Bis N Norgliovictin

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European Journal of Pharmacology ] (]]]]) ]]]–]]]

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmacology

0014-29

http://d

n Corr

Laborat

Univers

E-m

Pleasinfla

journal homepage: www.elsevier.com/locate/ejphar

Immunopharmacology and inflammation

Bis-N-norgliovictin, a small-molecule compound from marine fungus,inhibits LPS-induced inflammation in macrophages and improvessurvival in sepsis

Yuxian Song a, Huan Dou a, Wei Gong a, Xianqin Liu a, Zhiguo Yu b, Erguang Li a,Renxiang Tan b,n, Yayi Hou a,b,n

a Immunology and Reproductive Biology Lab & Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing 210093, PR Chinab Institute of Functional Biomolecules & State Key Laboratory of Pharmaceutical Biotechnology, School of Lifesciences, Nanjing University, Nanjing 210093, PR China

a r t i c l e i n f o

Article history:

Received 23 September 2012

Received in revised form

4 February 2013

Accepted 7 February 2013

Keywords:

Bis-N-norgliovictin

Sepsis

Toll-like receptor 4

Macrophage polarization

MyD88

TRIF

99/$ - see front matter & 2013 Published by

x.doi.org/10.1016/j.ejphar.2013.02.008

esponding authors at: Institute of Functiona

ory of Pharmaceutical Biotechnology, Scho

ity, Nanjing 210093, PR China

ail addresses: [email protected] (R. Tan), yayi

e cite this article as: Song, Y., et al.,mmation in macrophages and impro

a b s t r a c t

Sepsis is a highly lethal disorder characterized by systemic inflammation, and Toll-like receptor 4

(TLR4) in macrophages plays a crucial role in modulating innate immune response and outcome of

sepsis. During the screening of natural products against inflammation, we identified bis-N-norglio-

victin, a small-molecule compound isolated from marine-derived fungus, significantly inhibited

lipopolysaccharide (LPS, ligand of TLR4)-induced tumor necrosis factor-a (TNF-a) production in

RAW264.7 cells. In this study, we evaluated the effect of bis-N-norgliovictin on TLR4-mediated

inflammation in mouse macrophages and LPS-induced sepsis model. In RAW264.7 and mouse

peritoneal macrophages, bis-N-norgliovictin dose-dependently inhibited LPS-induced production of

TNF-a, interleukin-6 (IL-6), interferon-b (IFN-b) and monocyte chemoattractant protein (MCP-1), but

without suppressing cell viability. The anti-inflammatory effect was attributed to the down-regulation

of TLR4-triggered myeloid differentiation primary response protein 88 (MyD88)-dependent and

TIR-containing adapter inducing interferon-b (TRIF)-dependent signaling pathways, including p38

and c-Jun N-terminal kinase (JNK) of mitogen-activated protein kinases (MAPKs), nuclear factor-kB

(NF-kB) and interferon regulatory factor 3 (IRF3) cascades. Importantly, bis-N-norgliovictin also

protected mice against LPS-induced endotoxic shock. Intravenous injection of bis-N-norgliovictin 1 h

before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-a, IL-6,

MCP-1 and IL-10, attenuated liver and lung injury and diminished M1 macrophage polarization in liver.

Our results demonstrate that bis-N-norgliovictin exhibit potent anti-inflammatory effect both in vitro

and in vivo. These findings suggest that bis-N-norgliovictin can be a useful therapeutic candidate for the

treatment of sepsis and other inflammatory diseases.

& 2013 Published by Elsevier B.V.

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1. Introduction

Sepsis, a systemic inflammatory disease, is characterized by aninitial intense inflammatory response or ‘‘cytokine storm’’ thatoccurs during severe infection (Abraham and Singer, 2007;Rittirsch et al., 2008). Lacking of specific remedies, death fromseptic shock has increased over the past few decades (Angus et al.,2001; Martin et al., 2003). Fortunately, the identification of Toll-like receptors (TLRs), most notably TLR4, has sparked greatinterest in therapeutic manipulation of sepsis (Lemaitre et al.,1996; Romagne, 2007; Saturnino and Andrade, 2007). The

8283848586

Elsevier B.V.

l Biomolecules & State Key

ol of Lifesciences, Nanjing

[email protected] (Y. Hou).

