High-mobility group box 1 suppresses resolvin D1-induced phagocytosis via induction of resolvin D1-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase

Biochimica et Biophysica Acta 1852 (2015) 1981–1988

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High-mobility group box 1 suppresses resolvin D1-induced phagocytosis via induction of resolvin D1-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase Gyeoung-Jin Kang a, Hye-Ja Lee a, Yun Pyo Kang b, Eun Ji Kim a, Hyun Ji Kim a, Hyun Jung Byun a, Mi Kyung Park a, Hoon Cho c, Sung Won Kwon b, Chang-Hoon Lee a,⁎ a b c

BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea Department of Polymer Science & Engineering, Chosun University, Gwangju 501-759, Republic of Korea

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Article history: Received 25 November 2014 Received in revised form 14 June 2015 Accepted 8 July 2015 Available online 11 July 2015 Keywords: Resolution of inflammation Phagocytosis HMGB1 Resolvin D1 15-PGDH

a b s t r a c t High-mobility group box 1 (HMGB1) enhances inflammatory reactions by potentiating the activity of proinflammatory mediators and suppressing the phagocytosis of apoptotic neutrophils. However, the effects of HMGB1 on phagocytosis induced by pro-resolving mediators, such as resolvins, have not been studied up until this point. In this study, we investigated the effects and underlying mechanism of HMGB1 on resolvin D1-induced phagocytosis of MDA-MB-231 cells, which were selected as a model system based on their phagocytic capability and ease of transfecting them with a plasmid or siRNA in several cancer cell lines. Then we confirmed effects of HMGB1 in THP-1 cells. Resolvin D1 (RvD1) enhanced phagocytosis in MDA-MB-231 and THP-1 cells. HMGB1 suppressed RvD1-induced phagocytosis in MDA-MB.231 and THP-1 cells. HMGB1 dose-dependently induced the expression of 15hydroxyprostaglandin dehydrogenase (15-PGDH), the inactivating enzyme in pro-resolving lipid mediators such as RvE1 and RvD1. Involvement of 15-PGDH in-HMGB-1-induced suppression of phagocytosis was examined using siRNA of 15-PGDH or 15-PGDH inhibitor, TD23. Surprisingly, the silencing of 15-PGDH increased phagocytotic activity of MDA-MB-231 cells. TD23 also enhanced phagocytosis of MDA-MB-231 and THP-1 cells. In conclusion, the release of HMGB1 during the inflammatory phase induces 15-PGDH expression, which suppresses the phagocytotic activity of macrophages. These processes might be involved in the mechanism that blocks the resolution of inflammation, thereby allowing acute inflammation to progress to chronic inflammation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Inflammation is an essential protective mechanism that removes harmful stimuli, as microorganisms and damaged tissues [1]. Successful clearance of infectious microbes and damaged tissue followed by the disappearance of the inflammatory exudate is a process known as resolution [2]. However, nonresolving inflammation is a major driver of severe diseases, including cancer and autoimmune diseases [3]. In order to achieve normal resolution of inflammation, the body employs a substantial number of lipid mediators (LMs), which are derived from both ω-6 polyunsaturated fatty acid and the ω-3 polyunsaturated fatty acids (PUFAs) EPA and DHA, including lipoxins (LXs), prostaglandins (PGs), Protectins (Ps), resolvins (Rvs), to name a few [4]. These mediators display potent anti-inflammatory, pro-resolving, immunoregulatory and neuroprotective properties through multiple ⁎ Corresponding author. E-mail address: [email protected] (C.-H. Lee).

http://dx.doi.org/10.1016/j.bbadis.2015.07.005 0925-4439/© 2015 Elsevier B.V. All rights reserved.

actions, such as arresting the recruitment of polymorphonuclear leukocytes (PMN), blocking leukotrienes (LTs) and PGs, and reducing cytokine release and the recruitment of monocytes [5,6]. Phagocytic removal of apoptotic leukocytes is a prerequisite for the restoration of normal tissue function, and plays a critical role in the resolution of inflammation [2,7,8]. In this process, Rvs, for example, promote resolving phagocytosis [5]. RvE1 and PD1 in nanogram ranges promote phagocyte removal during acute inflammation, via regulation of leukocyte infiltration, increasing macrophage ingestion of apoptotic PMNs in vivo and in vitro, and enhancing the appearance of phagocytes carrying engulfed zymosan in lymph nodes and spleen [9]. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors [10]. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G protein-coupled receptors such as ChemR23 [11]. High-mobility group box 1 (HMGB1) is a nuclear non-histone DNA-binding protein that binds to specific DNA targets. HMGB1 is

