The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

The nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of “M2c” anti-inflammatory cells. We cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils. We found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206+ CD163+ CD16+) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206+ CD209+ CD163− MerTK−) to M2c (CD206high CD209− CD163+ MerTK+) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability. Our results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.

In the present study, we investigated the effects of PPAR-γ and LXRs in differentiation of M2c macrophages and induction of the MerTK/Gas6 axis. We found that PPAR-γ obstructs whereas LXRs promote MerTK upregulation. Importantly, MerTK expression induced by the PPAR-γ antagonist GW9662 is associated with M2c polarization, whereas LXR induction of MerTK occurs regardless of M2c phenotype acquisition. GW9662-driven M2c-like cells also release high amounts of Gas6 and low levels of TNF-α, but differ from conventional M2c cells by not showing enhanced clearance of ACs. These data contribute to better define the role of PPAR-γ and LXRs in human macrophage activation, and point out the existence of distinct regulation patterns for MerTK expression. The unexpected finding that PPAR-γ negatively controls the expansion of a discrete subset of anti-inflammatory macrophages may also have clinical implications.

Cell cultures
Monocytes from buffy coats of healthy blood donors were isolated by Ficoll-Paque™ Plus gradient (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and magnetic separation, using a kit for human monocyte enrichment by negative selection (EasySep™, StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer's instruction. CD14+ cells were cultured at 0.8×10 6 cells/ml in nontissue culture treated 24-well plates in X-Vivo™15 medium (Lonza, Walkersville, MD, USA) at 37°C in 5% CO 2 for 4 days, in the presence of rosiglitazone (PPAR-γ agonist, 0.1-10 μM), GW9662 (PPAR-γ antagonist, 0.01-10 μM) or T0901317 (LXR agonist, 0.001-1 μM) (Cayman Chemical, Ann Arbor, MI, USA). GW9662 (lot 0417082-20) was reconstituted in ethanol 2 mg/ml. T0901317 and rosiglitazone were reconstituted in dimethylsulfoxide (DMSO) 5 and 10 mg/ml, respectively. Serial dilutions were performed using culture medium. Cells were ultimately exposed to working solutions containing non-cytotoxic amounts of ethanol or DMSO (≤0.1%). In some experiments using high concentrations of reagent (i.e., rosiglitazone 50-100 μM) and vehicle (i.e., DMSO > 0.15%), vehicle controls were included. When specified, cells were differentiated in the presence of GM-CSF 100 ng/ml (Peprotech, Rocky Hill, NJ, USA) or IFN-γ 2.5 ng/ml (R&D Systems, Minneapolis, MN, USA) for M1 differentiation, IL-4 20 ng/ml (Novus Biologicals, Littleton, CO, USA) for M2a differentiation, and dexamethasone 100 nM (Sigma-Aldrich, St. Louis, MO, USA) for M2c differentiation. In some experiments, cells were coincubated with low doses of LPS (50 ng/ml; extracted from E. Coli 026:B6, Sigma-Aldrich) to stimulate cytokine secretion. After differentiation, cells were incubated for 20 minutes at 37°C with a detaching buffer containing EDTA 10 mM and lidocaine 15 mM in sterile Phosphate Buffered Saline (PBS). Cells were then washed and harvested by centrifugation. Pellets were resuspended in PBS containing 2% bovine serum albumin and freshly analyzed by flow cytometry. Supernatants were collected and immediately stored at −20°C before being tested by ELISA. Participants gave informed consent to donate their blood samples. The study was approved by the Institutional Review Board of Temple University.

ELISA
Gas6, IL-10 and TNF-α levels were measured in supernatants of cell cultures using sandwich ELISA according to standard procedure [32]. Briefly, 96-well plates were precoated overnight with a capture antibody. Samples from cell culture supernatants were applied to precoated plates in duplicate. Serial dilutions of purified recombinant rhGas6 (R&D Systems) were used to construct a standard curve. Blank wells received serum-free X-Vivo™15 medium. A purified goat polyclonal anti-human Gas6 antibody (R&D Systems) was used for capture. Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection. The plates were developed with 3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped with 2 N sulfuric acid. Absorbance was detected at 450 nm and read with a reference wavelength set at 570 nm using a VersaMAX ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density for each point was the average of duplicate samples. Concentrations were determined using SoftMax software (Molecular Devices) by applying four-parameter logistic regression to the standard curve. IL-10 and TNF-α levels were measured using human IL-10 ELISA MAX Standard kit and TNF-α ELISA MAX Standard kit (Biolegend), following the manufacturer's instructions.