Bis-N-norgliovictin, a smalves.... Eur J Pharmacol (201

lipopolysaccharide (LPS)-initiated TLR4 signaling leads to theactivation of nuclear factor-kB (NF-kB), mitogen-activated pro-tein kinases (MAPKs) and interferon regulatory factor 3 (IRF3),and the subsequent production of a variety of inflammatorymediators (Akira and Takeda, 2004; O’Neill and Bowie, 2007).Since most of the clinical trials targeting single inflammatorycytokine in the treatment of sepsis failed, therapeutic targeting ofTLR4 signaling looks promising (Wittebole et al., 2010).

Macrophages are proved to express most of functional TLRsincluding TLR4. They are the most efficient pathogen scavengersand the predominant source of cytokines and chemokines such astumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and mono-cyte chemoattractant protein (MCP-1). Nevertheless, excessive orprolonged production of inflammatory mediators leads to seriousconsequences including septic shock (Cohen, 2002). Macrophagesexhibit remarkable plasticity that allows them to modulate their

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l-molecule compound from marine fungus, inhibits LPS-induced3), http://dx.doi.org/10.1016/j.ejphar.2013.02.008i

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Fig. 1. Bis-N-norgliovictin did not affect cell viability. (A) Chemical structure of bis-N-norgliovictin. (B) Effect of bis-N-norgliovictin on cell viability. RAW264.7 cells

were treated with various concentrations of bis-N-norgliovictin or equal volume of DMSO (Con) for the indicated times. Cell viability was determined by CCK-8 assay as

described in materials and methods. The result expressed as mean7S.E.M (n¼5) is one of the independent experiments.

Y. Song et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]2

phenotype and serve distinct functions when respond to environ-mental signals (Gordon and Taylor, 2005; Sica and Mantovani,2012). Accordingly, they are classified into classically (M1) andalternatively (M2) activated macrophages (Gordon and Martinez,2010; Stout and Suttles, 2004). M1 macrophages promote inflam-matory process by up-regulating proinflammatory cytokines andactivating iNOS. In contrast, M2 macrophages dampen the inflam-matory process by producing anti-inflammatory factors andactivating arginase 1 (Liao et al., 2011; Porcheray et al., 2005).Accumulating evidence has suggested that M1 program inductionis related to sepsis severity (Benoit et al., 2008; Mehta et al.,2004). In patients with severe sepsis, high circulating concentra-tions of M1-type cytokines are highly correlated with mortality(Bozza et al., 2007; Lopez-Bojorquez et al., 2004). Therefore,modulating macrophage polarization is a new opportunity tothe therapeutics of severe sepsis (Hams et al., 2011; O’Neillet al., 2009).

In our screening for novel anti-inflammatory agents amongnatural products, a small-molecule compound, bis-N-norgliovic-tin, caught our eyes. It was isolated from marine-derived endo-phytic fungus S3-1-c and was an analogue of gliotoxin. Gliotoxinis one of the well-known members of the epipolythiodioxopiper-azine class of fungal metabolites, and shows anti-inflammatoryeffect both in vitro and in vivo (Mullbacher and Eichner, 1984;Pahl et al., 1996; Sutton et al., 1994). Whether bis-N-norgliovictinhas the similar anti-inflammatory effect as gliotoxin is stillunknown. Therefore, in this study, we evaluate the anti-inflammatory property of bis-N-norgliovictin in macrophagesand LPS-induced sepsis model and try to elucidate its plausiblemechanisms of action.

