Advanced Glycation End Products Enhance Macrophages Polarization into M1 Phenotype through Activating RAGE/NF-κB Pathway

Atherosclerotic lesions are accelerated in patients with diabetes. M1 (classically activated in contrast to M2 alternatively activated) macrophages play key roles in the progression of atherosclerosis. Since advanced glycation end products (AGEs) are major pathogenic factors and active inflammation inducers in diabetes mellitus, this study assessed the effects of AGEs on macrophage polarization. The present study showed that AGEs significantly promoted macrophages to express IL-6 and TNF-α. M1 macrophage markers such as iNOS and surface markers including CD11c and CD86 were significantly upregulated while M2 macrophage markers such as Arg1 and CD206 remained unchanged after AGEs stimulation. AGEs significantly increased RAGE expression in macrophages and activated NF-κB pathway, and the aforementioned effects were partly abolished by administration of anti-RAGE antibody or NF-κB inhibitor PDTC. In conclusion, our results suggest that AGEs enhance macrophage differentiation into proinflammatory M1 phenotype at least partly via RAGE/NF-κB pathway activation.


Introduction
Diabetes mellitus (DM) increases the risk of cardiovascular disease by 2-4-fold through accelerated atherosclerosis [1]. However, the exact mechanisms underlying accelerated atherosclerosis in DM patients are not fully understood.

BioMed Research International
Advanced glycation end products (AGEs), a heterogeneous group of molecules formed by irreversible nonenzymatic reactions between proteins and sugar residues in a hyperglycemic milieu, augment cardiovascular risk in patients with diabetes by stimulating inflammation and promoting atheroma formation [13]. In addition to macrophages, the crucial role of AGEs in the pathogenesis of atherogenesis has been established [14]; however, the interaction between these two players has not been fully explored. The present study therefore tests the hypothesis that AGEs enhance macrophage polarization into the M1 phenotype as a potential key mechanism underlying the increased inflammation and accelerated atherosclerosis observed in DM patients.

Materials and Methods
2.1. Mice. Male C57BL/6 mice (8 to 10 weeks old) were purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, China) and housed under specific pathogen-free conditions. All animal experiments were approved by the Ethics Committee of Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine.

Preparation and Culture of Bone Marrow
Derived Macrophages. The mice were anesthetized by using pentobarbital (50 mg/kg, i.p.) to minimize suffering and killed by cervical dislocation without recovery from anesthesia. Tibias and femurs then were removed under sterile conditions. The bone ends were cut and marrow was flushed using PBS supplemented with 2% Fetal Bovine Serum (FBS, Gibco, Australia, Cat. number 10099-141). A single cell suspension was obtained by filtering the cells through a 40 m strainer (BD Falcon). Then the suspension was centrifuged at 1500 rpm for 5 min and the cell pellet was resuspended in culture medium. Bone marrow cells were cultured for 7 days in Dulbecco's modified Eagle's medium (DMEM) High Glucose (HyClone, Beijing, China, Cat. number SH30022.01B) supplemented with 10% FBS, 2 mM L-glutamine (Sigma-Aldrich, Missouri, USA, Cat. number 59202C), 100 U/mL penicillin and 100 g/mL streptomycin (Beyotime, Jiangsu, China, Cat. number C0222), and 50 ng/mL M-CSF (PeProtech, New Jersey, USA, Cat. number 315-02). Cells were harvested on day 7 for further experiments. Macrophages (>95%) were identified by flow cytometry with anti-CD11b-APC and anti-F4/80-FITC (eBioscience, California, USA, Cat. numbers 17-0112 and 11-4801) staining.

Cell Viability Assay.
For cell viability detection, a CCK-8 assay was performed. Bone marrow derived macrophage (BMDM) cells (5 × 10 4 /well) were seeded in 96-well plates, cultured overnight, and then incubated with different concentrations (20, 100, 250, and 500 g/mL) of AGE-BSA (Anyan-bio Technology, Shanghai, China, Cat. number AY-4710P). BSA (500 g/mL, Sigma-Aldrich, Missouri, USA, Cat. number A1933) was used as control vehicle. Each concentration group was tested in triplicate in three replicate wells. After incubation for 24 h at 37 ∘ C with 5% CO 2 , 10 L CCK-8 solution (DOJINDO, Kumamoto, Japan, Cat. number CK-04) was added to each well and incubated for another 4 h. The optical density (OD) values were measured at 450 nm using a microplate reader (BioTek, USA). The cell viability rate of the treated cells was calculated as relative values to the control group.

