J Periodont Res 2015; 50: 737–747 All rights reserved

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12260

Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

E.-Y. Choi1, S.-H. Choe1, J.-Y. Hyeon1, J.-I. Choi2,3, I. S. Choi1, S.-J. Kim2,3,4 1 Department of Biological Science, College of Medical and Life Sciences, Silla University, Busan, Korea, 2Department of Periodontology, School of Dentistry, Pusan National University, Yangsan, Korea, 3Dental Research Institute, Pusan National University Dental Hospital, Yangsan, Korea and 4Institute of Translational Dental Sciences, Pusan National University, Yangsan, Korea

Choi E-Y, Choe S-H, Hyeon J-Y, Choi J-I, Choi IS, Kim S-J. Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages. J Periodont Res 2015; 50: 737–747. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Caffeic acid phenethyl ester (CAPE) has numerous potentially beneficial properties, including antioxidant, immunomodulatory and anti-inflammatory activities. However, the effect of CAPE on periodontal disease has not been studied before. This study was designed to investigate the efficacy of CAPE in ameliorating the production of proinflammatory mediators in macrophages activated by lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease. Material and Methods: LPS from P. intermedia ATCC 25611 was isolated by using the standard hot phenol–water method. Culture supernatants were assayed for nitric oxide (NO), interleukin (IL)-1b and IL-6. We used real-time polymerase chain reaction to quantify inducible NO synthase, IL-1b, IL-6, heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS) 1 mRNA expression. HO-1 protein expression and levels of signaling proteins were assessed by immunoblot analysis. DNA-binding activities of NF-jB subunits were analyzed by using the enzyme-linked immunosorbent assay-based kits. Results: CAPE exerted significant inhibitory effects on P. intermedia LPSinduced production of NO, IL-1b and IL-6 as well as their mRNA expression in RAW264.7 cells. CAPE-induced HO-1 expression in cells activated with P. intermedia LPS, and selective inhibition of HO-1 activity by tin protoporphyrin IX attenuated the inhibitory effect of CAPE on LPS-induced NO production. CAPE did not interfere with IjB-a degradation induced by P. intermedia LPS. Instead, CAPE decreased nuclear translocation of NF-jB p65 and p50 subunits induced with LPS, and lessened LPS-induced p50 binding activity. Further, CAPE showed strong inhibitory effects on LPS-induced signal transducer and activator of transcription 1 and 3 phosphorylation. Besides, CAPE

Sung-Jo Kim, DDS, PhD, Department of Periodontology, School of Dentistry, Pusan National University, 49 Busandaehak-ro, Mulgeum-eup, Yangsan 626-870, Korea Tel: +82 55 360 5201 Fax: +82 55 360 5194 e-mail: [email protected] Key words: caeic acid phenethyl ester; HO-1;

lipopolysaccharide; NF-jB; proinflammatory mediators; SOCS1; STAT Accepted for publication November 29, 2014

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significantly elevated SOCS1 mRNA expression in P. intermedia LPS-stimulated cells. Conclusion: Modulation of host response by CAPE may represent an attractive strategy towards the treatment of periodontal disease. In vivo studies are required to appraise the potential of CAPE further as an immunomodulator in the treatment of periodontal disease.

Growing evidence suggests that certain pharmacologic agents could be of therapeutic value as host response modulators in periodontal treatment. Caffeic acid phenethyl ester (CAPE), an active component of propolis obtained from honeybee hives, has been previously characterized as having numerous potentially beneficial properties, including antioxidant, anticarcinogenic, immunomodulatory and anti-inflammatory activities (1–3). However, to the best of our knowledge, the effect of CAPE on inflammatory periodontal disease has not been studied before. Periodontal disease is a chronic inflammatory pathology initiated by specific plaque bacteria that colonize the periodontal pocket, resulting in gradual attachment loss and destruction of alveolar bone (4). Current knowledge concerning the pathologic mechanisms of periodontal disease suggests that it results from the complex interaction between the specific plaque bacteria and the host immune– inflammatory responses that develop in response to bacteria and their byproducts. Recently, an alternative therapeutic strategy, in addition to traditional treatment targeting the reduction of bacteria in the plaque biofilm, has been evolved to modulate the host component of periodontal disease (5,6). Prevotella intermedia is a gramnegative periodontal pathogen believed strongly implicated in the etiology of chronic periodontitis (7,8). It is also particularly associated with the subgingival microflora of patients with necrotizing ulcerative gingivitis (9) and pregnancy gingivitis (10). Lipopolysaccharide (LPS) is a principal constituent of the cell wall of gramnegative bacteria, including P. inter-