Statistical analysis
Data are expressed as mean ± SEM. Statistical significance among different cell treatments was assessed by Student's paired t-test, or one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test if more than two treatment groups were compared. Statistical significance was defined as P <0.05. Analysis and graphing were performed using Prism™ software (GraphPad Software, La Jolla, CA, USA).

Results
The PPAR-γ antagonist GW9662 inhibits IL-4-driven macrophage alternative activation by inducing a phenotypic M2a-to-M2c switch Many of IL-4's effects on macrophage alternative (M2) differentiation are mediated by PPAR-γ via STAT6 [7,8,12,17,19]. IL-4 specifically induces the "M2a" phenotype, which is characterized by expression of the universal M2 marker CD206, high levels of CD209 and low membrane expression of CD163 and MerTK [4]. IL-4 hinders induction of the "M2c" phenotype, which is instead characterized by high levels of CD206, CD163 and MerTK and low levels of CD209 [4,33]. Here, we used flow cytometry to assess the effects of PPAR-γ inhibition on the phenotype of IL-4 exposed cells. Differentiation of monocytes/macrophages in the presence of IL-4 and the PPAR-γ antagonist GW9662 resulted in brighter expression of CD206, inhibition of CD209 induction, and upregulation of CD163 and MerTK. GW9662 effects were dose-dependent ( Figures 1A-B). Thus, blocking PPAR-γ activation during M2a differentiation provokes a phenotypic switch from M2a (CD206 M1 polarizing agents IFN-γ and GM-CSF act to oppose GW9662 effects on macrophage phenotype M1 stimulating cytokines such as IFN-γ and GM-CSF downregulate membrane expression of MerTK and CD163 [4]. IFN-γ also hinders dexamethasone induction of the M2c phenotype [33]. Unlike IFN-γ, GM-CSF is able to upregulate CD206 [4]. Herein, we investigated the effects of GW9662 in the presence of either IFN-γ or GM-CSF. We found that both IFN-γ and GM-CSF were permissive for GW9662 induction of the M2c receptor CD163, resulting in its significant upregulation. In parallel to what was reported above in the presence of IL-4 ( Figure 1), in the presence of GM-CSF, GW9662 also led to significantly enhanced expression of CD206. However, in the presence of IFN-γ, CD206 expression did not increase, and neither IFN-γ nor GM-CSF allowed significant MerTK upregulation (Figure 2A-B). Therefore, similarly to what is observed for dexamethasone [33], the presence of M1 cytokines obstructs the effects of GW9662 on the M2c phenotype.

GW9662 induces M2c-like cells that express MerTK and produce the MerTK ligand Gas6
Subsequently, we examined the M2c polarizing effects of GW9662 on otherwise untreated cells (M0 conditions). Inhibition of PPAR-γ resulted in a dose-dependent upregulation of MerTK along with a strongly significant induction of the M2c-associated receptors CD163 and CD16 ( Figure 3A-C). Addition of rosiglitazone (1 μM) exerted suppressive effects which were inversely proportional to the strength of GW9662 induction: in fact, rosiglitazone was able to neutralize the effects of GW9662 on MerTK and, partially, on CD16 expression, but failed to reverse the more robust upregulation of CD163 ( Figure 3A-C).
Furthermore, we looked by ELISA at the effects of GW9662 on macrophage production of the MerTK ligand Gas6. Gas6 was previously shown to be released by both M2c and M2a macrophages [4]. In line with its role favoring M2c polarization, GW9662 strongly increased Gas6 protein levels in supernatants of otherwise untreated cells ( Figure 3D). Of note, GW9662 induction of Gas6 was neutralized by LPS ( Figure 3D), suggesting an inhibitory role of TLR signaling on Gas6 production. Remarkably, opposite effects were observed for GW9662 on Gas6 production in M2a conditions. In line with its inhibitory role on M2a polarization, in fact, GW9662 suppressed Gas6 production by IL-4 stimulated cells ( Figure 3E).
The PPAR-γ agonist rosiglitazone attenuates M2c polarization induced by dexamethasone We cultured monocytes/macrophages in the presence of dexamethasone (M2c conditions), with or without the PPAR-γ agonist rosiglitazone. Consistent with the induction of M2c polarization by its antagonist GW9662, rosiglitazone (50-100 μM) was found to impede the upregulatory effects of dexamethasone (1 nM) on MerTK and CD163 expression ( Figure 4A-C). Inhibitory effects were not due to vehicle toxicity (i.e., DMSO 0.18% for rosiglitazone 50 μM and DMSO 0.36% for rosiglitazone 100 μM) ( Figure 4A), nor to significant effects of rosiglitazone on cell viability and morphology, as assessed by forward and side scatter by flow cytometry ( Figure 4D). However, no significant inhibitory effects of rosiglitazone were seen in the presence of higher concentrations of dexamethasone (10 nM) (not shown) and/or lower doses of rosiglitazone (1-10 μM).
In otherwise untreated cells (M0 conditions), addition of rosiglitazone alone did not significantly change the expression of MerTK and CD163, although it modestly increased CD16 expression ( Figure 5A-B).