Real Time PCR Analysis.
Total RNA was extracted from the cells using TRIzol reagent (Takara, Liaoning, China, Cat. number 9109) according to the manufacturer's instructions, and 1 g of total RNA was reverse transcribed to cDNA using the PrimeScript RT Master Mix kit (Takara, Liaoning, China, Cat. number RR036A) following the manufacturer's instructions. Real time PCR array analysis was performed by using the SYBR Premix Ex TaqTM kit (Takara, Liaoning, China, Cat. number RR420A) in a total volume of 20 L with 2 L of cDNA primers (0.2 mM each), 10 L of SYBR Green, and 0.4 L Rox Dye II. The standard PCR conditions consisted of 95 ∘ C for 30 sec, followed by 40 cycles of 95 ∘ C for 5 sec and 60 ∘ C for 34 sec, with a final dissociation stage, and the samples were run on an ABI 7500 detector (Applied Biosystems, California, USA). The amounts of target genes were determined and normalized to the amount of GAPDH cDNA (Sangon Biotech, Shanghai, China, Cat. number B661304). The sequences of the primers (Sangon Biotech, Shanghai, China) for the target genes are as follows:

Cytokines Detected by Enzyme-Linked Immunospecific
Assays (ELISA). After adjusting a single cell suspension of BMDM to a density of 1 × 10 6 cells/mL, 1 mL cell suspension was added into each well of 12-well plates. BMDM cells were then treated with different concentrations of AGEs (20, 100, and 500 g/mL) on the next day. The cells incubated with 500 g/mL BSA were as control groups. After another 24 h incubation, the supernatant fluid was collected and levels of TNF-, IL-6, and IL-10 were determined by commercially available ELISA kits (Neobioscience, Guangdong, China) according to the manufacturer's instructions.

Statistical Analysis.
All the results were expressed as mean ± SD. Student's -test was used for comparison between two groups. One-way analysis of variance (ANOVA) was used to assess the differences between multiple groups, and post hoc testing was completed by Fisher's least significant differences (LSD). In addition, the difference in NF-B activation triggered by AGEs with or without anti-RAGE antibody pretreatment was examined by a two-way ANOVA analysis with absence/presence of anti-RAGE antibody and time points as factors. Analysis was performed by SPSS software 19.0 (SPSS Inc., Chicago, USA) for Windows. A value of < 0.05 was considered statistically significant.

Effects of AGEs on Cell
Viability. BMDM cells were incubated with different concentrations of AGEs (20, 100, 250, and 500 g/mL) for 24 h. Cells incubated with 500 g/mL BSA were used as control groups. In CCK-8 assays, AGEs (20-500 g/mL) did not affect cell viability at 24 h ( Figure 1). Based on these results, concentrations of AGEs among the 20-500 g/mL range were chosen for subsequent experiments.

Effects of AGEs on Macrophage Production of IL-6, TNF-,
and IL-10. M1 macrophages usually predominantly produce proinflammatory cytokines such as IL-6 and TNF-, while M2 macrophages always secrete more anti-inflammatory cytokines such as IL-10. Thus mRNA levels of proinflammatory cytokines (IL-6, TNF-) and anti-inflammatory cytokine (IL-10) in macrophages were detected after incubation with different concentrations of AGEs (20, 100, and 500 g/mL) or BSA (500 g/mL) for 24 h. of AGEs induced IL-10 secretion (Figure 2(f)). These data demonstrate that AGEs predominantly induce macrophages to secrete proinflammatory cytokines.