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2. Materials and methods

2.1. Reagents

LPS (from Escherichia coli O111:B4), fluorescein isothiocyanate-conjugated LPS (FITC-LPS, from E. coli O111:B4), dexamethasone(Dex), monensin and saponin were purchased from Sigma-Aldrich(St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), peni-cillin G, streptomycin and fetal bovine serum (FBS) were purchasedfrom Gibco Inc. (Grand Island, NY). Mouse TNF-a, IL-6 and MCP-1ELISA kits, Alex Fluor 647-conjugated anti-Mouse CD206 and itsisotype control were from Biolegend (San Diego, CA). PE-conjugatedanti-Mouse F4/80, FITC-conjugated anti-Mouse CD11c, PE-Cy7-conjugated anti-Mouse TLR4/MD-2, APC-conjugated anti-MouseCD14 and their isotype controls were all obtained from eBioscience(San Diego, CA). FITC-conjugated anti-Mouse IFN-b was purchasedfrom R&D systems (Minneapolis, MN). Anti-phospho-JNK, anti-JNK,

Please cite this article as: Song, Y., et al., Bis-N-norgliovictin, a smalinflammation in macrophages and improves.... Eur J Pharmacol (201

anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-IkBa, anti-IkBa, anti-phospho-IRF3, anti-IRF3, anti-NF-kB/p65, and anti-b-tubulin were all purchased from Cell SignalingTechnology (Danvers, MA). HRP- conjugated goat anti-rabbit IgGand HRP-conjugated goat anti-Mouse IgG were purchased fromBeyotime Institute of Biotechnology (Jiangsu, China). Texas red-conjugated goat anti-rabbit IgG was purchased from Santa CruzBiotechnology Inc. (Santa Cruz, CA). CCK-8 was purchased fromDojinDo Molecular Technologies. Inc (Kyushu, Japan). pNF-kB-luc,pAP-1-luc, pRL-TK-Renilla and pGL6 plasmids were purchased fromBeyotime Institute of Biotechnology (Jiangsu, China). Endotoxin-freeplasmids extraction kit was from Biomiga (San Diego, CA).

2.2. Extraction and isolation of bis-N-norgliovictin

The culture broth of a marine derived fungus named S3-1-c wassubjected on a silica gel column eluted with CHCl3–MeOH (100:4).The MeOH solvent partition fraction was repeatedly chromato-graphed over silica gel and Sephadex LH-20 to afford a compoundidentified as bis-N-norgliovictin, whose structure (Fig. 1A) waselucidated on the basis of high resolution-electrospray ionization(HR-ESI)–MS and NMR data by comparison with literature values(Kirby et al., 1988). The purity of bis-N-norgliovictin was 499% byHPLC analysis.

2.3. Animals

Specific pathogen-free (SPF) male BALB/c mice (5–6-week-old)were purchased from Model Animal Genetics Research Center ofNanjing University (Nanjing, China). Animal welfare and experi-mental procedures were carried out strictly in accordance withthe Guide for the Care and Use of Laboratory Animals (NationalInstitutes of Health, the United States) and the related ethicalregulations of our university. All mice were housed in SPFcondition with a 12:12 h light–dark cycle, and received waterand food ad libitum. Mice were acclimated for at least a weekbefore use.

2.4. Cells

The mouse macrophage cell line RAW264.7 was purchasedfrom ATCC (American Type Culture Collection, Manassas, VA).RAW264.7 cells were cultured in endotoxin-free DMEM mediumcontaining 10% heat-inactivated FBS. The medium indicatedabove were all contained 100 U/ml penicillin G and 100 mg/mlstreptomycin. Male BALB/c mice were used for the preparation ofprimary mouse macrophages. Thioglycolate-elicited mouse peri-toneal macrophages were prepared as described (Liu et al., 2008).The cells were cultured in DMEM with 10% heat-inactivated NCS


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Table 1Primers used for real-time quantitative PCR analysis.