media. It is known to stimulate the secretion of proinflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-a), interleukin (IL)-1b and IL-6 in a variety of immune cells (11). Periodontal disease is characterized by the increased production of inflammatory mediators, and it has been reported that a variety of proinflammatory mediators, such as NO, IL-1b and IL-6, may be relevant to tissue breakdown characteristic of periodontal disease. These mediators are clearly elevated in sites with periodontal disease and closely correlated with clinical signs of disease (12–16). In addition, it has been suggested that NO, IL-1b and IL-6 have a strong potential to induce bone resorption in periodontal disease (17–19). Therefore, host modulatory agents that block these inflammatory mediators could be beneficial in blocking the development and progression of periodontal disease. Heme oxygenase-1 (HO-1), the inducible isoform of heme oxygenase that catalyzes the degradation of heme to bilirubin/biliverdin, free iron and carbon monoxide (CO), has been shown to have numerous potentially beneficial properties including antiinflammatory and antioxidant effects (20–22). While HO-1-deficient mice exhibited severe inflammation, HO-1 overexpression exerted anti-inflammatory effects (21,23). The p38, JNK, NF-jB and Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathways play a role in P. intermedia LPS-induced production of IL-6 and NO in RAW264.7 cells (24). These pathways, except JNK, also mediate P. intermedia LPS-induced production of IL-1b (Fig. S1). In addition, the

STAT3 pathway also involved in the production of NO, IL-1b and IL-6 induced by P. intermedia LPS (Fig. S2). Suppressors of cytokine signaling (SOCS) proteins, which act as a family of negative feedback regulators of cytokine signaling to attenuate proinflammatory cytokine production, consists of eight members, including SOCS1–7 and cytokine-inducible Src homology 2 domain-containing protein (CIS) (25,26). They are induced by a range of microbial factors, including LPS, and cytokines (27,28). We here demonstrate that CAPE inhibits P. intermedia LPS-induced production of NO, IL-1b and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and inhibition of NF-jB, STAT1 and STAT3 activation, which is related to the activation of SOCS1 signaling.

Material and methods Reagents

CAPE was the product of SigmaAldrich (St. Louis, MO, USA). Primary antibodies against JNK, phospho-JNK, p38, phospho-p38, IjB-a, STAT1, phospho-STAT1, STAT3 and phospho-STAT3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against HO-1, NF-jB p65, NF-jB p50, b-actin and PARP-1 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tin protoporphyrin IX (SnPP) was obtained from Frontier Scientific Inc. (Logan, UT, USA). Except where otherwise noted, all other chemicals and reagents used were obtained from Sigma-Aldrich.

Effect of caffeic acid phenethyl ester on periodontal disease Bacterial culture and isolation of lipopolysaccharide

P. intermedia ATCC 25611 was cultured anaerobically in GAM broth (Nissui, Tokyo, Japan) containing 1 lg/mL menadione and 5 lg/mL hemin as described previously (24). LPS was prepared from freeze-dried P. intermedia cells using the standard hot phenol–water procedure followed by treatment with DNase, RNase and proteinase K as described in our previous work (24).

and CAPE for 24 h (for IL-6) or 48 h (for IL-1b), after which the amounts of IL-1b and IL-6 released into the culture supernatants were analyzed

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with commercial ELISA kits (OptEIA; BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions.

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Cell culture and cytotoxicity assay

RAW264.7 cells (American Type Culture Collection, Rockville, MD, USA), a murine macrophage cell line, were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 lg/ mL streptomycin) at 37°C in a humidified atmosphere containing 5% CO2 as previously reported (24). The extent of cytotoxicity from CAPE was evaluated using the MTT assay in accordance with the manufacturer’s protocols.