Effects of AGEs on the Expression of iNOS and Arg1 in
Macrophages. It is widely accepted that iNOS is a marker for M1 while Arg1 is a marker for M2 macrophages [7]. To elucidate the effects of AGEs on iNOS and Arg1 expression in BMDM, mRNA levels of iNOS or Arg1 in AGEs stimulated macrophages were assessed by real time PCR. 24 hours after stimulation, mRNA level of iNOS was dose dependently upregulated while Arg1 mRNA expression remained unchanged (Figures 3(a) and 3(b)). Expressions of iNOS and Arg1 in macrophages incubated with AGEs 500 g/mL for 24 h were determined by FCM. Similarly, AGEs still significantly upregulated iNOS production without affecting Arg1 expression (Figures 3(c) and 3(d)).

Effects of AGEs on the Expression of Surface Markers on
Macrophages. M1 macrophages usually express a higher level of cell surface markers such as CD11c and CD86, while M2 macrophages express a higher level of mannose receptor (CD206) [6,7]. We thus evaluated the impacts of AGEs on the expressions of these surface markers by FCM. As expected, expressions of CD11c and CD86 were significantly upregulated on macrophages after AGEs stimulation (Figures 4(a) and 4(b)). Nevertheless, the level of CD206 remained unchanged (Figure 4(c)). Double staining with F4/80 and CD11c further indicated that AGEs increased CD11c expression on F4/80 + macrophages (Figure 4(d)).    Figure 4: Effect of AGEs on expressions of surface markers. Cells were cultured with AGEs or BSA 500 g/mL for 24 h. The markers were measured by FCM. AGEs increased CD11c (a) and CD86 (b) expression but did not affect CD206 expression (c). Double staining with CD11c and F4/80 indicated that AGEs increased F4/80 + CD11c + double positive macrophages (d). * < 0.05, and * * < 0.01 compared with control. MFI: mean fluorescence intensity. Each bar represents the mean value ± SD of triplicate determinations, representative of three independent experiments.

Role of RAGE/NF-B Pathway on Macrophage Polarization after AGEs
Stimulation. RAGE is the most important and common receptor which interacts with AGEs. What is more, AGEs/RAGE/NF-B axis plays critical roles in inducing various kinds of cells to inflammatory conditions [17,18]. Thus we hypothesized that the axis might be involved in AGEs induced macrophage polarization. To confirm the hypothesis, firstly we assayed the effect of AGEs on expression of RAGE by real time PCR and FCM. As a result, AGEs treatment significantly upregulated not only mRNA level but also protein level of RAGE expression in macrophages (Figures 5(a) and  5(b)).
Next, we evaluated the effect of AGEs on activation of NF-B pathway in macrophages. Because activation of NF-B pathway is associated with phosphorylation of NF-B, phospho-NF-B-p65 (p-p65) and NF-B-p65 (p65) were measured as mean fluorescence intensity (MFI) by FCM in different time points after AGEs stimulation. The p-p65 level increased within 15 min after treatment with AGEs. Moreover, the effect peaked at 60 min and decreased thereafter. In contrast, the total NF-B-p65 (p65) level remained unchanged over the time after AGEs treatment. Furthermore, the p-p65/p65 ratio increased significantly shortly after AGEs stimulation, peaked at 60 min, and then decreased ( Figure 6(a)). These data indicate that AGEs can activate the NF-B pathway.
To further identify the role of AGEs/RAGE axis on activation of NF-B, RAGE neutralizing antibody was used to block RAGE receptors. BMDM cells were pretreated with anti-RAGE antibody (50 g/mL) for 60 min; then AGEs were added into the culture medium to stimulate the cells. FCM results indicated that p-p65 still increased within 15 min and peaked at 60 min and decreased thereafter, while total p65 levels remained unchanged after AGEs treatment. The p-p65/p65 ratio changed in the same way as p-p65 level ( Figure 6(b)). The results demonstrate that AGEs can still activate the NF-B pathway even with RAGE receptor blockade. Of note, the peak of p-p65/p65 ratio was significantly lower and the changes of p-p65/p65 ratios were somehow less in the RAGE blocking group than in the group without anti-RAGE antibody pretreatment (Figure 6(c)). These data show that anti-RAGE antibody can partly inhibit the activation of NF-B which can be activated by AGEs.
The experiments above demonstrate that AGEs can induce NF-B activation at least partly through RAGE signaling. Furthermore, the inability of RAGE blocking pretreatment to completely inhibit the NF-B activation suggests AGEs might interact with other receptors to initiate NF-B activation.
The role of RAGE/NF-B pathway on M1 markers after AGEs stimulation was then investigated. Anti-RAGE antibody and a specific inhibitor of NF-B (PDTC) were used to block the RAGE/NF-B pathway, respectively. The expressions of M1 markers including iNOS and CD86 were evaluated by FCM, and the mRNA levels of IL-6 and TNF-were evaluated by real time PCR. As shown in Figure 7(a), AGEs significantly upregulated iNOS and CD86 expression and these effects were significantly attenuated by blocking RAGE with anti-RAGE antibody. Moreover, PDTC also significantly inhibited AGEs induced iNOS and CD86 expression.

