Gr-1⁺ CD11b⁺ Myeloid-derived Suppressor Cells Suppress Inflammation and Promote Insulin Sensitivity in Obesity^*

Mice and Tissues

Wild-type and ob/+ mice on the C57BL/6 background were purchased from The Jackson Laboratory, fed with regular LFD (Harlan) with 11% energy derived from fat, and bred in our facility. To induce obesity, 6-week-old wild-type mice were fed with 60% HFD (Research Diets, Inc., D12492). Mice were sacrificed by cervical dislocation. Tissues such as epididymal WAT, liver, spleen, bone marrow, and blood were either immediately processed for single cell suspension or snap-frozen in liquid nitrogen (16). Frozen tissues were stored at −80 °C. All animal procedures were approved by the Cornell Institutional Animal Care and Use Committee.

Preparation of Single-cell Suspension from Tissues

1) SVF of WAT: Cells were prepared as we described previously (16). Briefly, epididymal WAT was collected and minced with razor blades. After two washes with KRBH buffer (10 mm HEPES (pH7.4), 15 mm NaHCO₃, 120 mm NaCl, 4 mm KH₂PO₄, 1 mm MgSO₄, 1 mm CaCl₂, and 2 mm sodium pyruvate), fat pads were digested with 1.5 mg/ml of type II collagenase (Sigma) in KRBH buffer for 45 min with gentle shaking at 37 °C. The cell suspension was filtered through a 250-μm nylon mesh (Small Parts, Inc.), spun at 300 × g for 10 min to separate floating adipocytes from the SVF pellet, and then isolated SVF were resuspended in PBS. 2) Bone marrow: Cells were collected by flushing cells from the marrow cavity of the femur and tibia with PBS. 3) Spleen: Isolated spleens were placed in sterile cold PBS and disrupted by gentle crushing with the end of a syringe plunger through a strainer with 70-μm pores (BD Biosciences). 4) Blood: Whole blood was collected from the tail or orbital sinus and transferred into heparin tubes. 5) Non-parenchymal hepatic cells were purified as described (17–19). Briefly, minced liver pieces were digested with 1 mg/ml of type IV collagenase (Sigma) in RPMI1640 medium. Non-parenchymal cells were enriched by density centrifugation using 30 and 70% Percoll at 1200 × g for 20 min at 4 °C. The buffy layer over the 70% Percoll was harvested, washed, and resuspended in PBS. This method showed a similar extent of increase of Gr-1⁺ CD11b⁺ cells in obesity as that using the perfusion method, where mice were perfused via the hepatic vein with 30 ml of PBS prior to collagenase digestion. In all cases, erythrocytes were lysed using cell lysis buffer (BD Biosciences) prior to staining or purification.

Flow Cytometry

Following incubation with anti-CD16/CD32 antibody to block Fc receptors, 2 × 10⁵ single cell suspensions of various tissues were incubated with 20 μl of antibodies diluted at optimal concentrations (supplemental table S1) in a 96-well plate for 20 min at 4 °C. Cells were washed three times with PBS and then resuspended in 400 μl of PBS for analysis using the FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using the CellQuest software (BD Biosciences). Isotype controls were included in all experiments to ensure proper gating of the population for the liver and adipose SVF (supplemental Fig. S1). The methods to analyze tissue samples by flow cytometry, either as percent of live cells or percent of total CD45⁺ cells, are illustrated in supplemental Fig. S1A. Detailed information for antibodies used in flow cytometry, magnetic purification, cell depletion, and Western blot analysis are provided in supplemental table S1.

Magnetic Bead Purification

A single cell suspension from either the SVF of adipose tissue or spleen was prepared as described above. After two washes with 10 ml PBS containing 1 mm EDTA and 2% BSA, 10⁸ cells were incubated with 400 μl of biotin-conjugated antibody specific for Gr-1 and CD8 at 4 °C for 20 min followed by incubation with 100 μl of streptavidin particles at 4 °C for 20 min. Following two washes with PBS, cells were passed through a 30-μm nylon mesh (Small Parts, Inc.) and then transferred to a 12 × 75 mm tube that was inserted into a cell separation magnet apparatus (BD Biosciences) for 5 min. Cells were washed three times with PBS and then resuspended in 1 ml DMEM containing 10% FBS. Purity was normally over 90% (supplemental Fig. S4A).