The LXR agonist T0901317 upregulates MerTK independently of M2c phenotype acquisition
To investigate the potential role of the nuclear receptors LXRs in M2c differentiation, we cultured monocytes in the presence or absence of the LXR agonist T0901317. In accord with results previously obtained in mice [20], T0901317 was found to upregulate MerTK expression, with significant effects already occurring at low doses (0.01 μM) ( Figure 5C-D). Surprisingly, MerTK upregulation by T0901317 was not associated with the acquisition of the M2c phenotype. Indeed, the M2c surface markers CD163 and CD16 ( Figure 5C-D), as well as the M2 receptor CD206 (not shown), were downregulated in a dose-dependent manner. Therefore, in contrast to what is observed for dexamethasone, M-CSF plus IL-10 [4] or GW9662, the MerTK expression pattern regulated by LXRs is uncoupled to M2c differentiation.
Similarly to dexamethasone, GW9662 inhibits in vitro macrophage production of TNF-α and IL-10 Since GW9662 appears to stimulate the expansion of regulatory M2c cells, we sought to examine its effects on macrophage production of proinflammatory and antiinflammatory cytokines. For this purpose, we measured by ELISA TNF-α and IL-10 levels released in supernatants of cells cultured with GW9662, without or after stimulation with low doses of LPS, and compared the effects with those obtained from dexamethasone cultures. In our conditions, LPS was able to significantly augment cell release of IL-10, but not TNF-α ( Figure 6). GW9662 significantly reduced spontaneous as well as LPS-triggered release of both TNF-α ( Figure 6A) and IL-10 ( Figure 6B). Dexamethasone treatment yielded similar results ( Figure 6C-D), in agreement with previous in vitro data [34,35].
Altogether, GW9662 and dexamethasone exert analogous effects: both induce differentiation toward the M2c phenotype, upregulate the MerTK/Gas6 pathway, and inhibit release of both TNF-α and IL-10 in vitro. With regard to cytokine production, GW9662 and dexamethasone driven cell populations differ from the M2c subset differentiated in the presence of M-CSF, which was instead previously shown to produce low levels of TNF-α but high levels of IL-10 in vitro [4].