Discussion
In response to hyperglycemia in patients with diabetes, various metabolic mechanisms contribute to the pathogenesis of diabetic complications. The close participation of AGEs in vascular complications has been well documented. In the present study, we find that AGEs not only predominantly induce macrophages to secrete inflammatory cytokines but also induce M1 polarization as evidenced by the iNOS and expressions of surface markers on macrophages. Moreover, AGEs stimulation activates RAGE/NF-B pathway and blockade of RAGE or NF-B can attenuate the AGEs effects on macrophage polarization. Taken together, our results indicate that AGEs can induce macrophages polarization into M1 phenotype partly through RAGE/NF-B pathway and this might be one of the mechanisms responsible for the diabetic vascular complications.
Advanced glycation end products play an important role in the pathogenesis of diabetic complications by inducing inflammation, especially in atherosclerosis, while macrophages and their polarization status dominate the process of atheroma formation and progression. Therefore, investigating the interaction between AGEs and the polarization of macrophages might further reveal the underlying mechanism of diabetic accelerated atherosclerosis. Previous studies have shown that AGEs not only promoted macrophages to secret proinflammatory cytokines [19,20] but also enhanced their migration ability [21], while Choi et al. recently proved that AGEs enhanced iNOS expression in macrophages [22]. In the present study, we also found AGEs upregulated iNOS production. As to the cytokines expressions, although our results showed the highest concentration of AGEs slightly increased IL-10 production, the expressions of proinflammatory cytokines such as IL-6 and TNF-increased more sharply and dominated the macrophage cytokine expression profile. Thus, our results are in line with previous studies which indicate AGEs induce macrophages into proinflammatory status by enhancement on proinflammatory molecules production. As M1 macrophages are characterized by "proinflammatory" function, the above mentioned data suggest AGEs might prime macrophages into M1 phenotype.
However, it is essential to conduct the surface marker study to confirm the macrophages polarization. Unfortunately, no previous studies have evaluated the impact of AGEs on macrophage surface markers alteration. We thus carried out related experiments to elucidate the surface markers shift on macrophages to further ascertain the polarization changes induced by AGEs. As a result, we found the expressions of M1 surface markers including CD11c and CD86 were upregulated, while M2 surface marker CD206 remained unchanged after AGEs treatment. What is more, double staining with CD11c and F4/80, a macrophage-restricted surface marker [23], further suggested that AGEs induce macrophage polarization into M1 phenotype. Taken together, AGEs increased proinflammatory mediators and M1 surface markers on macrophages; these data indicate that AGEs prime macrophage into M1 phenotype.
The underlying mechanism of AGEs mediated macrophage M1 polarization remains largely unknown now. It is known that receptor for AGEs (RAGE) is a multiligand receptor expressed on many cells and mediates the most biological effects of AGEs [24]. Previous study showed that AGEs-RAGE interaction could enhance macrophages migration [21] and promote proinflammatory mediators production such as IL-1 , IL-6, and TNF- [25]. However, the role of AGEs-RAGE axis on macrophage polarization was not reported. Consistent with previous studies, we found AGEs-RAGE interaction participated in the signal transduction to increase expressions of IL-6 and TNF-since anti-RAGE antibody pretreatment could partly abate AGEs induced cytokines production. Furthermore, we also found AGEs increased M1 markers expression via RAGE signaling since pretreatment with anti-RAGE antibody attenuated M1 markers expression. Thus, our data prove