Gene Sequence

TNF-a Forward: 50-CCCTCACACTCAGATCATCTTCT-30

Reverse: 50-GCTACGACGTGGGCTACAG-30

IL-6 Forward: 50-TAGTCCTTCCTACCCCAATTTCC-30

Reverse: 50-TTGGTCCTTAGCCACTCCTTC-30

IFN-b Forward: 50-CAGCTCCAAGAAAGGACGAAC-30

Reverse: 50-GGCAGTGTAACTCTTCTGCAT-30

MCP-1 Forward: 50-TTAAAAACCTGGATCGGAACCAA-30

Reverse: 50-GCATTAGCTTCAGATTTACGGGT-30

IL-10 Forward: 50-GCTCTTACTGACTGGCATGAG-30

Reverse: 50-CGCAGCTCTAGGAGCATGTG-30

iNOS Forward: 50-CCAAGCCCTCACCTACTTCC-30

Reverse: 50-CTCTGAGGGCTGACACAAGG-30

Arg-1 Forward: 50-CTCCAAGCCAAAGTCCTTAGAG-30

Reverse: 50-AGGAGCTGTCATTAGGGACATC-30

GAPDH Forward: 50-GGTGAAGGTCGGTGTGAACG-30

Reverse: 50-CTCGCTCCTGGAAGATGGTG-30

Y. Song et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]] 3

(PAA Laboratories, Pasching, Austria). After 2 h, nonadherent cellswere removed and the adherent monolayer cells were used asperitoneal macrophages. Cells were maintained at 37 1C in a 5%CO2/air environment. All cell culture media and all other reagentswere entirely free of endotoxin, as checked by tachypleusamebocyte lysate assay (Xiamen Houshiji, Ltd., Fujian, China).

2.5. Cell viability assay

The cytotoxicity of bis-N-norgliovictin was determined usingthe Cell Counting Kit-8 (CCK-8) according to the manufacturer’sprotocol. Briefly, RAW264.7 cells or thioglycolate-elicited mouseperitoneal macrophages were seeded in 96-well plates at adensity of 1�104 cells/well. After overnight culturing, the cellswere incubated with various concentrations of bis-N-norgliovictinor DMSO (as control) for 8 h, 24 h or 48 h, respectively. Subse-quently, 10 ml CCK-8 was added to each well and incubated foranother 3 h. The absorbance at 450 nm was measured usingmicroplate reader (Synergy HT, Bio-Tek). The average opticaldensity formed in control cells was taken as 100% viability, andthe results of treatments were expressed as a percentage of thecontrol.

2.6. Enzyme-linked immunosorbent assay

For TNF-a, IL-6 and MCP-1 measurements, RAW264.7 cells orthioglycolate-elicited mouse peritoneal macrophages werepretreated with or without bis-N-norgliovictin for 1 h before LPSstimulation for another 20 h. The levels of proinflammatorymediators in the culture media were determined using TNF-a,IL-6 and MCP-1 ELISA kit according to the manufacturer’s instruc-tions, respectively. The lower detection limits using standardcurves were 7.8 pg/ml for TNF-a and IL-6, and 62.5 pg/ml forMCP-1, respectively.

2.7. Intracellular staining or cell surface staining and flow

cytometric analysis

RAW264.7 cells or thioglycolate-elicited mouse peritonealmacrophages were cultured in 24-well plates at a density of2�105 cells/well. The cells were pretreated with bis-N-norglio-victin or DMSO (as control) for 1 h, then stimulated with 3 mMmonensin (an inhibitor of intracellular protein transport) and100 ng/ml LPS for another 6 h. Collected Cells were fixed with 4%paraformaldehyde (PFA) for 20 min and permeabilized with 0.1%saponin for 15 min. After incubating with purified anti-CD16/CD32 antibody (Fc blocker) for 10 min, cells were stained withFITC-conjugated anti-Mouse IFN-b for 30 min at 4 1C. FITC-conjugated anti-Rat IgG,k was used as isotype control. Afterwashing for three times with phosphate buffered saline (PBS),the cells were analyzed by BD FACS Calibur flow cytometer(Bedford, MA). For TLR4/MD-2 and CD14 analysis, cell surfacewere stained with PE-Cy7-conjugated anti-Mouse TLR4/MD-2 andAPC-conjugated anti-Mouse CD14 directly without fixing orpermeabilizing.