T Cell Suppression Assays

In a U-bottom 96-well cell culture plate coated with 5 μg/ml anti-CD3 antibody, 2 × 10⁵ purified CD8⁺ T cells were cultured in DMEM supplemented with 10% FBS, 5 μg/ml anti-CD28 antibody, and/or 10 units/ml IL-2 (BD Biosciences) for 96 h. In some wells, CD8⁺ T cells were cocultured with 2–4 × 10⁵ purified Gr-1⁺ MDSCs from obese or lean animals. In some experiments, the inducible nitric oxide synthase inhibitor l-N-monomethyl arginine (0.5 mm, Calbiochem), the arginase-specific inhibitor N-hydroxy-nor-arginine (0.5 mm, Calbiochem), or an IFN-γ blocking antibody was added to cultures (19). In some experiments, a transwell system was used, where Gr-1⁺ cells were plated in the upper chamber.

CFSE labeling

CD8⁺ T cells were purified from spleens using the CD8 magnetic beads. Following two washes with PBS, purified CD8⁺ T cells were adjusted to 2 × 10⁶ cells/ml and mixed with an equal volume of 10 μm carboxyfluorescein succinimidyl ester solution (VWR International) for 10 min. The reaction was terminated by the addition of 10 volumes of cold RPMI 1640/10% FBS. Labeled cells were washed twice with PBS/2% FBS and adjusted to 1 × 10⁶ cells/ml.

Intracellular Cytokine Staining

After purification, cells were adjusted to 1 × 10⁶ cells/ml and cultured for 6 h in 2.0 μm monensin (GolgiStop, BD Biosciences). Cells were then stained for cell surface markers followed by immunostaining using anti-IFN-γ-FITC or anti-IL-10-FITC according to the supplier's protocol (GolgiStop, BD Biosciences).

Wright's Staining

FACS-sorted cells were adjusted to 2 × 10⁵ cells/ml, and 200 μl of cell suspension was applied to a clean slide (VWR International). After air-drying at room temperature, 1 ml of the Wright's stain (Sigma) was applied to the slide for 2 min followed by the addition of 2 ml of distilled water for 2 more min. Slides were rinsed twice with water and photographed with an inverted Nikon microscope.

Adipose Tissue Explants and Conditioned Medium

Conditioned medium was obtained as described (20) with the following modifications: Epididymal fat pads were harvested from lean and obese animals, weighed, washed twice in 200 ml of PBS, and sliced into 1-mm-size pieces. Tissue chunks were cultured in 3 ml of DMEM with 10% FBS in 6-cm dishes. Medium was collected the next day, spun to eliminate contaminating cells, and used as conditioned medium.

In Vitro Differentiation of Gr-1⁺CD11b⁺ Myeloid Cells

2 × 10⁵ Gr-1⁺ CD11b⁺ myeloid cells purified from spleens of obese mice were cultured in DMEM-conditioned medium from obese or lean adipose tissue explants. Five days later, cells were collected, and cell surface expression levels of I-A^b, F4/80, and CD11b were analyzed by flow cytometry. Markers of M1 and M2 macrophages were measured by quantitative-PCR.

Q-PCR and Western Blot Analysis

These experiments were carried out as described (21). Q-PCR data were normalized to the ribosomal l32 gene in the corresponding sample. Antibodies and primer sequences are listed in supplemental tables 1 and 2, respectively.

In Vivo Studies

Mice fed with HFD for 12–20 weeks were used in the following in vivo studies. 1) Loss-of-function antibody-mediated ablation of Gr-1⁺ cells: HFD mice were injected intraperitoneally with Gr-1-specific or isotype IgG2b antibody at 6 μg/g body weight every 3–4 days. 2) Gain-of-function adoptive transfer study: Gr-1⁺ cells were purified from the spleen and bone marrow of donor HFD mice using magnetic beads and adjusted to 2 × 10⁷/ml. Recipient HFD mice were injected i.v. with 1 × 10⁷ cells in 500 μl of saline three times. In some experiments, Gr-1⁺ cells were labeled with CFSE prior to the transfer. Detailed information for antibodies was provided in supplemental table S1.

