6 did not differ between lean and obese mice (Fig. 4E, 4F).

To test which cytokine could be a required stimulus of ATM proliferation in obesity, we depleted AT explants by using neutralizing Abs against IL-4, IL-13, and also IL-6. Of note, IL-6 promotes M2 macrophage polarization by increasing IL-4Rα expression (8). Importantly, only IL-6 depletion led to a significant reduction of BrdU incorporation by ATMs (Fig. 5A, 5B). Gene expression analysis verified that IL-6 neutralization blunted Il4ra expression (Fig. 5C). Furthermore, IL-6 was also produced by M2-polarized ATMs in obese mice (Fig. 5D).

We found that mRNA expression of Il-6 and its receptor was elevated in obesity (Fig. 4A, 4B). We thus asked whether IL-6 serves as an upstream regulator of IL-4R–dependent ATM proliferation. Therefore, we tested the effect of IL-6 neutralization on ATM proliferation and macrophage polarization. Again, flow cytometry revealed that the inhibition of IL-6 signaling led to a significant decrease in the proliferation rate of ATMs (Fig. 6A, 6C), without affecting leukocyte or ATM viability (Fig. 6B). Additional stimulation of IL-6–depleted AT explants with Th2 cytokines, such as IL-4, IL-13, and GM-CSF, revealed an attenuation of the established effect of IL-13 and IL-4 on BrdU incorporation upon IL-6 neutralization (Fig. 6A, 6C). Furthermore, IL-6 depletion shifted the M1-to-M2 ratio of ATMs toward a more classically activated M1 phenotype (Fig. 6D, 6E). Importantly, proliferation and polarization of ATMs upon treatment with GM-CSF, which does not use IL-4R signaling, were unaffected by IL-6 neutralization (Fig. 6A, 6C–E).

AT of obese individuals exhibits a chronic low-grade inflammatory state, which is linked to a whole plethora of obesity-associated diseases. Obesity-associated inflammation is characterized by increased numbers of ATMs (4). The rising number of ATMs in obese AT was originally thought to exclusively result from monocyte recruitment from the circulation (26). Monocyte migration into AT is dependent on the release of MCP-1 (CCL2) from hypertrophic adipocytes and CCR2 expression on the surface of blood monocytes (26, 27). We and others have recently shown that ATMs, apart from being replenished by monocytes, also proliferate locally within AT (17, 19). This local proliferation was predominantly found in macrophages of the alternatively activated M2 phenotype. In the present study, we used an organotypic organ culture model of AT to identify cytokines that promote local ATM proliferation. Notably, macrophage proliferation takes at least 1–2 d (28), whereas monocyte recruitment appears as early as 60 min after cytokine stimulation (29). Therefore, in animals, distinguishing between local proliferation of mature tissue macrophages and infiltration of monocytes descending from proliferating bone marrow precursors is challenging.

In this study, we show that AT explants from obese mice sustain their proinflammatory macrophage phenotype and the high ATM proliferation rate for at least 48 h ex vivo, as compared with AT explants from lean littermates. Thus, using AT explants from obese mice allowed us to study the influence of several cytokines on proliferation of mature tissue macrophages without the bias of early monocyte recruitment. Of note, the concentration that we used for ex vivo stimulation of AT explants (50 ng/ml) reflects a compromise between commonly used cell culture concentrations (10–50 ng/ml) (8) and higher cytokine concentrations, commonly used for i.p. injections of living animals (5 μg per mouse) (30, 31).

However, in AT explants, polarization of ATM subtypes can be provoked by Th2 cytokine stimulation, as shown for IL-4, IL-13, and GM-CSF. Although GM-CSF is frequently used to induce M1 polarization in human macrophages (5, 32), in our hands, GM-CSF was also a potent inducer of M2 ATM polarization. Notably, GM-CSF has been shown to stimulate IL-13 expression in macrophages in vitro and in vivo (33–35), as well as the expression of CD206 (36), a surface marker of M2 macrophages.

Most importantly, Th2 cytokines enhanced the local proliferation of ATMs as shown by BrdU incorporation. In contrast, stimulation with proinflammatory molecules, such as TNF-α and LPS, promoted M1 polarization and decreased ATM proliferation. Furthermore, our findings were independently confirmed by Ki67 staining, an additional marker for cell proliferation, thereby excluding BrdU incorporation by pinocytosis. Thus, ATM proliferation capacity strongly depends on the polarization state of ATMs. These findings are in line with data from Jenkins et al. (30, 37) who reported that IL-4 signaling is a key player of proliferation in resident tissue macrophages. In obesity, Zheng et al. (31) showed that proliferation and M2 polarization of ATMs are regulated by STAT6, a downstream molecule in the IL-4Rα signaling cascade. Most recently, osteopontin has been reported as a required factor in local ATM proliferation (38). In line with this, osteopontin also increases M2 markers, such as CD163 and CD206, and reduces inflammatory properties, such as TNF-α expression, in human macrophages (39).