2.8. Reverse transcription and real-time quantitative PCR

Total RNA was extracted using Trizol reagent (Invitrogen, USA)following the manufacturer’s instructions. The concentration andpurity of the RNA were determined by measuring the absorbanceat 260 nm and 280 nm using spectrophotometer (Smart Spec Plus,Bio-Rad). Total RNA (1 mg) was reverse-transcribed using RevertAid

TM

First Strand cDNA Synthesis Kit (Fermentas) in a totalvolume of 20 ml.


After reverse transcription of the RNA, real-time quantitativePCR was performed using SybrGreen PCR Master Mix (with Rox)(Invitrogen, USA) and 7300 Real-Time PCR System (AppliedBiosystems, Foster City, CA). The sequences of the primers areshown in Table 1. The following cycle parameters were used:55 1C for 2 min, 95 1C for 10 min, and then 40 cycles of 95 1C for30 s, 60 1C for 30 s. The relative expression levels of the targetgenes against that of the GAPDH was calculated as 2�DDCt

according to the manufacture’s specification. Samples were per-formed in triplicate and every experiment was performed at leastthree times.

2.9. Analysis of LPS binding to macrophages

The binding of FITC-LPS to RAW264.7 cells or thioglycolate-elicited mouse peritoneal macrophages was monitored accordingto Julia Carracedo’s method (Carracedo et al., 2002). In brief, cells(5�105) were pre-incubated in the absence or presence of bis-N-norgliovictin for 1 h at 37 1C in RPMI-1640 containing 10% FBSbefore incubated with FITC-LPS for 1 h. After washing twice withPBS, the binding of FITC-LPS was analyzed by flow cytometry.

2.10. Western blot analysis

Proteins were extracted in lysis buffer (20 mM Tris–HCl, pH7.6, 250 mM NaCl, 0.5% NP-40, 3 mM EDTA, 1.5 mM EGTA, 10 g/ml Aprotinin, 10 g/ml Leupeptin, 1 mM DTT, 1 mM PNPP and0.1 mM Na3VO4 as protease and phosphatase inhibitor). Theprotein concentration of each sample was determined using aBCA protein assay kit (Pierce Chemical, Rockford, IL). Appropriateamount of total proteins (40–50 mg) were electrophoresed on SDSpolyacrylamide gels with Tris-glycine running buffer and electri-cally transferred onto 0.45 mm PVDF membranes. After blockingwith 5% (w/v) bovine serum albumin (BSA) in Tris buffered saline(TBS) for 1 h, the membrane was washed and then incubated withdifferent primary antibodies (dilution at 1:1000) over night at4 1C. After washing, the membrane was incubated at roomtemperature with horseradish peroxidase-conjugated anti-ratIgG (dilution at 1:3000) for 1 h and then visualized by using theECL Plus western blotting detection reagents (Millipore, USA).Protein b-tubulin was used as an internal control. The densities ofbands were scanned and quantified using Image J software.

2.11. Nuclear translocation of NF-kB

RAW264.7 cells (2�105/well) were pretreated withbis-N-norgliovictin for 1 h before stimulation with 100 ng/ml


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LPS for 1 h. The cells were then fixed with 4% PFA for 30 min andpermeabilized with 0.1% Triton X-100 for 15 min. After 1 hincubation with blocking buffer (3% BSA in PBS), cells wereincubated with anti-NF-kB/p65 antibody (dilution at 1:100) over-night at 4 1C, washed, and then incubated with Texas red-conjugated anti-rabbit IgG (dilution at 1:400) for 1 h. In order toidentify the nucleus, samples were stained with DAPI (500 ng/ml)

Fig. 2. Bis-N-norgliovictin inhibited production of pro-inflammatory mediators in vitro. (A

1 h before LPS (100 ng/ml) stimulation for another 20 h or 6 h. Productions of TNF-a, IL-6 a

in the cells was determined by intracellular staining and flow cytometric analysis. Dat

(MFI7S.E.M) of three independent experiments. (B) RAW264.7 cells were treated with or w

for another 6 h. Expression levels of TNF-a, IL-6, MCP-1 and IFN-b mRNA were tested by r

Con (control), equal volume of DMSO; Dex (Dexamethasone, 10 mg/ml) was used as positiv

to LPS alone.


for 10 min. Fluorescent images were observed under Nikonfluorescence microscope (Tokyo, Japan).