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secreted by activated macrophages, NK cells, dendritic cells, as well as necrotic and apoptotic cells in response to infection, injury, and inflammation [12–15]. HMGB1 was released by cultured macrophages more than 8 h after stimulation with endotoxin, TNF-α, or IL-1 and mice showed increased serum levels of HMGB-1 between 8–32 h after endotoxin exposure [12]. Macrophages that ingest apoptotic cells begin to secret HMGB1 [16]. Unlike in sepsis, HMGB1 levels are increased as early as 1 h after ischemia/reperfusion injury and then increase in a time-dependent manner for up to 24 h [17]. 15-PGDH is an enzyme degrading several lipid mediators such as PGE1, LxA4, RvD1, and RvE1 [18–21]. 15-PGDH decrease was reported in lung, gastric, thyroid cancers [22–24]. Reduced expression of 15PGDH is observed in the systemic inflammatory response and inflammatory bowel disease [25,26]. The time course of 15-PGDH induction by phorbol 12-myristate 13-acetate (PMA) was reported in U937 cells. Maximum induction was observed at 24–36 h following the addition of PMA [27]. This induction by PMA was inhibited by the concurrent addition of dexamethasone [27]. We are interested in the reports that chronic inflammation might be due to failure of resolution and antiresolution factor might be existed to interfere the resolution response [28,29]. We speculated that antiresolution factor might be delayed expression pattern in inflammation and suppress efferocytosis [12,30]. Many recent reports have indicated that HMGB1 enhances and maintains inflammatory reactions by potentiating the activity of pro-inflammatory mediators such as LPS and cytokines, and by suppressing the phagocytosis of apoptotic neutrophils [30–32]. However, no studies up to this point have focused on understanding the relationship between HMGB1 and pro-resolving mediators such as Rvs. We speculated that HMGB1 might block the action of proresolving mediators. In this study, we examine the effects and underlying mechanism of HMGB1 on RvD1-induced phagocytosis in MDA-MB-231 cells which were selected as model system based on their phagocytic capability and ease of transfecting them with a plasmid or siRNA in several cancer cell lines [33–35]. We also confirmed the effects of HMGB1 on phagocytosis of THP-1 cells. We found that HMGB1 suppresses RvD1induced phagocytosis via induction of the RvD1 inactivation enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-PGDH is important in suppression of phagocytosis. 2. Materials and Methods 2.1. Chemical and reagents RPMI-1640 medium, high-glucose Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and antibiotics (penicillin and streptomycin) were purchased from Welgene Inc. (Korea). Resolvin D1 was gained from Cayman Chemical (Ann Arbor, MI). Recombinant protein HMGB1 and 15PGDH were obtained from R&D Systems (Minneapolis, MN). RvD1 was acquired from Cayman Chemical (Ann Arbor, MI). Anti-HMGB1 antibody was obtained from Cell Signaling (Beverly, MA), anti-15-PGDH antibody from Abcam (Cambridge, MA), and β-actin antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-labeled zymosan was purchased from Invitrogen (Carlsbad, CA). All of the other chemicals were of reagent grade. 2.2. Cell culture The human cell lines THP-1, HT1080, and Jurkat human cell lines were cultured in RPMI1640 medium and MDA-MB-231, MCF7, and HaCaT human cell lines were high glucose DMEM. All cell lines were cultured with 10% FBS and 1% antibiotics in a humidified CO2 incubator. Human THP-1 cells differentiated into macrophage-like cells by treatment with TPA (12-O-tetradecanoylphorbol-13-acetate).

2.3. RNA preparation and polymerase chain reaction (PCR) Total RNA was prepared using TRIzol® RNA Isolation Reagents (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed with a first strand cDNA synthesis kit (Promega, Madison, WI). The reverse transcription PCR reaction was performed in the 96-Well GeneAmp® PCR System 9700 (Applied Biosystems, Piscataway, NJ) using AccuPower® HotStart PCR PreMix (Bioneer, Korea) with the appropriate sense and antisense primers. The primer sequence was as follows: 15PGDH (F 5′-CCAATTTTGCCACAGCCACA-3′, R 5′-CTTCAAGCTGGGAGGT CTGG-3′; 274 bp), HMGB1 (F 5′-AGCACCCAGATGCTTCAGTC-3′, R 5′CCTCTTGGGTGCATTGGGAT-3′; 203 bp), and β-actin (F 5′-TGAAGCTGAGGGAGCCACAGC-3′, R 5′-GGGTTCTCCCTGGGCACCAA-3′; 540 bp). The reaction products were visualized by electrophoresis on a 1.2% agarose gel (Invitrogen) under UV light illumination after staining with SafePinky DNA Gel staining solution (GenDEPOT, Inc., Barker, TX). Real-time quantitative PCR was performed with an iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA) with a CFX384 Real-Time PCR (Bio-Rad). Real-time PCR for the relative quantification of target gene copy numbers in relation to GAPDH expression was conducted using the following primers; 5′-CAGGTCCGGAAGGCAAAGAT-3′ (forward) and 5′-GGGGAGGAAACTGTC AAGCC-3′ (reverse) for 15-PGDH, 5′CCTCAAGATCATCAGCAATG-3′ (forward) and 5′AGTCCTTCCACGATACCA-3′ (reverse) for GAPDH. Real-time PCR results were expressed using the CFX Manager software (Bio-Rad) that measures amplification of the target and the endogenous control in experimental samples and in a reference sample. Measurements were normalized using the endogenous control. 2.4. Western blot analysis Cells were washed twice with ice-cold PBS and disrupted in RIPA buffer with protease inhibitor cocktail and Xpert Phosphatase Inhibitor Cocktail Solution (GenDEPOT, Inc.) on ice for 30 min. Cell lysates were centrifuged at 15,000 rpm for 15 min at 4 °C, and the resultant supernatants were subjected to Western blotting. The total protein concentration was quantified using the Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). Proteins were separated by electrophoresis on a 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), after which samples were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with 5% skim milk for 1 h and incubated overnight at 4 °C with the appropriate primary antibodies. After TBST washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 90 min at room temperature. The proteins were visualized with PowerOpti-ECL detection reagent (Animal Genetics Inc., Korea) using the ChemiDoc™ XRS+ System (Bio-Rad). Proteins in the cell supernatants were precipitated with MeOH/CHCL3 and resuspended in sample buffer and separated by SDS-PAGE. 2.5. Gene silencing HMGB-1 siRNA (sequence (I): 5′-AAGCACCCAGAUGCUUCAGU-3′, (II):5′-CUGCGAAGCUGAAGGAAAA-3′) and 15-PGDH siRNA (sequence (I): 5′-GCGGCAUCAUUAUCAAUAU-3′, (II): 5′-GCUGGUGCAUUGGAAU CUU-3′) was purchased from Santa Cruz Biotechnology and ST Pharm (Korea). Cells were transfected with siRNA using Lipofectamine™ 2000 Transfection reagent (Invitrogen) following the manufacturer's protocol. The ratio of siRNA versus Lipofectamine reagent was 1:1.15. After 48 h of transfection, the cells were used in further experiments. 2.6. Phagocytosis assay FITC-labeled zymosan A particles were pre-treated with opsonizing reagent (Invitrogen) according to the manufacturer's instructions. To