The main focus of the present study was to define microenvironmental cues in AT from obese mice that promote M2 polarization and ATM proliferation in obese mice in vivo. First, we verified the previously documented obesity-induced increase of TNF-α and MCP-1 expression that is indicative for the proinflammatory environment present in insulin-resistant individuals (4, 26). Notably, also the gene expression of Il-13 and Il-10 and their respective receptors were elevated in AT of obese mice, both of which represent classical anti-inflammatory cytokines. We further report an enhanced STAT6 phosphorylation of AT from obese mice after IL-4 stimulation, which might reflect higher tissue expression of the IL-4Rα or an increased susceptibility to IL-4 stimulation. Of note, the IL-4Rα–chain is essential for signal transduction upon binding of either IL-4 or IL-13. Hence, also molecules involved in Th2 immunity and M2 polarization are increased in obesity. However, we suggest a different M2 phenotype in lean and obese AT. M2 macrophages in lean AT seem to represent a resting, tissue-resident M2 phenotype, characterized by a low proliferation rate, whereas M2 macrophages in obese AT might represent a locally stimulated M2 phenotype. This stimulated M2 phenotype in obesity is characterized by high local proliferation.

We further analyzed the underlying mechanism behind this high proliferation rate of M2 macrophages in obesity. We found that gene expression of both Il-6 and its receptor is significantly elevated in AT of obese mice. Importantly, IL-6 was recently shown promoting M2 polarization of ATMs via a direct upregulation of IL-4Rα expression and an enhanced IL-4 response in macrophages (8). Accordingly, we tested the impact of IL-6 signaling on local macrophage proliferation. Because of high abundance of IL-6 in AT explants, we used a neutralizing Ab against IL-6. We found that the proliferation of ATMs is significantly decreased by IL-6 depletion. Importantly, IL-4 and IL-13 stimulation of ATM proliferation can also be suppressed by blocking IL-6 signaling. Additionally, gene expression analysis of AT explants treated with the IL-6 Ab revealed a downregulation of the Il4ra subunit. Hence, the effect of cytokine stimulation via the IL-4Rα/STAT6 axis is attenuated by IL-6 inhibition. In line with this, GM-CSF stimulation of ATM proliferation was unaffected by IL-6 depletion, because GM-CSF signals via the CSF-2 receptor and not by the IL-4 receptor or a STAT6-dependent mechanism (40). Therefore, we conclude that although several Th2 cytokines are sufficient to increase local ATM proliferation, only IL-6 is a required factor for sustaining local ATM proliferation.

However, in this study we were unable to provide direct evidence for an involvement of IL-13 in local ATM proliferation. Although Il-13 expression is enhanced in obesity, IL-13 depletion by using neutralizing Abs (clone eBio1316H) does not alter ATM proliferation. Furthermore, IL-13 stimulation of STAT6 phosphorylation is not as efficient as IL-4 in AT explants from obese mice. Therefore, the ligand of the IL-4Rα in obesity is still ill-defined. Importantly, we found that Il13ra2, an exclusive IL-13 receptor, was also strongly enhanced in obesity. Of note, IL-13Rα2 is considered to be a decoy receptor with a short intracellular domain only. Furthermore, signaling activity of this receptor is discussed controversially (34, 37). Therefore, high-affinity binding of IL-13 to IL-13Rα2 can avoid IL-13 binding to the IL-4Rα and may be responsible for a lower efficacy of IL-13 in comparison with IL-4. Because we show in the present study that Il13ra2 expression is preferentially found on adipocytes, whereas Il-13 is preferentially expressed in ATMs, accumulation of macrophages around dead adipocytes in CLS could favor IL-13 binding to IL-4Rα on macrophages, rather than high-affinity binding of IL-13 to IL-13Rα2 on surrounding healthy adipocytes. However, whether IL-13 directly stimulates the IL-4Rα via an increased local concentration within CLS needs to be addressed in further studies. Then, more sophisticated models, including ATM-specific knockdown approaches of IL-13 (41) or by using IL-13 knockout mice (42), may overcome the technical limitations of using neutralizing Abs, used in the present study. Of note, also a basal activity of the IL-4Rα has been reported (43). Most importantly, a reduced ATM proliferation in obese STAT6 knockout mice (31, 44) also point to an involvement of the IL-4Rα in obesity-associated ATM proliferation in vivo.

However, we provide evidence in this study for a distinct M2 phenotype of ATMs in obesity, characterized by an enhanced Il4ra expression and a high proliferation rate. Of note, IL-6 is also produced by M2-polarized ATMs in obesity and increases ATM proliferation as well as M2 polarization, presumably due to upregulation of IL-4Rα expression. Because IL-6 has been shown previously to increase systemic glucose tolerance and insulin sensitivity (8–11), the role of IL-6 as a proinflammatory cytokine and its prognostic value in obesity need to be reconsidered. Therefore, IL-6 or the IL-4 receptor could be potential targets to sustain insulin sensitivity in obesity.

We are grateful for the excellent technical assistance of Angela Ehrlich and Claudia Merkwitz. We also thank Martin Krüger and Marco Koch for helpful discussions and Kathrin Jäger and Andreas Lösche from the FACS core unit (all Leipzig University).

The authors have no financial conflicts of interest.