Metabolic Phenotyping

Mice were phenotyped 1 day after the last injection as we described previously (16). Fasting glucose was measured after a 16-h fast. For the glucose tolerance test, mice were fasted for 16–20 h followed by injection of glucose (Sigma) at 1 g/kg body weight. For the insulin tolerance test, mice were fasted for 4–6 h followed by injection of insulin (Sigma) at 1 unit/kg body weight. Blood glucose was monitored using a One-Touch Ultra glucometer (LifeScan, Inc.). Circulating insulin, IL-6, and TNF-α were measured using ELISA kits from Millipore and eBiosciences according to the suppliers' protocols.

Statistical Analysis

Results are expressed as mean ± S.E. Comparisons between groups were made by unpaired Student's t test or two-way ANOVA. p < 0.05 was considered as statistically significant.

Gr-1⁺CD11b⁺ MDSCs Accumulate in Peripheral Tissues of Obese Mice

In the genetic obese mouse model deficient for the leptin gene (ob/ob), their body weight and fasting glucose were increased with age (Fig. 1A), as was the percent of circulating Gr-1⁺ CD11b⁺ cells (B). In adult ob/ob mice, the percent of MDSCs was significantly elevated (2∼3-fold) in the spleen, liver, and SVF of epididymal WAT compared with age-matched controls (ob/+) (Fig. 1, C and D). Indeed, the total number of Gr-1⁺ cells in SVF of one epididymal fat pad increased over 30-fold (mean ± S.E. of ob/+ versus ob/ob: 0.11 ± 0.01 × 10⁵ to 3.65 ± 0.10 × 10⁵) (Fig. 1E). In contrast, the percentage of these cells in the bone marrow was comparable between lean and obese animals (Fig. 1, C and D), suggesting that the biogenesis of Gr-1⁺ CD11b⁺ cells may not be altered during obesity.

To rule out the contribution of leptin and further determine the temporal accumulation of these Gr-1⁺ CD11b⁺ cells in metabolic tissues, we placed 6-week-old B6 mice on LFD or HFD for a total of 21 weeks, in which 11 or 60% of calories were derived from fat. Expectedly, mice maintained on HFD gained more weight than mice on LFD and exhibited fasting hyperglycemia upon 8 weeks of feeding (Fig. 1F). Indeed, the levels of Gr-1⁺ CD11b⁺ MDSCs increased with time in several tissues, including the liver, WAT, and spleen upon HFD feeding (Fig. 1G). The difference in the percent of these cells between HFD and LFD cohorts became apparent upon 8 weeks of feeding. By week 21, the percent of these cells increased by 4-fold in the liver and over 2-fold in the WAT of HFD mice (Fig. 1G). It is interesting to note that the timing of MDSC accumulation at 8-week HFD in both the liver and WAT is similar to that of infiltration by macrophages and CD8⁺ T cells (6). Taken together, our data show that MDSCs are enriched in the liver and WAT at an early stage of obesity.

Gr-1⁺CD11b⁺ MDSCs from Obese Mice Consist of Two Major Subsets

To gain further insight into the maturation stage and lineage commitment, we characterized the morphology and lineage markers of these cells from the liver (Fig. 2) and spleen (supplemental Fig. S2) using FACS and three-color flow cytometry, respectively. Similar to what had been reported in tumor-induced MDSCs (22, 23), the Gr-1⁺ CD11b⁺ cells from the livers of obese animals were a heterogeneous population consisting of two major subsets with different Gr-1 expression levels, termed Gr-1^hi and Gr-1^med, with distinct side-scatter characteristics, a measure of cell granularity (Fig. 2A). Morphologically, sorted Gr-1^med CD11b⁺ cells from the livers of obese mice had large smooth bean- or kidney-shaped nuclei typical of monocyte/macrophage lineage, whereas Gr-1^hi CD11b⁺ cells had segmented nuclei, characteristic of granulocyte/neutrophil lineage (Fig. 2B).

In line with their morphology, Gr-1^hi CD11b⁺ cells expressed the common neutrophil marker Ly6G, whereas Gr-1^med CD11b⁺ cells expressed the typical monocyte/macrophage marker F4/80 (Fig. 2C) (13). Importantly, confirming their status as immature myeloid cells and distinguishing them from mature myeloid cells such as dendritic cells and macrophages, the Gr-1^med CD11b⁺ subset expressed low levels of both MHC class II (I-A^b) and the dendritic cell marker CD11c (Fig. 2C). Thus, although a subset of Gr-1⁺ CD11b⁺ cells express the typical macrophage marker F4/80, they are distinct from mature macrophages.