GW9662 does not enhance macrophage phagocytosis of apoptotic cells
Since conventional M2c macrophages, induced by dexamethasone or M-CSF and IL-10, are characterized by augmented capability to phagocytose ACs (efferocytosis) [4,33], we looked at the potential effects of GW9662 on macrophage phagocytosis of apoptotic neutrophils. For this purpose, CD14-labeled macrophages, differentiated in the presence or absence of IL-4 (20 ng/ml), with or without GW9662 (10 μM), were coincubated with CFSElabeled apoptotic neutrophils at a 1:5 ratio for 30 minutes, and analyzed by flow cytometry. Consistent with previous studies supporting a central role for PPAR-γ in efferocytosis [26][27][28][29], we did not observe increased amounts of total (CFSE+) or highly (CFSE bright ) efferocytic macrophages among GW9662-treated cells. Indeed, in otherwise untreated cells (M0 conditions), GW9662 significantly decreased efferocytosis, while in the presence of IL-4 (M2a conditions), no significant change was observed ( Figure 7A-B). Therefore, GW9662-driven M2c-like cells differ from conventional M2c macrophages [4,33] because they do not show enhanced efferocytic properties.
(See figure on previous page.) Figure 3 GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.
LXRs are known to mediate MerTK expression in murine macrophages exposed to ACs. Cholesterol and oxysterol species contained in ingested ACs drive LXR induction of MerTK, resulting in enhancement of AC clearance and transrepression of macrophage inflammation in response to phagocytosis [20]. In human macrophages, we confirm that the LXR agonist T0901317 upregulates MerTK. However, LXR induction of MerTK is unexpectedly dissociated from acquisition of the M2c phenotype; indeed, CD206, CD163 and CD16 are downregulated. In light of these data, we hypothesize that MerTK regulation follows at least two expression patterns: one linked to the M2c phenotype, driven by M-CSF and IL-10, glucocorticoids or PPAR-γ antagonists; and another one independent from M2c polarization, driven by LXRs through AC-derived oxysterols and nonsteroidal LXR agonists.
The present study adds to our previous research on characterization of anti-inflammatory M2c macrophages in humans [4,33]. We recently described the M2c subset as CD206 + CD163 + CD16 + MerTK + M2 macrophages, well distinguished from IL-4-induced CD206 + CD209 + M2a cells, able to release high levels of the MerTK ligand Gas6 and specialized in phagocytosis of early apoptotic cells via MerTK [4]. Differentiation of monocytederived macrophages in the presence of M-CSF plus serum, M-CSF plus IL-10, or glucocorticoids gives rise to the M2c phenotype [4]. However, some differences exist among different stimulations. For instance, M2c cells induced by M-CSF and IL-10 highly express CD14, while dexamethasone-driven M2c cells express CD14 levels comparable to baseline. Moreover, M2c cells obtained in the presence of M-CSF produce high levels of IL-10, which are further amplified by Gas6 via MerTK [4], whereas dexamethasone, as shown here, inhibits IL-10 production in vitro. Although classified as M2c stimuli [2], we previously observed that TGF-β gives a different phenotype, characterized by CD206 and CD16 induction, but inhibition of CD163, MerTK and Gas6, whereas IL-10 without M-CSF gives only a partial phenotype, characterized by upregulation of CD163 and Gas6, but not CD206, Figure 6 GW9662, like dexamethasone, inhibits in vitro production of TNF-α and IL-10. TNF-α (A and C) and IL-10 (B and D) production levels were quantified by ELISA in culture medium of cells incubated for 3 days in the presence or absence of GW9662 (2.5-10 μM) (n = 3) (A and B) or dexamethasone (1-100 nM) (n = 4) (C and D). When specified, LPS (50 ng/ml) was added in the last 48 hours. Data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Comparison between spontaneous and LPS-triggered cytokine release was performed using Student's paired t-test. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons respect to the relative GW9662 or dexamethasone untreated control. Each set of data is representative of three to four independent experiments. CD16 and MerTK [4]. Herein, we further describe a new variant of M2c-like cells induced by GW9662, reporting similarities and differences with conventional M2c subsets. Such heterogeneity among macrophage populations recently spurred the proposal to adopt in the near future revised macrophage nomenclature more strictly linked to the activation standards [37].
Other authors previously investigated GW9662 and rosiglitazone effects on human macrophage phagocytosis of ACs and phenotype. In agreement with our findings, Bouhlel and colleagues [19] observed that rosiglitazone alone did not affect macrophage differentiation in basal conditions, yet it amplified M2a polarization induced by IL-4, including CD163 downregulation. In accord with our results, Majai and colleagues [28] found that GW9662 decreased AC phagocytosis, owing to downregulation of efferocytic molecules such as CD36, transglutaminase-2 and AXL, and inhibited LPS-induced IL-10 production; also, dexamethasone was shown to share with GW9662 down-regulation of CD36, transglutaminase-2 and IL-10. In contrast with our observations, however, these authors failed to find significant changes in MerTK, Gas6, CD206 or CD16 expression upon GW9662 treatment, nor did they observe GW9662 inhibition of TNF-α production. It is important to note that Majai et al. [28] cultured monocytes in medium containing M-CSF and human serum, a combination that we demonstrated already promotes M2c polarization in vitro [4]; thus, their experimental conditions may have masked the M2c polarizing effects of GW9662 that we observed. Our finding that PPAR-γ inhibition leads to differentiation of M2c-like monocytes/ macrophages is then novel, and may have significant implications in several fields focusing on macrophage biology, including studies on adipose tissue-associated macrophages, macrophages of atherosclerotic plaques, macrophage activation in chronic inflammatory diseases like systemic lupus erythematosus (SLE), and tumorassociated macrophages.