G.-J. Kang et al. / Biochimica et Biophysica Acta 1852 (2015) 1981–1988

induce phagocytosis of the zymosan particles, MDA-MB-231 cells were pre-exposed to RvD1 for 10 min at 37 °C, and stimulated with HMGB1. After 30 min of stimulation, FITC-zymosan particles were added and incubated for 24 h. THP-1 cells were differentiated to human macrophagelike cells by treatment of TPA (20 nM) for 48 h. And then cells were pre-exposed to RvD1 for 10 min at 37 °C, and the cells were additionally incubated with FITC-zymosan particles for 30 min in a presence of RvD1. Also, the cells were pre-stimulated with HMGB1 for 24 h and then exposed to RvD1 and FITC-zymosan particles. After washing with PBS, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min. To determine the number of fluorescent-green particles, counts were conducted in four randomly chosen fields under ×10 magnification (Leica, Germany) or flow cytometric analysis (FACSAria™ III, BD Biosciences, San Jose, CA). FACSDiva software 6.1.3 used for data analysis. In MDAMB-231 cells, Number of fluorescent particles was counted the fluorescent particles in the image of fluorescence microscope. In THP-1 macrophages, FITC-positive cells were calculated as the total number of cells with at least two beads as a percentage of the total number of cells counted from the image of fluorescence microscope. 2.7. Enzyme-linked immunosorbent assay The production of TNF-α, IL-6, and TGF-β proteins in the supernatant of the cultured cells was measured using ELISA kits (R&D Systems Inc.) according to the manufacturer's instructions. 2.8. Enzyme assay Substrate mixture containing RvD1 (50 μM) and NAD+ (1 mM, Sigma) in 100 μL assay buffer (50 mM Tris, 0.1 mM Dithiothreitol (DTT), pH 7.5) was incubated with recombinant 15-PGDH (0.1 μg) or HMGB1 (10 or 100 ng, 30 min)-pretreated 15-PGDH. 15-PGDH activity was monitored spectrophotometrically by the formation of NADH from NAD+ for 45 min at 37 °C (340 nm, SpectraMax M3, Molecular Devices, Sunnyvale, CA). 2.9. Identification of Oxo-RvD1 by LC-MS The sample preparation for analysis of RvD1 and RvD1 metabolites were described in previous studies [18,36]. Briefly, after reaction terminated with addition of 200 μL of cold MeOH, the sample mixtures were centrifuged with 16,000 g at 4 °C in 15 min. After addition of 1 mL water to the supernatant and pH adjustment to 3.5 by 1 M HCl, the acidified samples were extracted and purified with C18 based solid phase extraction (SPE) method [36]. The final product was analyzed by HPLC-PDAMS/MS system. Liquid chromatography were performed using Agilent 1290 UPLC system equipped with C18 based Brownlee SPP column (2.7 μm, 2.1 mm × 75 mm, PerkinElmer, Branchburg, NJ, USA). The isocratic mobile phase (methanol:water:acetic acid, 65:35:0.01, v/v/v) was eluted at a 0.2 ml/min flow rate and UV spectrum was acquired by 1290 Infinity Diode Array Detector (Agilent, CA, USA) before MS/ MS analysis. The MS/MS analysis were carried out using a 6460 triple quadruple MS/MS system (Agilent, CA, USA) at negative mode. For MS/MS scan, 100 to 400 m/z was set as scan rage and the 110 and 120 of fragment voltages were used for fragment ion scan of the 373 and 375 m/z, respectively. For multiple reaction monitoring (MRM), 10 and 13 eV were used for monitoring of 231 m/z from 373 m/z of parent ions (oxo-RvD1s) and 233 m/z for 375 m/z of parent ions (RvD1), respectively. 2.10. Statistical analysis The student's t-test was used to determine the statistical significance of the differences between the experimental and control group values. The data presented represent the mean ± standard deviation.

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3. Results 3.1. RvD1 enhanced phagocytosis of MDA-MB-231 cells Usually, cells of hematopoietic origin, such as THP-1 cells or primary macrophage cells, were used to evaluate the phagocytic capability but transfecting them with a plasmid or siRNA was difficult. So, we selected MDA-MB-231 cancer cells since several researchers reported that these cells have phagocytic capability [37,38]. We first confirmed the phagocytic activity of MDA-MB-231 cells and examined the effects of RvD1 on the phagocytic activity of MDA-MB-231 cancer cells (Figs. 1, 1S). We found that RvD1 enhanced phagocytosis of MDA-MB-231 in time dependent manner (Fig. 1B). We also confirmed that RvD1 dose dependently enhanced phagocytosis of THP-1 cells (Fig. 1C). RvD1 enhanced phagocytosis of raw264.7 and murine peritoneal macrophages (Fig. 2S). 3.2. HMGB1 suppressed the RvD1-enhanced phagocytosis HMGB1 is known to suppress the phagocytosis of several cells, including macrophages and neutrophils [30–32]. So we examined whether HMGB1 suppressed the RvD1-induced phagocytosis of MDAMB-231 cells. HMGB1 suppressed the phagocytosis of MDA-MB-231 cells in the presence and absence of RvD1 (Fig. 2A). HMGB1 also suppressed the RvD1-enhanced phagocytosis of THP-1 cells (Fig. 2B). We also examined the effects of HMGB1 on the inflammatory cytokines such as TNF-α, and IL-6 and anti-inflammatory cytokine, TGF-β1 in MDA-MB-231 cancer cells and THP-1 cells by ELISA. In THP-1 cells, HMGB-1 induced expression of TNF-α and IL-6 (Fig. 3S). However, TNF-α and IL-6 were not detected in MDA-MB-231 cells (data not shown). HMGB-1 reduced expression of TGF-β1 in THP-1 and MDAMB-231 cells (Fig. 3S). 3.3. HMGB1 induced the expression of 15-PGDH HMGB1 treatment suppressed RvD1-induced phagocytosis and gene silencing of HMGB1 induced phagocytosis and that led us to investigate the mechanism of HMGB1's suppression of RvD1-induced phagocytosis of MDA-MB-231 cells. We initially speculated that HMGB1 might inactivate RvD1. Therefore, we examined the effects of HMGB1 on the RvD1-inactivating enzyme, 15-PGDH. HMGB1 dose-dependently induced 15-PGDH mRNA expression (Fig. 3A). After treating cells with HMGB1 for 24 h, the level of 15-PGDH was examined by Western blot analysis. HMGB1 induced 15-PDGH protein expression in both MDAMB-231 and THP-1 cells (Fig. 3B). HMGB-1 induced 15-PGDH expression is also observed in various cells (Fig. 4S). Gene silencing of HMGB1 reduced 15-PGDH expression at the mRNA (Fig. 3C) and protein levels (Fig. 3D). We also confirmed that HMGB-1 increased the activity of 15-PGDH by measuring the NADH produced in enzyme reaction (Fig. 5S). RvD1-derived oxoRvD1 was confirmed in assay mixtures containing RvD1, NAD+, and cell lysates from HMGB1-treated MDAMB-231 cells (Fig. 6S). These results indicate that HMGB1 induces expression and activity of 15-PGDH. 3.4. Involvement of 15-PGDH in phagocytosis of MDA-MB-231 cells To investigate 15-PGDH's involvement in HMGB-1-suppressed phagocytosis, we examined the effects of 15-PGDH gene silencing on HMGB1-induced phagocytosis of MDA-MB-231 cells since THP-1 cells have low efficiency of transfection and require additional differentiation step induced by TPA. Gene silencing of 15-PGDH is confirmed by Western blot (Fig. 4A). HMGB1 in control-siRNA-transfected cells suppressed RvD1-induced phagocytosis, whereas gene silencing of 15-PGDH increased phagocytosis, even without HMGB1 and RvD1 treatment (Fig. 4B, C). So, we also confirmed the effects of 15-PGDH on phagocytosis of MDA-MB-231 cells and THP-1 cells using TD23,