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Measurement of nitric oxide production

NO production was determined by an assay of the culture media for nitrite (NO2 ), a stable oxidative metabolite of NO. Briefly, cells incubated with different doses of P. intermedia LPS and CAPE for 24 h, and 100 lL aliquots of culture supernatants were allowed to react with an equal volume of Griess reagent at room temperature for 10 min. Optical densities were determined by measuring the absorbance at 540 nm with a Spectra Max 250 enzyme-linked immunosorbent assay (ELISA) Reader (Molecular Devices, Sunnyvale, CA, USA), and nitrite concentrations were quantified by using a standard curve prepared with serial dilutions of NaNO2. Measurement of interleukin-1b and 6 production

RAW264.7 cells were incubated with different doses of P. intermedia LPS

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Fig. 1. Effects of CAPE on Prevotella intermedia LPS-induced production of NO (A), IL1b (B) and IL-6 (C) in RAW264.7 cells. Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 24 h (for NO and IL-6) or 48 h (for IL-1b). The concentrations of NO, IL-1b and IL-6 released into the culture medium were analyzed by enzyme-linked immunosorbent assay. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone. CAPE, caffeic acid phenethyl ester; IL, interleukin; LPS, lipolysaccharide; NO, nitric oxide.

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Real-time polymerase chain reaction analysis

Cells were plated in 100 mm tissue culture dishes at a density of 1 9 107 cells/dish and incubated with different concentrations of P. intermedia LPS and CAPE for the indicated times. Following incubation, they were washed twice with phosphate-buffered saline and harvested by centrifugation. Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols. The reverse transcription of 1 lg of total RNA into cDNA was conducted using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). A real-time polymerase chain reaction (PCR) was performed using the CFX96 real-time PCR detection system (Bio-Rad) with specific primers for murine inducible NO synthase (iNOS), IL-1b, IL-6, HO-1 and SOCS1. The expression of b-actin was used as an endogenous control. PCR was carried out with SsoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer’s instructions. Thermocycling conditions were as follows: After denaturing at 98°C for 30 s, PCR was performed for 45 cycles, each of which consisted of denaturing at 95°C for 1 s, annealing/extending at 60°C for 5 s. The PCR oligonucleotide primers used were: iNOS (130 bp) forward, 50 -GCACCACCCTCCTCGTTCAG-30 and reverse, 50 -TCCACAACTCGC TCCAAGATTCC-30 ; IL-1b (131 bp) forward, 50 -TTCAGGCAGGCAGTA TCA-30 and reverse, 50 -AGGATGG GCTCTTCTTCAA-30 ; IL-6 (162 bp) forward, 50 -GCCAGAGTCCTTCAGAGAGATACAG-30 and reverse, 50 GAATTGGATGGTCTTGGTCCTT AGC-30 ; HO-1 (149 bp) forward, 50 CAATGTGGCCTTCTCTCTGT-30 and reverse, 50 -TTTTGGTGAGGGA ACTGTGT-30 ; SOCS1 (133 bp) forward, 50 -CACTTCTGGCTGGAG ACC-30 and reverse, 50 -TGGA GAGGTAGGAGTGGAA-30 ; b-actin (149 bp) forward, 50 -TGAGAGGGA AATCGTGCGTGAC-30 and reverse, 50 GCTCGTTGCCAATAGTGA TGACC-30 . Each assay was normalized to the level of b-actin mRNA.

Immunoblot analysis

Cells were seeded in 60 mm tissue culture dishes at a density of 4 9 106 cells/dish and incubated with the different doses of P. intermedia LPS and CAPE for the indicated times. Immunoblot analysis was performed according to the procedures, which were used in our previous study (24). Briefly, whole cell lysates and nuclear extracts were prepared using lysis buffer (50 mM Tris Cl, 150 mM NaCl, 0.002% sodium azide, 0.1% sodium dodecyl sulfate and 1% Nonidet P-40) and the nuclear extract kit (Active Motif, Carlsbad, CA, USA), respectively. The protein samples (30 lg for each) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane by electroblotting, probed with the appropriate primary antibodies, and then incubated with horseradish peroxidase-coupled secondary antibodies. Immunoreactive bands were visualized by the reaction with an enhanced chemiluminescence detection system (Cell Signaling Technology) according to the manufacturer’s procedures. Nuclear factor-jB DNA-binding activity assay

Cells were plated in 60 mm tissue culture dishes at a density of 4 9 106 cells/dish and incubated with the indicated doses of P. intermedia LPS and CAPE for the different periods of time. After preparing the nuclear protein extracts from cells as described above, the DNA-binding activity of NF-jB p65 or p50 in the nuclear

extracts was measured using the ELISA-based NF-jB transcription factor assay kit (Active Motif) as previously described (24). Statistical analysis

Data are expressed as means  SD and statistical analysis was performed using Student’s t-test with p < 0.05 considered statistically significant.