PPAR-γ and M2 polarization in macrophages of atherosclerotic plaques
The role of PPAR-γ in macrophages from atherosclerotic lesions is controversial. TZDs were shown to inhibit atherogenesis in LDL-R KO and ApoE KO mouse models [44,45] and to reduce carotid artery wall thickness in diabetic patients [46]. However, in vivo impact of TZDs in atherosclerosis depends on mechanisms involving multiple cell targets apart from macrophages, such as inhibition of endothelial activation [45], inhibition of vascular smooth muscle cell proliferation [47], reduction of vascular resistance and blood pressure [48], increased insulin sensitivity and adiponectin production [8], and anti-oxidant properties [49]. Moreover, although PPAR-γ may limit atherogenesis at initial phases, serious doubts arise about its role in plaque instability. In fact, rosiglitazone was ultimately reported to increase the risk of myocardial infarction in diabetic patients [50,51], leading to its withdrawal from the market in several countries. Plaque instability is favored by enlargement of necrotic core in atherosclerotic lesions. Cholesterol-laden macrophages undergo apoptosis, and apoptotic macrophages turn into secondary necrotic cells if not promptly cleared [52,53]. Macrophagespecific PPAR-γ might have atherogenic potential by driving phagocytosis of oxLDLs via CD36. PPAR-γ is in fact inducible by oxLXLs themselves, and is expressed in M1-like macrophage foam cells of human atherosclerotic lesions [23]. On the other side, LXRs, MerTK and CD163 seem to prevent plaque instability. LXRs protect against foam cell formation, by inducing ABC transportermediated cholesterol efflux [9] and by upregulating MerTK in mice [20] and in humans (as shown in this paper). MerTK, in turn, inhibits uptake of lipoproteins [54] shifting phagocytosis activity toward efficient and non-inflammatory clearance of cholesterol-laden apoptotic macrophages [52]. Additionally, both LXRs and MerTK exert anti-apoptotic effects on macrophages [31,33,55]. CD163 exerts beneficial effects owing to upregulation of heme oxygenase-1 in response to hemoglobin-haptoglobin complexes, which ultimately results in iron clearance and prevention of oxidative reactions, along with release of IL-10 and anti-inflammatory heme metabolites [56]. LXR-α and MerTK were demonstrated to be atheroprotective in LDL-R KO and ApoE KO mouse models [52,53,57]. In humans, non-foamy protective M2-like CD206 + macrophages expressing high levels of MerTK [26,58] and CD163 [59,60] have been described in areas of plaques far from the necrotic core and close to microvessels or microhemorrhages, respectively. We hypothesize that the potential atherogenic role of PPAR-γ may become explicit in the presence of IL-4 or other PPAR-γ agonists like TZDs. IL-4 can in fact amplify PPAR-γ expression induced by oxLDLs [13], and at the same time downregulate LXR expression, so that PPAR-γ activation of LXRs is impaired [26]. In this setting, macrophage uptake of lipoproteins is not followed by cholesterol efflux, thereby facilitating foam cell formation. Moreover, chronic stimulation with IL-4 and PPAR-γ activation induce apoptosis in macrophages [33,61], while IL-4 down-regulation of MerTK and CD163 [4] may interfere with the clearance of apoptotic macrophages and iron, respectively. In fact, in both LDL-R KO and ApoE KO mice, IL-4 proved to extend the size of atherosclerotic lesions [62,63]. Taking together our present findings and previously reported data, we suggest that new PPAR-γ agonists not affecting macrophage-specific PPAR-γ might overcome controversial effects and cardiovascular safety concerns of TZDs. On the other hand, treatments apt to elicit the expansion of MerTK + and CD163 + cells (e.g., M2c polarizing agents and IL-4/STAT-6 inhibitors) may help against atherosclerosis progression.