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(A)

MDA-MB-231 Zymosan-FITC RvD1 (10 nM)

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Zymosan-FITC only

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**

40 30 20

10 0 RvD1 (10 nM)

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MDA-MB-231

TPA-induced human macrophage-like THP-1 cells *

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RvD1 10 nM

80

*

60

**

40 20

FITC-positive cells (%)

Number of fluorescent particles

Fluorescence microscopy

0

20.0

*** **

15.0 10.0 5.0 0.0

1

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12

24

RvD1

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1

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10

20

(nM)

Time (hr)

RvD1 10 min pre-treated Fig. 1. RvD1 induces phagocytic activity. A. Effects of RvD1 on phagocytosis in MDA-MB-231 cancer cells. B. Time dependent effects of RvD1 on phagocytosis in MDA-MB-231 cancer cells. C. Dose-dependent effects of RvD1 on phagocytosis of TPA-human macrophage-like THP-1 cells. In (A) and (B), human MDA-MB-231 metastatic breast cancer cells were pre-treated with RvD1 in serum free conditions for 10 min. The cells were incubated with zymosan particles conjugated with FITC for 24 h (A) or indicated time (B). In (C) THP-1 macrophage-like cells were pre-treated with RvD1 in a dose-dependent manner (for 10 min). And then the cells were incubated with zymosan particles conjugated with FITC (4.0 × 104 particles/μL) for 30 min. The cells were fixed and observed by fluorescence microscopy. Engulfed zymosan particles were counted from four random fields under magnification. Values are mean ± SD (n = 4). *, p b 0.05; **, p b 0.01; ***, p b 0.001.

a well-known 15-PGDH inhibitor [39]. TD23 increased phagocytosis (Fig. 4D). These results suggest that HMGB1 inhibits phagocytosis of MDAMB-231 cells through the regulation of 15-PGDH. 15-PGDH itself is important in regulation of phagocytosis. 4. Discussion In this study, we showed that HMGB1 inhibits RvD1-induced phagocytosis via the induction of 15-PGDH enzyme expression, which inactivates PGE2, LxA4, RvD1 and RvE1 [18–21]. RvD1 is one of wellcharacterized pro-resolving lipid mediators that induces efferocytosis of macrophage during the resolution stage of inflammation [5,40,41]. It is known that pro-resolving lipid mediators, including RvD1, begin to be synthesized in the early phase of inflammation, although they did not induce resolution such as efferocytosis in the early phase [7]. Recent reports have revealed that HMGB1 plays an important role in maintaining inflammation. It can be actively released from various immune cells such as macrophages, monocytes, NK cells, dendritic cells, and endothelial cells, as well as from necrotic cells [42–44]. The most familiar HMGB1 mechanism for inflammation is the induction of inflammatory cytokine release [45–47]. Recent studies have shown that extra- or intracellular HMGB1 can inhibit the efferocytosis of macrophages, which is an important process of inflammation resolution [30–32].

We hypothesized that some players that have an important role in the maintenance of inflammation, and might act as antiresolution factor and inhibit the efferocytosis induced by pro-resolving lipid mediators such as RvD1 [28–30]. HMGB1 is the best candidate since it suppressed efferocytosis [30,31]. Therefore, we examined the effects of HMGB1 on RvD1-enhanced phagocytosis of MDA-MB231 cancer cells which were chosen as a model phagocytic cell system because they were easy to handle and transfect. MDA-MB-231 cancer cells are known to have phagocytic activity [33–35], and RvD1 enhanced the phagocytosis of MDA-MB-231 cells (Figs. 1, 1S). This suggested that MDA-MB-231 cancer cells might be a good model system for evaluating the activity of the pro-resolving lipid. In addition, we also confirmed effects of RvD1 on phagocytosis of THP-1 cells and other cells such as raw264.7 and peritoneal macrophages from mice (Figs. 1C, 2S). In healthy animals and normal human subjects, HMGB1 is present at an undetectable plasma level of – 5 ng/ml, but HMB1 increase to an average of 25.2 and 83.7 ng/ml in survivors and non-survivors among septic patients, respectively [12]. The plasma HMGB1 median concentration on the day of admission increases to 190 ng/ml in patients with community-acquired pneumonia [48]. Analysis of synovial fluid samples from rheumatoid arthritis patients further confirmed the extracellular presence of HMGB1; 14 of 15 samples had HMGB-1 concentrations of 1.8–10.4 μg/ml [49]. We use 3.0 μg/ml HMGB1 according to protocols of Liu et al., [30].