Results Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced production of nitric oxide and interleukins 1b and 6

Treatment of RAW264.7 cells with P. intermedia LPS (10 lg/mL) brought about strong elevations in the levels of NO, IL-1b and IL-6 compared to the control, and CAPE exerted significant inhibitory effects on the release of these proinflammatory mediators from cells stimulated with LPS (Fig. 1). Notably, at the highest concentration of 10 lM, CAPE suppressed NO and IL-6 release approximately by 86% and 72%, respectively. In addition, CAPE caused 77% inhibition of IL-1b production at a concentration of 5 lM. It was found that CAPE at the concentrations used in this study did not show cytotoxicity in the presence of P. intermedia LPS as assessed by MTT assay, indicating that the decrease in LPS-induced production of inflammatory mediators by addition of CAPE was not due to its nonspecific cytotoxic effects (data not

Fig. 2. Effect of CAPE on Prevotella intermedia LPS-induced iNOS protein expression in RAW264.7 cells. Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 24 h. iNOS protein synthesis was measured by immunoblot analysis of cell lysates using iNOS-specific antibody. A representative immunoblot from three separate experiments with similar results is shown. CAPE, caffeic acid phenethyl ester; iNOS, inducible nitric oxide synthase; LPS, lipolysaccharide.

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Real-time polymerase chain reaction analysis

Cells were plated in 100 mm tissue culture dishes at a density of 1 9 107 cells/dish and incubated with different concentrations of P. intermedia LPS and CAPE for the indicated times. Following incubation, they were washed twice with phosphate-buffered saline and harvested by centrifugation. Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols. The reverse transcription of 1 lg of total RNA into cDNA was conducted using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). A real-time polymerase chain reaction (PCR) was performed using the CFX96 real-time PCR detection system (Bio-Rad) with specific primers for murine inducible NO synthase (iNOS), IL-1b, IL-6, HO-1 and SOCS1. The expression of b-actin was used as an endogenous control. PCR was carried out with SsoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer’s instructions. Thermocycling conditions were as follows: After denaturing at 98°C for 30 s, PCR was performed for 45 cycles, each of which consisted of denaturing at 95°C for 1 s, annealing/extending at 60°C for 5 s. The PCR oligonucleotide primers used were: iNOS (130 bp) forward, 50 -GCACCACCCTCCTCGTTCAG-30 and reverse, 50 -TCCACAACTCGC TCCAAGATTCC-30 ; IL-1b (131 bp) forward, 50 -TTCAGGCAGGCAGTA TCA-30 and reverse, 50 -AGGATGG GCTCTTCTTCAA-30 ; IL-6 (162 bp) forward, 50 -GCCAGAGTCCTTCAGAGAGATACAG-30 and reverse, 50 GAATTGGATGGTCTTGGTCCTT AGC-30 ; HO-1 (149 bp) forward, 50 CAATGTGGCCTTCTCTCTGT-30 and reverse, 50 -TTTTGGTGAGGGA ACTGTGT-30 ; SOCS1 (133 bp) forward, 50 -CACTTCTGGCTGGAG ACC-30 and reverse, 50 -TGGA GAGGTAGGAGTGGAA-30 ; b-actin (149 bp) forward, 50 -TGAGAGGGA AATCGTGCGTGAC-30 and reverse, 50 GCTCGTTGCCAATAGTGA TGACC-30 . Each assay was normalized to the level of b-actin mRNA.