that AGEs-RAGE interaction plays a pivotal role in AGEs induced M1 polarization. Former research has established that AGEs-RAGE axis could activate several central transcription factors including nuclear factor-(NF-) B, cAMP-response-element-binding protein-(CREB-) 1, early growth response-(EGR-) 1, and activator protein-(AP-) 1 [26]. Among them, NF-B is the key target of AGEs/RAGE signaling in atherosclerosis. It could mediate proinflammatory response in various kinds of cells [18,27], as well as in macrophages [28]. In the present study, we found AGEs activated NF-B and its activation could be partly inhibited by RAGE blocking pretreatment. That means AGEs can induce NF-B activation partly via RAGE signaling. It should be noted that NF-B has been found to participate in several molecules induced macrophage M1 polarization, for example, C reactive protein (CRP) and Maltosebinding protein (MBP) [29,30]. Thus NF-B probably will be implicated in AGEs-RAGE axis induced M1 polarization. As we found in this study, administration of anti-RAGE antibody partly suppressed NF-B activation, and pretreatment with anti-RAGE antibody or PDTC abated effects of AGEs on priming macrophages to the M1 phenotype. These results demonstrate AGEs induce macrophages into the M1 phenotype at least partly through the RAGE/NF-B pathway.
Of note, in addition to RAGE, AGEs also act as ligands for several other receptors such as AGE-receptor complex (AGE-R1/OST-48, AGE-R2/80K-H, and AGE-R3/galectin-3) and toll-like receptor-4 (TLR-4) [31,32]. Furthermore, there is evidence that AGEs can induce NF-B activation through TLR4 signaling [32], and TLR4 signaling can mediate M1 polarization [33,34]. In the current experiment, we found the RAGE blockade pretreatment could not totally abolish the activation of NF-B triggered by AGEs. Thus, this phenomenon suggested, besides RAGE, AGEs probably could interact with other receptors to initiate cell signaling and then induced NF-B activation to mediate macrophage M1 polarization. What is more, previous studies have demonstrated that several other pathways, such as C-Jun N-terminal kinase (JNK), Notch, and JAK/STAT signaling pathway, are involved in macrophage polarization [35]. Thus, further work is warranted to reveal other receptors and pathways underlying AGEs induced macrophage polarization.
As is well known, macrophages respond to an extraordinary diversity of environmental stimuli, including different microbial products and various cytokines. Although M1 and M2 macrophages coexist in the atherosclerotic lesions, a large body of experimental evidence supports the contribution of M1 macrophages in atherosclerosis progression and lesion destabilization [12,36]. Very recently, Fadini et al. found an increased M1/M2 polarization ratio in prediabetic and diabetic patients [37,38] suggesting an unfavorable M1/M2 polarization might be a crucial underlying mechanism responsible for accelerated atherosclerosis in diabetic patients. Although hyperglycemia was found to induce macrophages to produce proinflammatory molecules [39,40], it remains unknown whether it can also affect macrophage polarization. In the present study, we found that AGEs induced macrophages polarization into M1 phenotype. Such findings suggest that AGEs, the main pathogenic factor of diabetes, may be one of the chief culprits to disturb the M1/M2 ratio. By promoting macrophage polarization into M1 phenotype, AGEs might act as a major player linking diabetes and accelerated atherosclerosis.

Conclusion
The present study suggests that AGEs enhance mouse bone marrow derived macrophage polarization toward a proinflammatory M1 phenotype at least partly via activating the RAGE/NF-B pathway. These data thus underscore a potential role of AGEs on affecting macrophage polarization. Strategies which might block or even reverse the effect of AGEs on macrophage M1 polarization might be novel ways to alleviating the atherosclerosis progression in diabetic patients.