G.-J. Kang et al. / Biochimica et Biophysica Acta 1852 (2015) 1981–1988

(A)

MDA-MB-231 P=0.005 (**)

Number of fluorescent particles

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P=0.011 (*)

P=0.009 (**)

40

P=0.036 (*)

30 20 10 0 RvD1

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TPA-induced human macrophage-like THP-1 cells FITC-positive cells (%)

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Fig. 2. HMGB1 reduces the phagocytic activity. A. Effect of HMGB1 on RvD1-induced phagocytosis in MDA-MB-231 cells. Human MDA-MB-231 breast cancer cells were pretreated with RvD1 under serum free conditions for 10 min. The cells were incubated with zymosan particles conjugated with FITC in a presence of HMGB1 for 24 h. B. Effect of HMGB1 on RvD1-induced phagocytosis in THP-1 cells. TPA-induced human THP-1 macrophage-like cells were stimulated with HMGB1 for 24 h. And then the cells were treated with RvD1 for 10 min and additionally incubated with zymosan particles conjugated with FITC for 30 min. The method of zymosan uptake assay is the same that described above. Values are mean ± SD (n = 4). *, p b 0.05; **, p b 0.01; ***, p b 0.001.

HMGB1 suppressed RvD1-enhanced phagocytosis of MDA-MB-231 cancer cells and gene silencing of HMGB1 restored the phagocytic capability of MDA-MB-231 cells (Fig. 2). This original finding suggests that HMGB1 might block the action of RvD1, leading to the maintenance of inflammation. In accordance with our results, HMGB1 played an important role in inflammation [50]. HMGB1 consists of three cysteine residues: Cys23, Cys45, and Cys106 [51]. Especially, Cys23 and Cys45 in the A box undergo a conformational change in response to oxidation stress [51]. Recombinant HMGB1 used, was shown to induce the inflammatory cytokines in THP1 macrophages (Fig. 3S). This result indicated that the HMGB1 might have been disulfide form [51]. Therefore, our HMGB1 was regarded as disulfide form. HMGB1 suppresses phagocytosis by binding to the RAGE. The amino acid residues responsible for RAGE binding are 150–183 residues in HMGB1 [51]. So phagocytosis seems to be independent of redox state of cysteine residues of HMGB1 since DTT-treated and non-treated HMGB1s did not showed the different activities in phagocytosis (Fig. 7S). Exactly how HMGB1 suppresses phagocytosis is still not fully understood. The effects of HMGB1 on 15-PGDH, which inactivates pro-

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resolving mediators such as PGE2, LxA4, RvE1, and RvD1, have not been studied at all [18,19,23]. We demonstrated that HMGB1 induced 15-PGDH, the enzyme catalyzing the oxidation and inactivation of RvD1 (Fig. 3) [18]. HMGB1-induced expression of 15-PGDH is also confirmed in other cell lines including HaCat, Jurkat, HT1080, and MCF7 cells (Fig. 4S). It is likely that HMGB1 suppresses phagocytosis induced by RvD1 by increasing the expression and activity of 15-PGDH (Figs. 3, 5S). We also confirmed the oxo-RvD1 in assay mixture of 15-PGDH containing RvD1, NAD, and cell lysates from HMGB-1-treated MDAMB-231 cells (Fig. 6S). The time course of 15-PGDH induction by phorbol 12-myristate 13-acetate (PMA) was reported in U937 cells. Maximum induction was observed at 24–36 h following the addition of PMA [27]. 15-PGDH decrease has been reported in lung, gastric, thyroid cancers [22–24]. Reduced expression of 15-PGDH is observed in the systemic inflammatory response and inflammatory bowel disease [25,26] To confirm the involvement of 15-PGDH in phagocytosis of MDAMB-231 cells, we silenced the 15-PGDH gene, which increased phagocytic activity even without RvD1 treatment (Fig. 4C). TD23, a 15-PGDH inhibitor, also increased phagocytosis (Fig. 4D) [39]. These results suggest that 15-PGDH is a key player in phagocytosis. Therefore, inhibition of 15-PGDH might be useful to induce the resolution phase by inhibiting the inactivation of LxA4, RvD1 or RvE1. It is still not clear why 15-PGDH gene silencing or inhibitor increased phagocytosis. If we focus on the substrate of 15-PGDH, we might explain the role of 15-PGDH in phagocytosis. 15-PGDH inactivated the PGE2, LxA4, RvE1, and RvD1. PGE2 impairs, whereas Lxs, and Rvs, enhances phagocytosis [40,52–54]. 15-PGDH gene silencing might increase PGE2, LxA4, RvD1, and RvE1, which are substrates of this enzyme. Interestingly, PGE2 suppresses phagocytosis, and LxA4, RvD1, and RvE1 increase phagocytosis. So, one possible explanation is that 15-PGDH-gene-silencing-induced increases of phagocytosis are furthered by increased amounts of LxA4, RvD1, and RvE1. Interestingly, RvE1 is a stronger inducer of phagocytosis than RvD1. Thus, it is possible that 15-PGDH-gene-silencing-induced increases of LxA4, RvD1, and RvE1 might evoke phagocytosis. However, these speculations need to be proven by experimentation. To this end, we are now, as part of a new project, analyzing metabolites under the 15-PGDH gene silencing condition. In a study by Murakami et al., they found that serum HMGB1 levels were markedly elevated 5 h after the LPS/N-galactosamine-challenge in mice, and it were significantly reduced by RvD1 [55]. Contrastingly, in our results, HMGB1 suppressed the RvD1-induced phagocytosis by enhancing expression of 15-PGDH, which inactivates RvD1 [18]. So, these two results reflect the fact that in the course of inflammation, either ongoing inflammation or resolution might be determined by combat between HMGB1 and Rvs such as RvD1. In summary, HMGB1 suppresses RvD1-induced phagocytosis via the expression of 15-PGDH. These mechanisms might explain how HMGB1 maintains the inflammatory phase, and demonstrates that 15-PGDH might be an important target for inducing the resolution of inflammation. Transparency Document The transparency document associated with this article can be found, in the online version. Conflict of Interest Nothing to declare Acknowledgments This study was supported by grants from the National Research Foundation grant (No. 2011-0022074), the Korea Healthcare Technology R&D project (No. A101836), and the Basic Science Research Program, through the NRF (NRF-2014R1A2A1A01004016).