PPAR-γ and M2 polarization in systemic lupus erythematosus (SLE)
Pathogenic macrophage populations in SLE are traditionally considered to be M1 oriented [64], in accord with increased production of IFN-γ during disease exacerbations [65]. More recently, SLE macrophages were classified as M2b cells [66]. In addition to enhanced expression of iNOS and proinflammatory cytokines, in fact, they also express high levels of IL-10 and relatively low levels of IL-12 [66]. Excessive immune complexes and TLR signaling occurring in SLE may in fact stimulate M2b polarization [2], which may partially account for increased IL-10 levels in SLE serum [65]. By contrast, serum levels of IL-4 are not significantly changed in SLE, and M2a macrophages do not seem to be expanded [64,65]. We and others recently reported higher circulating levels of MerTK and CD163 cleavage products in active SLE patients, thereby suggesting increased turnover of M2c cells and relative insufficiency of M2c and MerTK activity [32,67,68]. In fact, lupus-like syndromes arise because of defective clearance of ACs owing to impaired expression or function of efferocytic molecules, as occurs in mice lacking functional MerTK [69]. The therapeutic utility of glucocorticoids in SLE may be at least in part attributable to M2c induction, MerTK upregulation and enhanced macrophage phagocytosis of ACs [4,5,67]. Previous studies support a protective role for both LXRs and PPAR-γ in SLE: LXR-α/β and PPAR-γ KO mice accumulate ACs in spleens, produce anti-nuclear autoantibodies and develop lupus-like syndrome with glomerulonephritis [20,29]. Also, TZDs were reported to ameliorate murine lupus [48,70,71]. However, beneficial effects of TZDs were only observed at early stages and were mostly associated with vascular and insulin-sensitizing effects (i.e., inhibition of endothelin production, vasodilation, reduced blood pressure, improvement of lipid metabolism) rather than to direct anti-inflammatory effects on macrophages [48,70,71]. Likewise, reduced renal inflammation and reduced macrophage activation appeared to be an indirect effect secondary to adiponectin induction [71]. Although a rationale for using TZDs in SLE autoimmunity would be conversion of M2b macrophages into M2a cells [38] and promotion of PPAR-γ-dependent efferocytosis [26][27][28][29], the inhibitory role herein shown on M2c differentiation and on MerTK expression should be taken into account.

PPAR-γ and M2 polarization in tumor-associated macrophages (TAMs)
Tumor-associated macrophages (TAMs) are generally considered M2c-like macrophages, expressing CD206 and CD163 [72]. As M2c cells, TAMs also release Gas6, which facilitates tumor cell proliferation and probably immune tolerance to cancer [4,73]. Besides direct antiproliferative effects on certain tumor cells [23], PPAR-γ may exert anti-tumoral effects by acting on macrophages through a dual mechanism. On the one hand, PPAR-γ inhibits inflammation-driven carcinogenesis by suppressing NF-κB and proinflammatory genes in M1-like cells. On the other hand, PPAR-γ restores anti-tumor cytotoxic T lymphocyte activity by inhibiting tolerogenic M2c-like TAMs [74]. Our finding that GW9662 inhibition of PPAR-γ generates M2c-like macrophages and Gas6 release is in fact consistent with previously reported GW9662 suppression of anti-tumor immune responses [74].

Conclusions
This study extends our knowledge of the role of PPAR-γ and LXRs receptors in human macrophage activation. We show that blocking PPAR-γ during monocyte-tomacrophage maturation elicits differentiation of M2c-like CD206 + CD163 + cells and upregulation of the MerTK/ Gas6 axis. Although PPAR-γ signaling may reduce M1 (and M2b) inflammatory cytokine production and potentiate M2a alternative activation, results suggest that it also impedes M2c polarization and restoration of fully antiinflammatory conditions in chronic settings like metabolic syndrome and autoimmune diseases. Therapeutic advantages might derive from the use of more selective PPAR-γ agonists, targeting adipose tissue PPAR-γ2 isoform rather than ubiquitously expressed PPAR-γ1 isoform [75], with the aim of exploiting beneficial effects of PPAR-γ on insulin-sensitivity and adiponectin secretion while minimizing controversial effects on macrophages. Finally, we point out that LXR stimulation upregulates MerTK independently of M2c polarization, thus revealing the existence of different regulation patterns for MerTK expression.