Interestingly, obesity had no effect on the expression of any of the markers tested here, including the two activation markers CD115 and CD62L in the liver (Fig. 2C). This is reminiscent of what has been reported in cancer, where tumor-induced MDSCs have similar levels of cell surface activation markers as Gr-1⁺ CD11b⁺ cells from naïve mice, despite functional differences (24). Taken together, our data demonstrate that two major subsets of Gr-1⁺ CD11b⁺ MDSCs with granulocytic and monocytic characteristics are highly enriched in peripheral tissues in obesity and are immature in nature.

Ablation of Gr-1⁺ Cells Promotes Glucose Intolerance and Insulin Resistance in Obese Mice

To determine the metabolic function of these cells in vivo, we carried out loss and gain of function analyses using antibody-mediated depletion and adoptive transfer, respectively. First we depleted mice for Gr-1⁺ cells using Gr-1-specific antibody (supplemental Fig. S3A), widely used to target Gr-1⁺ cells in cancer models (25–31). Circulating Gr-1⁺ cells were significantly reduced 24 h following the injection of the Gr-1-specific antibody, remained low for 3 days, and started to recover on days 3–4 (Fig. 3A). Gr-1⁺ cells were reduced significantly in the liver and WAT following antibody injection (Fig. 3B). Demonstrating the specificity of antibody depletion, levels of Gr-1⁻ CD11b⁺ cells as well as B220⁺ B cells in both the liver and WAT were not affected (Fig. 3B and supplemental Fig. S3B). Hence, we developed a regimen where HFD mice were injected with either Gr-1 or isotype control IgG2b antibody every 3–4 days for 2 weeks (Fig. 3C).

Although antibody injection had no effect on body nor fat weight (Fig. 3D and not shown), obese mice injected with Gr-1 antibody exhibited elevated fasting glucose (E) and insulin levels (F). Furthermore, glucose tolerance and insulin sensitivity significantly deteriorated upon Gr-1 depletion as measured by glucose and insulin tolerance tests (Fig. 3, G and H). As Gr-1⁺ CD11b⁺ cells represent over 90% of total Gr-1⁺ cells (the rest are Gr-1⁺ CD11b⁻ granulocytes) in HFD mice (Fig. 3B and supplemental Fig. S3A), we reasoned that alterations in metabolic phenotypes upon Gr-1 depletion are likely attributed to Gr-1⁺ CD11b⁺ MDSCs. Thus, these data suggest that depletion of Gr-1⁺ cells in obese animals increases insulin resistance and glucose intolerance.

Elevation of Gr-1⁺ CD11b⁺ MDSCs Improves Glucose Tolerance in Obese Animals

Next, we adoptively transferred purified Gr-1⁺ CD11b⁺ cells with over 90–95% purity into HFD mice (supplemental Fig. S4A). Twenty-four hours following cell transfer of CFSE-labeled Gr-1⁺ CD11b⁺ cells, CFSE⁺ cells predominantly homed to the liver of recipients, as shown previously in naïve and tumor-bearing animals (17) (supplemental Fig. S4, B and C). Upon three cell transfers (Fig. 4A), the levels of Gr-1⁺ CD11b⁺ MDSCs in the HFD + Gr-1 cohort increased about 50% in the liver (HFD + Gr-1 versus HFD: 9.2 ± 0.3% versus 6.1 ± 0.2%, p < 0.05) (Fig. 4B).

In line with the results from the loss of function study, the HFD + Gr-1 recipients exhibited reduced fasting glucose and insulin levels when compared with the control HFD cohort (Fig. 4, E and F), whereas their body and fat pad weights were not affected (C and D). Furthermore, glucose tolerance was significantly improved following three Gr-1 cell transfers (Fig. 4G). In line with improved glucose homeostasis, expression of a key gluconeogenic enzyme G6pase (encoding glucose-6-phosphatase) was significantly reduced in the liver of the HFD + Gr-1 cohort (Fig. 4H). Taken together, we conclude that elevation of Gr-1⁺ CD11b⁺ MDSCs attenuates glucose intolerance in obese mice.