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(A)

(C)

MDA-MB-231

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3.0 2.0 1.0 0.0

HMGB1

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5 10 20 40 mRNA level (qPCR)

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15-PGDH

274 bp

HMGB1

203 bp

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β-actin

43 kDa

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Fig. 3. HMGB1 induces the expression of 15-PGDH. A. Effects of HMGB-1 on mRNA expression of 15-PGDH in MDA-MB-231 cells. B. Effects of HMGB-1 on protein expression of 15-PGDH in MDA-MB-231 cells. C. Effects of HMGB1 gene silencing on mRNA expression of 15-PGDH analyzed by RT-PCR (top) and qPCR (bottom). D. Effects of HMGB1 gene silencing on protein expression of 15-PGDH in MDA-MB-231 (top) and THP-1 cells (bottom). In (A), human MDA-MB-231 cells were pre-incubated for 18 h and then treated with HMGB1 under serum free conditions for 24 h in dose-dependent manner (5, 10, 20, 40 nM). The mRNA levels of 15-PGDH was determined from the cells by qRT-PCR. In (B), human MDA-MB-231 cells (top) and THP-1 macrophage-like cells (bottom) were treated with HMGB1 (20 nM) under serum free conditions for 24 h. And then, the protein expression was determined from cell lysate by Western blot method. In (C), human MDA-MB-231 cells were transfected with control siRNA or HMGB1 siRNA for 48 h. The mRNA levels of 15-PGDH and HMGB1 were determined from the siRNA transfected cells by RT-PCR or qRT-PCR. In (D), the protein expression of 15-PGDH were identified in HMGB1 siRNA-transfected MDA-MB-231 (top) and THP1 (bottom) cells by western blot method. These data are representative of three independent experiments. Band density was measured relative to β-actin using the Image J program. Values are mean ± SD (n = 3). *, p b 0.05; **, p b 0.01.

G.-J. Kang et al. / Biochimica et Biophysica Acta 1852 (2015) 1981–1988

(B)

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Fig. 4. 15-PGDH is involved in phagocytosis. A. Effects of 15-PGDH siRNA on 15-PGDH expression. B. Effects of 15-PGDH gene silencing on phagocytosis of MDA-MB-231 cells treated with RvD1 and HMGB1. C. Effects of 15-PGDH siRNA on phagocytosis. D. Effects of 15-PGDH inhibitor, TD-23, on the phagocytosis of MDA-MB-231 cells and THP-1 macrophage-like cells with or without RvD1. In (A), human MDA-MB-231 metastatic breast cancer cells were transfected with control siRNA or 15-PGDH siRNA (I) for 48 h. The image was acquired with ×10 objective. In (B) effects of 15-PGDH on RvD1-induced phagocytosis of MDA-MB-231 cells. The cells were treated with RvD1 in the presence of HMGB1 for 24 h. The method of zymosan uptake assay is the same that described above. Values are mean ± SD (n = 4). In (C), MDA-MB-231 cells were transfected with control siRNA or 15-PGDH siRNA (I) or (II) for 48 h. (A) MDA-MB-231 cells were washed with PBS and fixed by 4% PFA. Flow cytometric analysis was performed and FACSDiva software 6.1.3 used for data analysis. In (D), human MDA-MB-231 cells (top) and THP-1 macrophage-like cells (bottom) were pre-treated with RvD1 (10 nM) and/or TD23 (10 μM) under serum free conditions. And then the cells were additionally incubated with zymosan particles conjugated with FITC in the presence of RvD1 and TD23. Cells were washed with PBS and fixed by 4% PFA. Flow cytometric analysis was performed for MDA-MB-231 cells and zymosan uptake assay in THP-1 cells is the same that described above. Values are mean ± SD (n = 4). *, p b 0.05; **, p b 0.01; ***, p b 0.001.

Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbadis.2015.07.005.

References [1] F.R. Cochran, M.B. Finch-Arietta, Regulation of interleukin-1 beta and tumor necrosis factor secretion by the human monocytic leukemia cell line, THP-1, Agents Actions 27 (1989) 271–273.

[2] D.W. Gilroy, T. Lawrence, M. Perretti, A.G. Rossi, Inflammatory resolution: new opportunities for drug discovery, Nat. Rev. Drug Discov. 3 (2004) 401–416. [3] C. Nathan, A. Ding, Nonresolving inflammation, Cell 140 (2010) 871–882. [4] G.L. Bannenberg, Therapeutic applicability of anti-inflammatory and proresolving polyunsaturated fatty acid-derived lipid mediators, ScientificWorldJournal 10 (2010) 676–712. [5] C.H. Lee, Resolvins as new fascinating drug candidates for inflammatory diseases, Arch. Pharm. Res. 35 (2012) 3–7. [6] J.M. Schwab, C.N. Serhan, Lipoxins and new lipid mediators in the resolution of inflammation, Curr. Opin. Pharmacol. 6 (2006) 414–420. [7] T. Lawrence, D.A. Willoughby, D.W. Gilroy, Anti-inflammatory lipid mediators and insights into the resolution of inflammation, Nat. Rev. Immunol. 2 (2002) 787–795.