Immunoblot analysis

Cells were seeded in 60 mm tissue culture dishes at a density of 4 9 106 cells/dish and incubated with the different doses of P. intermedia LPS and CAPE for the indicated times. Immunoblot analysis was performed according to the procedures, which were used in our previous study (24). Briefly, whole cell lysates and nuclear extracts were prepared using lysis buffer (50 mM Tris Cl, 150 mM NaCl, 0.002% sodium azide, 0.1% sodium dodecyl sulfate and 1% Nonidet P-40) and the nuclear extract kit (Active Motif, Carlsbad, CA, USA), respectively. The protein samples (30 lg for each) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane by electroblotting, probed with the appropriate primary antibodies, and then incubated with horseradish peroxidase-coupled secondary antibodies. Immunoreactive bands were visualized by the reaction with an enhanced chemiluminescence detection system (Cell Signaling Technology) according to the manufacturer’s procedures. Nuclear factor-jB DNA-binding activity assay

Cells were plated in 60 mm tissue culture dishes at a density of 4 9 106 cells/dish and incubated with the indicated doses of P. intermedia LPS and CAPE for the different periods of time. After preparing the nuclear protein extracts from cells as described above, the DNA-binding activity of NF-jB p65 or p50 in the nuclear

extracts was measured using the ELISA-based NF-jB transcription factor assay kit (Active Motif) as previously described (24). Statistical analysis

Data are expressed as means  SD and statistical analysis was performed using Student’s t-test with p < 0.05 considered statistically significant.

Results Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced production of nitric oxide and interleukins 1b and 6

Treatment of RAW264.7 cells with P. intermedia LPS (10 lg/mL) brought about strong elevations in the levels of NO, IL-1b and IL-6 compared to the control, and CAPE exerted significant inhibitory effects on the release of these proinflammatory mediators from cells stimulated with LPS (Fig. 1). Notably, at the highest concentration of 10 lM, CAPE suppressed NO and IL-6 release approximately by 86% and 72%, respectively. In addition, CAPE caused 77% inhibition of IL-1b production at a concentration of 5 lM. It was found that CAPE at the concentrations used in this study did not show cytotoxicity in the presence of P. intermedia LPS as assessed by MTT assay, indicating that the decrease in LPS-induced production of inflammatory mediators by addition of CAPE was not due to its nonspecific cytotoxic effects (data not

Fig. 2. Effect of CAPE on Prevotella intermedia LPS-induced iNOS protein expression in RAW264.7 cells. Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 24 h. iNOS protein synthesis was measured by immunoblot analysis of cell lysates using iNOS-specific antibody. A representative immunoblot from three separate experiments with similar results is shown. CAPE, caffeic acid phenethyl ester; iNOS, inducible nitric oxide synthase; LPS, lipolysaccharide.

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Choi et al. degrading IjB-a (Fig. 6A). We then analyzed the effects of CAPE on the translocation of NF-jB subunits to the nucleus in P. intermedia LPS-treated cells. Immunoblot analysis of nuclear fraction was carried out using antibodies against NF-jB p65 and p50. Nuclear translocation of NF-jB p65 and p50 subunits induced by P. intermedia LPS was significantly repressed by CAPE treatment (Fig. 6B). Finally, we determined whether CAPE could affect the binding of NF-jB to DNA. The DNAbinding activity of NF-jB in nuclear fraction was measured with an ELISA-based NF-jB transcription factor assay kit. We observed that the DNA-binding activities of NF-jB p65 and p50 subunits were markedly upregulated in response to P. intermedia LPS treatment (Fig. 6C). Whereas CAPE significantly lessened LPSinduced NF-jB p50 binding activity, it did not affect p65 binding (Fig. 6C).

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Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced phosphorylation of STAT1 and STAT3

Fig. 4. Involvement of HO-1 in the inhibitory effects of CAPE on Prevotella intermedia LPS-induced production of NO in RAW264.7 cells. (A,B) Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 6 h. (A) HO-1 protein synthesis was measured by immunoblot analysis of cell lysates using HO-1specific antibody. A representative immunoblot from three separate experiments with similar results is shown. (B) Real-time polymerase chain reaction was performed with EvaGreen Supermix, b-actin being used as an endogenous control. The results are means  SD of three independent experiments. *p < 0.05 vs. P. intermedia LPS alone; **p < 0.01 vs. P. intermedia LPS alone. (C) Cells were treated with CAPE (10 lM) and P. intermedia LPS (10 lg/mL) for 24 h in the presence of different doses of SnPP, after which the culture supernatants were assayed for NO. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone; ††p < 0.01 vs. P. intermedia LPS plus CAPE. CAPE, caffeic acid phenethyl ester; HO-1, heme oxygenase-1; LPS, lipolysaccharide; SnPP, tin protoporphyrin IX.

Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced nuclear factor-jB transcriptional activity

We next investigated whether the inhibitory effects of CAPE on P. intermedia LPS-induced production of NO,

IL-1b and IL-6 are associated with the NF-jB signaling pathway. To determine the effect of CAPE on the degradation of IjB-a induced by P. intermedia LPS, we measured the cytoplasmic levels of IjB-a protein. Immunoblot analysis showed that CAPE failed to prevent LPS from

Next, we tested the hypothesis that CAPE downregulates P. intermedia LPS-induced production of NO, IL-1b and IL-6 via inhibition of STAT signaling cascade. CAPE showed strong dose-dependent inhibitory effects on LPS-induced STAT1 and STAT3 phosphorylation in RAW264.7 cells (Fig. 7). Effect of caffeic acid phenethyl ester on SOCS1 expression in cells activated by P. intermedia lipopolysaccharide

We also examined the possibility that CAPE would modify the SOCS signaling that is involved in cytokine signaling as negative regulators to inhibit proinflammatory cytokine production. Real-time PCR analysis revealed that CAPE dose-dependently upregulated the mRNA level of SOCS1 in RAW264.7 cells treated with P. intermedia LPS (Fig. 8). By contrast, treatment with CAPE did

Effect of caffeic acid phenethyl ester on periodontal disease A

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Fig. 5. Effects of CAPE on Prevotella intermedia LPS-induced phosphorylation of p38 (A) and JNK (B) in RAW264.7 cells. Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 15 min (for p38) or 30 min (for JNK). Cells lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from three separate experiments with similar results is shown. CAPE, caffeic acid phenethyl ester; LPS, lipolysaccharide.

not result in the elevation of SOCS2 and SOCS3 mRNA levels in LPSstimulated cells (Fig. S4).

Discussion Previous studies indicate that P. intermedia LPS is a potent inducer of inflammatory mediators, including NO, IL-1b, IL-6 and TNF-a, in macrophages (24,29,30). In addition, LPS of this bacterium dose-dependently inhibited bone formation by reducing alkaline phosphatase activity and calcium incorporation, and induced the release of NO and IL-6 by fetal mouse osteoblasts in organoid culture (17). LPS from P. intermedia has been shown to be distinct from enterobacterial LPS in structure and function (31). In addition, unlike Salmonella LPS, P. intermedia LPS activates macrophages of classical LPS-refractory C3H/HeJ mice (32). Further, while the biological activity of LPS prepared from Enterobacteriaceae was repressed by polymyxin B, P. intermedia LPS was not prone to polymyxin B treatment (32). LPS from other bacterial pathogens such as Porphyromonas gingivalis might be more relevant to the pathogenesis of periodontal disease. However, there are strain-dependent diversities in the capacity of P. gingivalis LPS to induce the production of proinflammatory mediators. For instance, LPS isolated from P. gingivalis ATCC 33277 and W50 failed to induce the release of NO

by RAW264.7 cells (33,34). On the contrary, the findings of Shapira et al. (34) and our unpublished observations show that NO could be produced by murine macrophages when stimulated with LPS preparations from two strains of P. gingivalis, A7436 and 381, although NO induction by these LPS was far less potent than with LPS from enteric bacteria. For these reasons, the present work attempted to explore the effects of CAPE on the capacity of murine macrophages to produce NO, IL-1b and IL-6 in response to P. intermedia LPS and its mechanisms of action. The results of the present study show that CAPE significantly suppresses P. intermedia LPS-induced production of iNOS-induced NO, IL1b and IL-6 as well as their mRNA expression in RAW264.7 cells, indicating that it suppressed these proinflammatory mediators at both gene transcription and translation levels. We examined whether the repressive effect of CAPE on P. intermedia LPS-induced production of proinflammatory mediators are related to the induction of HO-1 expression. The results indicated that CAPE has an ability to induce HO-1 expression in cells treated with LPS, and CAPEinduced HO-1 mediates the inhibition of LPS-induced production of NO. By contrast, HO-1 was not involved in the CAPE-mediated inhibition of IL-1b and IL-6. It has been demonstrated that the anti-inflammatory effects of HO-1 are ascribed to the