Gr-1⁺ CD11b⁺ MDSCs Suppresses Inflammatory Response in Obese Animals

As inflammation contributes significantly to the pathogenesis of insulin resistance during obesity (1, 2), we next examined the status of inflammation in both in vivo studies. Pointing to a systemic effect of MDSCs on inflammation, circulating levels of the proinflammatory cytokines IL-6 and TNF-α were significantly decreased in the HFD + Gr-1 cohort (Fig. 4I). Conversely, Gr-1-depletion significantly elevated circulating levels of IL-6 but not TNF-α (Fig. 3I). Further supporting these observations, phosphorylation of JNK, a key player linking inflammation and insulin resistance (32), was increased in the liver upon Gr-1 depletion (Fig. 3J) and decreased upon Gr-1⁺ transfers (Fig. 4J). Thus, these in vivo data collectively suggest that Gr-1⁺ CD11b⁺ cells down-regulate systemic inflammation during obesity.

MDSCs Suppress CD8⁺ T Cell Activation in Obese Mice

Mature CD8⁺ T cells are known to stimulate inflammation and promote insulin resistance (6). Strikingly, Gr-1 depletion doubled the proportion of CD8⁺ T cells in WAT (Fig. 5A), whereas adoptive transfer of Gr-1⁺ CD11b⁺ cells had the opposite effect (B), suggesting that MDSCs are suppressors of CD8⁺ T cell activation in obesity. We next carried out coculture experiments using purified CD8⁺ T cells and MDSCs purified from lean and obese mice to delineate the effect of MDSCs on CD8⁺ T cells. Indeed, activation/proliferation of CD8⁺ T cells was greatly diminished when cocultured with Gr-1⁺ cells from obese animals relative to that with Gr-1⁺ cells from lean animals (Fig. 5C). The repressive effect of obese Gr-1⁺ CD11b⁺ cells on CD8⁺ T cells was contact-dependent (Fig. 5E) and required IFN-γ and iNOS, as addition of either an IFN-γ neutralizing antibody or the iNOS inhibitor L-NMMA to the coculture reversed the inhibitory effect, whereas addition of the arginase inhibitor nor-NOHA had no major effect (D). Further analysis revealed that IFN-γ is secreted by CD8⁺ T cells rather than MDSCs (supplemental Fig. S5A). Providing further support to the notion that Gr-1⁺ CD11b⁺ MDSCs suppress CD8⁺ T cell proliferation and activation in vivo, adoptive transfer of Gr-1⁺ CD11b⁺ cells increased apoptosis of CD8⁺ T cells (Fig. 5F and supplemental Fig. S5B). Consistently, in vivo MDSCs induced CD8⁺ T cell apoptosis largely via IFN-γ (Fig. 5G). Thus, our data show that MDSCs directly inhibit activation and induce apoptosis of CD8⁺ T cells in both the liver and WAT of obese mice, in part via the activities of IFN-γ and iNOS.

MDSCs Skew M1-M2 Macrophage Polarization to M2 in Obese Mice

Macrophages and their M1-M2 polarization in the liver and WAT have been implicated as a key effector mechanism for obesity-induced insulin resistance (7, 33–36). We next examined the relationship between MDSCs and macrophages. Upon a 5-day culture with macrophage colony-stimulating factor, Gr-1⁺ CD11b⁺ MDSCs from obese animals acquired I-A^b expression with elevated F4/80 levels to become F4/80^hi I-A^b+ macrophages (Fig. 5H), suggesting that MDSCs can differentiate into mature macrophages in vitro. Upon culturing MDSCs in conditioned medium from fat tissue slices or explants for 5 days (20, 37), we found that the expression levels of two M2 markers, Arg1 (encoding Arginase 1) and Chi3l3 (encoding chitinase 3-like 3) (13, 38), were significantly increased by 6∼7-fold in conditioned medium from obese adipose explants relative to those differentiated in conditioned medium from lean adipose explants (Fig. 5I). In contrast, levels of M1 markers such as Tnfa, Cxcl10 (encoding chemokine C-X-C motif ligand 10), and Nos2 (encoding iNOS) (13, 38) were either unaltered or significantly attenuated following the differentiation (Fig. 5I). Accordingly, depletion of Gr-1⁺ cells in vivo significantly increased the M1 polarization profile (e.g. Tnfa, Cxcl10, and Ccl5, encoding CCL5) (Fig. 5J). The pro-M2 effect of MDSCs in obesity may be due to their secretion of T_H2 cytokine IL-10 in obesity (Fig. 5K). Thus, these data suggest that MDSCs may promote the basis of M1-M2 toward M2 during obesity.