1988

G.-J. Kang et al. / Biochimica et Biophysica Acta 1852 (2015) 1981–1988

[8] P. Maderna, C. Godson, Phagocytosis of apoptotic cells and the resolution of inflammation, Biochim. Biophys. Acta 1639 (2003) 141–151. [9] J.M. Schwab, N. Chiang, M. Arita, C.N. Serhan, Resolvin E1 and protectin D1 activate inflammation-resolution programmes, Nature 447 (2007) 869–874. [10] S. Krishnamoorthy, A. Recchiuti, N. Chiang, S. Yacoubian, C.H. Lee, R. Yang, N.A. Petasis, C.N. Serhan, Resolvin D1 binds human phagocytes with evidence for proresolving receptors, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 1660–1665. [11] T. Ohira, M. Arita, K. Omori, A. Recchiuti, T.E. Van Dyke, C.N. Serhan, Resolvin E1 receptor activation signals phosphorylation and phagocytosis, J. Biol. Chem. 285 (2010) 3451–3461. [12] H. Wang, O. Bloom, M. Zhang, J.M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, K.R. Manogue, E. Faist, E. Abraham, J. Andersson, U. Andersson, P.E. Molina, N.N. Abumrad, A. Sama, K.J. Tracey, HMG-1 as a late mediator of endotoxin lethality in mice, Science 285 (1999) 248–251. [13] B. Degryse, T. Bonaldi, P. Scaffidi, S. Muller, M. Resnati, F. Sanvito, G. Arrigoni, M.E. Bianchi, The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells, J. Cell Biol. 152 (2001) 1197–1206. [14] S. Muller, P. Scaffidi, B. Degryse, T. Bonaldi, L. Ronfani, A. Agresti, M. Beltrame, M.E. Bianchi, New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal, EMBO J. 20 (2001) 4337–4340. [15] L. Falciola, F. Spada, S. Calogero, G. Langst, R. Voit, I. Grummt, M.E. Bianchi, High mobility group 1 protein is not stably associated with the chromosomes of somatic cells, J. Cell Biol. 137 (1997) 19–26. [16] S. Qin, H. Wang, R. Yuan, H. Li, M. Ochani, K. Ochani, M. Rosas-Ballina, C.J. Czura, J.M. Huston, E. Miller, X. Lin, B. Sherry, A. Kumar, G. Larosa, W. Newman, K.J. Tracey, H. Yang, Role of HMGB1 in apoptosis-mediated sepsis lethality, J. Exp. Med. 203 (2006) 1637–1642. [17] A. Tsung, R. Sahai, H. Tanaka, A. Nakao, M.P. Fink, M.T. Lotze, H. Yang, J. Li, K.J. Tracey, D.A. Geller, T.R. Billiar, The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia–reperfusion, J. Exp. Med. 201 (2005) 1135–1143. [18] Y.P. Sun, S.F. Oh, J. Uddin, R. Yang, K. Gotlinger, E. Campbell, S.P. Colgan, N.A. Petasis, C.N. Serhan, Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation, J. Biol. Chem. 282 (2007) 9323–9334. [19] M. Arita, S.F. Oh, T. Chonan, S. Hong, S. Elangovan, Y.P. Sun, J. Uddin, N.A. Petasis, C.N. Serhan, Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions, J. Biol. Chem. 281 (2006) 22847–22854. [20] C.B. Clish, B.D. Levy, N. Chiang, H.H. Tai, C.N. Serhan, Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/ leukotriene B4 12-hydroxydehydrogenase in inflammation, J. Biol. Chem. 275 (2000) 25372–25380. [21] H.H. Tai, C.M. Ensor, M. Tong, H. Zhou, F. Yan, Prostaglandin catabolizing enzymes, Prostaglandins Other Lipid Mediat. 68–69 (2002) 483–493. [22] M.G. Backlund, J.R. Mann, V.R. Holla, F.G. Buchanan, H.H. Tai, E.S. Musiek, G.L. Milne, S. Katkuri, R.N. DuBois, 15-hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer, J. Biol. Chem. 280 (2005) 3217–3223. [23] H.H. Tai, M. Tong, Y. Ding, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and lung cancer, Prostaglandins Other Lipid Mediat. 83 (2007) 203–208. [24] V. Quidville, N. Segond, S. Lausson, M. Frenkian, R. Cohen, A. Jullienne, 15hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line, Prostaglandins Other Lipid Mediat. 81 (2006) 14–30. [25] T. Otani, K. Yamaguchi, E. Scherl, B. Du, H.H. Tai, M. Greifer, L. Petrovic, T. Daikoku, S.K. Dey, K. Subbaramaiah, A.J. Dannenberg, Levels of NAD(+)-dependent 15hydroxyprostaglandin dehydrogenase are reduced in inflammatory bowel disease: evidence for involvement of TNF-alpha, Am. J. Physiol. Gastrointest. Liver Physiol. 290 (2006) G361–G368. [26] A.I. Ivanov, A.A. Romanovsky, Prostaglandin E2 as a mediator of fever: synthesis and catabolism, Front. Biosci. 9 (2004) 1977–1993. [27] M. Tong, H.H. Tai, Dexamethasone inhibits the induction of NAD(+)-dependent 15hydroxyprostaglandin dehydrogenase by phorbol ester in human promonocytic U937 cells, Biochim. Biophys. Acta 1497 (2000) 61–68. [28] T. Lawrence, D.W. Gilroy, Chronic inflammation: a failure of resolution? Int. J. Exp. Pathol. 88 (2007) 85–94. [29] D.L. Gilroy, T. Lawrence, The Resolution of Acute Inflammation: A ‘Tipping Point’ in the Development of Chronic Inflammatory Diseases, Birkhauser Basel, Boston, Berlin, 2007. [30] G. Liu, J. Wang, Y.J. Park, Y. Tsuruta, E.F. Lorne, X. Zhao, E. Abraham, High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine, J. Immunol. 181 (2008) 4240–4246. [31] S. Banerjee, A. de Freitas, A. Friggeri, J.W. Zmijewski, G. Liu, E. Abraham, Intracellular HMGB1 negatively regulates efferocytosis, J. Immunol. 187 (2011) 4686–4694.