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enzymatic by-products of heme degradation. These include CO, which has been shown to diminish the production of inflammatory mediators in LPS-activated macrophages, and bilirubin, which is a strong antioxidant (35–37). Hence, we suggest that HO-1 expression induced by CAPE might lead to the suppression of P. intermedia LPS-induced NO production via enhanced build-up of CO and bilirubin. The JNK and p38 pathways play a role in P. intermedia LPS-induced production of IL-6 and NO in RAW264.7 cells (24). The p38 pathway also mediate IL-1b production induced by P. intermedia LPS (Fig. S1). However, activation of JNK and p38 induced by P. intermedia LPS was not affected by CAPE treatment, implying that these pathways are not involved in the attenuation of P. intermedia LPS-induced production of NO, IL-1b and IL-6 by CAPE. NF-jB is a multi-unit transcription factor that has been shown to play pivotal roles in the regulation of diverse target genes related with inflammatory responses and bacterial LPS is one of the known inducers of NF-jB activity (38–40). In unstimulated cells, NF-jB presents in the cytoplasm in an inactive form linked to a group of inhibitors, called inhibitory jB (IjB) proteins. IjB undergoes phosphorylation, followed by ubiquitination and consequent proteasome-mediated degradation in response to a variety of stimuli, including LPS. Then, the free NF-jB is translocated to the nucleus, where it provokes activation of various genes associated with the inflammation (41,42). In this study, CAPE did not interfere with IjB-a degradation induced by P. intermedia LPS. Instead, nuclear translocation of p65 and p50 subunits induced by LPS was significantly repressed by CAPE treatment. In addition, CAPE significantly lessened LPS-induced NF-jB p50 binding activity, while it did not affect p65 binding. Unlike NF-jB p65, the p50 NF-jB subunit does not comprise a transactivation domain and, hence, usually develops a heterodimer with other NF-jB subunits to take part in target gene transcription (42–44).

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Choi et al. degrading IjB-a (Fig. 6A). We then analyzed the effects of CAPE on the translocation of NF-jB subunits to the nucleus in P. intermedia LPS-treated cells. Immunoblot analysis of nuclear fraction was carried out using antibodies against NF-jB p65 and p50. Nuclear translocation of NF-jB p65 and p50 subunits induced by P. intermedia LPS was significantly repressed by CAPE treatment (Fig. 6B). Finally, we determined whether CAPE could affect the binding of NF-jB to DNA. The DNAbinding activity of NF-jB in nuclear fraction was measured with an ELISA-based NF-jB transcription factor assay kit. We observed that the DNA-binding activities of NF-jB p65 and p50 subunits were markedly upregulated in response to P. intermedia LPS treatment (Fig. 6C). Whereas CAPE significantly lessened LPSinduced NF-jB p50 binding activity, it did not affect p65 binding (Fig. 6C).

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Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced phosphorylation of STAT1 and STAT3

Fig. 4. Involvement of HO-1 in the inhibitory effects of CAPE on Prevotella intermedia LPS-induced production of NO in RAW264.7 cells. (A,B) Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 6 h. (A) HO-1 protein synthesis was measured by immunoblot analysis of cell lysates using HO-1specific antibody. A representative immunoblot from three separate experiments with similar results is shown. (B) Real-time polymerase chain reaction was performed with EvaGreen Supermix, b-actin being used as an endogenous control. The results are means  SD of three independent experiments. *p < 0.05 vs. P. intermedia LPS alone; **p < 0.01 vs. P. intermedia LPS alone. (C) Cells were treated with CAPE (10 lM) and P. intermedia LPS (10 lg/mL) for 24 h in the presence of different doses of SnPP, after which the culture supernatants were assayed for NO. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone; ††p < 0.01 vs. P. intermedia LPS plus CAPE. CAPE, caffeic acid phenethyl ester; HO-1, heme oxygenase-1; LPS, lipolysaccharide; SnPP, tin protoporphyrin IX.

Effects of caffeic acid phenethyl ester on P. intermedia lipopolysaccharide-induced nuclear factor-jB transcriptional activity

We next investigated whether the inhibitory effects of CAPE on P. intermedia LPS-induced production of NO,

IL-1b and IL-6 are associated with the NF-jB signaling pathway. To determine the effect of CAPE on the degradation of IjB-a induced by P. intermedia LPS, we measured the cytoplasmic levels of IjB-a protein. Immunoblot analysis showed that CAPE failed to prevent LPS from