[32] A. Friggeri, Y. Yang, S. Banerjee, Y.J. Park, G. Liu, E. Abraham, HMGB1 inhibits macrophage activity in efferocytosis through binding to the alphavbeta3-integrin, Am. J. Physiol. Cell Physiol. 299 (2010) C1267–C1276. [33] M. Ghoneum, S. Gollapudi, Phagocytosis of Candida albicans by metastatic and nonmetastatic human breast cancer cell lines in vitro, Cancer Detect. Prev. 28 (2004) 17–26. [34] P. Montcourrier, P.H. Mangeat, C. Valembois, G. Salazar, A. Sahuquet, C. Duperray, H. Rochefort, Characterization of very acidic phagosomes in breast cancer cells and their association with invasion, J. Cell Sci. 107 (Pt 9) (1994) 2381–2391. [35] P. Montcourrier, P.H. Mangeat, G. Salazar, M. Morisset, A. Sahuquet, H. Rochefort, Cathepsin D in breast cancer cells can digest extracellular matrix in large acidic vesicles, Cancer Res. 50 (1990) 6045–6054. [36] Y. Lu, S. Hong, K. Gotlinger, C.N. Serhan, Lipid mediator informatics and proteomics in inflammation resolution, ScientificWorldJournal 6 (2006) 589–614. [37] P.J. Coopman, D.M. Thomas, K.R. Gehlsen, S.C. Mueller, Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells, Mol. Biol. Cell 7 (1996) 1789–1804. [38] P.J. Coopman, M.T. Do, E.W. Thompson, S.C. Mueller, Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity, Clin. Cancer Res. 4 (1998) 507–515. [39] Y. Wu, H.H. Tai, H. Cho, Synthesis and SAR of thiazolidinedione derivatives as 15PGDH inhibitors, Bioorg. Med. Chem. 18 (2010) 1428–1433. [40] H.N. Lee, J.K. Kundu, Y.N. Cha, Y.J. Surh, Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-alpha expression, J. Cell Sci. 126 (2013) 4037–4047. [41] H.M. Hsiao, R.E. Sapinoro, T.H. Thatcher, A. Croasdell, E.P. Levy, R.A. Fulton, K.C. Olsen, S.J. Pollock, C.N. Serhan, R.P. Phipps, P.J. Sime, A novel anti-inflammatory and pro-resolving role for resolvin D1 in acute cigarette smoke-induced lung inflammation, PLoS One 8 (2013) e58258. [42] P. Scaffidi, T. Misteli, M.E. Bianchi, Release of chromatin protein HMGB1 by necrotic cells triggers inflammation, Nature 418 (2002) 191–195. [43] H. Wang, J.M. Vishnubhakat, O. Bloom, M. Zhang, M. Ombrellino, A. Sama, K.J. Tracey, Proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes, Surgery 126 (1999) 389–392. [44] Q. Yang, X. Liu, Z. Yao, S. Mao, Q. Wei, Y. Chang, Penehyclidine hydrochloride inhibits the release of high-mobility group box 1 in lipopolysaccharide-activated RAW264.7 cells and cecal ligation and puncture-induced septic mice, J. Surg. Res. 186 (2014) 310–317. [45] J. Li, R. Kokkola, S. Tabibzadeh, R. Yang, M. Ochani, X. Qiang, H.E. Harris, C.J. Czura, H. Wang, L. Ulloa, H.S. Warren, L.L. Moldawer, M.P. Fink, U. Andersson, K.J. Tracey, H. Yang, Structural basis for the proinflammatory cytokine activity of high mobility group box 1, Mol. Med. 9 (2003) 37–45. [46] U. Andersson, H. Wang, K. Palmblad, A.C. Aveberger, O. Bloom, H. Erlandsson-Harris, A. Janson, R. Kokkola, M. Zhang, H. Yang, K.J. Tracey, High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes, J. Exp. Med. 192 (2000) 565–570. [47] Y. Sha, J. Zmijewski, Z. Xu, E. Abraham, HMGB1 develops enhanced proinflammatory activity by binding to cytokines, J. Immunol. 180 (2008) 2531–2537. [48] D.C. Angus, L. Yang, L. Kong, J.A. Kellum, R.L. Delude, K.J. Tracey, L. Weissfeld, I.M.S. Gen, Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated pneumonia and pneumonia with severe sepsis, Crit. Care Med. 35 (2007) 1061–1067. [49] R. Kokkola, E. Sundberg, A.K. Ulfgren, K. Palmblad, J. Li, H. Wang, L. Ulloa, H. Yang, X.J. Yan, R. Furie, N. Chiorazzi, K.J. Tracey, U. Andersson, H.E. Harris, High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis, Arthritis Rheum. 46 (2002) 2598–2603. [50] H. Yang, D.J. Antoine, U. Andersson, K.J. Tracey, The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis, J. Leukoc. Biol. 93 (2013) 865–873. [51] S.A. Lee, M.S. Kwak, S. Kim, J.S. Shin, The role of high mobility group box 1 in innate immunity, Yonsei Med. J. 55 (2014) 1165–1176. [52] D.M. Aronoff, C. Canetti, M. Peters-Golden, Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP, J. Immunol. 173 (2004) 559–565. [53] D. Prescott, D.M. McKay, Aspirin-triggered lipoxin enhances macrophage phagocytosis of bacteria while inhibiting inflammatory cytokine production, Am. J. Physiol. Gastrointest. Liver Physiol. 301 (2011) G487–G497. [54] D. El Kebir, P. Gjorstrup, J.G. Filep, Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 14983–14988. [55] T. Murakami, K. Suzuki, H. Tamura, I. Nagaoka, Suppressive action of resolvin D1 on the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice, Exp. Ther. Med. 2 (2011) 57–61.