Next, we tested the hypothesis that CAPE downregulates P. intermedia LPS-induced production of NO, IL-1b and IL-6 via inhibition of STAT signaling cascade. CAPE showed strong dose-dependent inhibitory effects on LPS-induced STAT1 and STAT3 phosphorylation in RAW264.7 cells (Fig. 7). Effect of caffeic acid phenethyl ester on SOCS1 expression in cells activated by P. intermedia lipopolysaccharide

We also examined the possibility that CAPE would modify the SOCS signaling that is involved in cytokine signaling as negative regulators to inhibit proinflammatory cytokine production. Real-time PCR analysis revealed that CAPE dose-dependently upregulated the mRNA level of SOCS1 in RAW264.7 cells treated with P. intermedia LPS (Fig. 8). By contrast, treatment with CAPE did

Effect of caffeic acid phenethyl ester on periodontal disease

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2007625).

Figure S1 Involvement of p38, JNK, NF-jB and JAK2/STAT1 pathways in Prevotella intermedia LPS-induced production of IL-1b in RAW264.7 cells. Cells were pretreated with various kinase inhibitors for 1 h or 30 min, and then stimulated with P. intermedia LPS (10 lg/mL) for 48 h. Supernatants were removed and assayed for IL-1b. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone. Figure S2 Involvement of STAT3 pathway in Prevotella intermedia LPSinduced production of NO (A), IL-1b (B) and IL-6 (C) in RAW264.7 cells. Cells were pretreated with STAT3 activation inhibitor (static) for 45 min, and then stimulated with P. intermedia LPS (10 lg/mL) for 24 h (for NO and IL-6) or 48 h (for IL-1b). Supernatants were removed and assayed for NO, IL-1b and IL-6. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone. Figure S3 Effects of SnPP on the CAPE-mediated inhibition of IL-1b (A) and IL-6 (B) in RAW264.7 cells activated by Prevotella intermedia LPS. Cells were treated with CAPE and P. intermedia LPS (10 lg/mL) for 24 h (for IL-6) or 48 h (for IL-1b) in the presence of different doses of SnPP, after which culture supernatants were assayed for IL-1b and IL6. Data are presented as percentage of P. intermedia LPS alone. The results are means  SD of three independent experiments. **p < 0.01 vs. P. intermedia LPS alone; ††p < 0.01 vs. P. intermedia LPS plus CAPE. Figure S4 Time course of SOCS2 (A) and SOCS3 (B) mRNA expression in RAW264.7 cells stimulated with Prevotella intermedia LPS (10 lg/mL) in the absence or presence of CAPE (10 lM). Real-time PCR was performed with EvaGreen Supermix, b-actin being used as an endogenous control. The results are means  SD of three independent experiments.

Supporting Information

References

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B

Fig. 7. Effects of CAPE on Prevotella intermedia LPS-induced phosphorylation of STAT1 (A) and STAT3 (B) in RAW264.7 cells. Cells were incubated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 4 h. Phosphorylation of STAT1 and STAT3 was measured by immunoblot analysis of cell lysates. A representative immunoblot from two separate experiments with similar results is shown. CAPE, caffeic acid phenethyl ester; LPS, lipolysaccharide.

Fig. 8. Effect of CAPE on SOCS1 expression in RAW264.7 cells activated by Prevotella intermedia LPS. Cells were treated with different doses of CAPE in the absence or presence of P. intermedia LPS (10 lg/mL) for 0.5 h. Real-time polymerase chain reaction was performed with EvaGreen Supermix, b-actin being used as an endogenous control. Data are presented as a percentage of P. intermedia LPS alone. The results are means  SD of three independent experiments. *p < 0.05 vs. P. intermedia LPS alone; **p < 0.01 vs. P. intermedia LPS alone.

modulation of the host response associated with periodontal disease. Overall, our study suggests that CAPE inhibits P. intermedia LPSinduced production of NO, IL-1b and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and inhibition of NF-jB, STAT1 and STAT3 activation, which is related to the activation of SOCS1 signaling. Modulation of host response by CAPE may represent an attractive strategy towards the treatment of periodontal disease. In vivo studies are underway to appraise further the potential of CAPE as an immuno-

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modulator in the treatment of periodontal disease.

Acknowledgements

Additional Supporting Information may be found in the online version of this article:

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Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

Caffeic acid phenethyl ester (CAPE) has numerous potentially beneficial properties, including antioxidant, immunomodulatory and anti-inflammatory acti...
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