2.12. Transfection and luciferase assay

RAW264.7 cells in 24-well plate were co-transfected with200 ng plasmids encoding

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) RAW264.7 cells were treated with or without bis-N-norgliovictin (0.5, 1, 2 mg/ml) for

nd MCP-1 (20 h) in culture supernatants were tested by ELISA. IFN-b production (6 h)

a presented as mean concentration (pg/ml7S.E.M) or mean fluorescence intensity

ithout bis-N-norgliovictin (0.5, 1, 2 mg/ml) for 1 h before LPS (100 ng/ml) stimulation

eal-time quantitative PCR. Data were expressed as relative fold expression to control.

e control. #Po0.05 compared to control; nPo0.05, nnPo0.01, nnnPo0.001 compared


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NF-kB luciferase or AP-1 luciferase, and 10 ng pRL-TK-Renillaluciferase. Total amounts of plasmid DNA were equalized withpGL6 (empty control vector). Lipofectamine LTX with PLUSreagent (Invitrogen, Carlsbad, CA) was used for tranfection(http://tools.invitrogen.com/content/sfs/manuals/lipofectamineLTX_and_PLUS_man.pdf). After 24 h, the cells were left untreated ortreated with different concentrations of bis-N-norgliovictin for 1 hbefore stimulated with LPS for another 5 h. Luciferase activitieswere measured using the Dual-Luciferase Reporter Assaysystem (Promega, Madison, WI) with a GloMax-96 MicroplateLuminometer (Promega, Madison, WI) according to the manufa-cturer’s instructions. Data are normalized for transfection effi-ciency by dividing Firefly luciferase activity with that of Renillaluciferase.

Fig. 3. Effects of bis-N-norgliovictin on LPS recognition and TLR4/MD-2/CD14 expressi

pretreated with bis-N-norgliovictin (0.5 mg/ml or 2 mg/ml) or Polymyxin B (PMB, 50 mg/

37 1C. Samples were measured by flow cytometry. ((C), (D)) RAW264.7 cells were treat

LPS (100 ng/ml) stimulation for another 24 h. Cell surface staining and flow cytometry

results were expressed as mean7S.E.M of three independent experiments. #Po0.05 co

a positive control.


2.13. In vivo LPS-induced sepsis model

LPS was dissolved in saline for administration to mice.Bis-N-norgliovictin was first dissolved in DMSO and then dilutedwith saline to indicated concentrations. For analysis of cytokineproduction in mice, mice were randomly divided into 6 groups (6mice per group). Group 1 mice were injected i.p. with DMSO insaline; group 2 mice were injected i.p. with bis-N-norgliovictin(30 mg/kg of body); group 3 mice were injected i.p. with DMSO insaline 1 h before administration of LPS (10 mg/kg); group 4–6mice were injected i.p. with bis-N-norgliovictin (3 mg/kg, 10 mg/kg or 30 mg/kg) 1 h before administration of LPS (10 mg/kg).Blood was collected (using the retrobulbar plexus route undersodium pentobarbital anesthesia) 2 h after LPS injection, and

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on. (A) RAW264.7 cells or (B) thioglycolate-elicited peritoneal macrophages were

ml) for 30 min and further incubated with 2 mg/ml FITC-conjugated LPS for 1 h at

ed with or without bis-N-norgliovictin (0.5 mg/ml, 1 mg/ml, 2 mg/ml) for 1 h before

was conducted to determine TLR4/MD-2 (C) and CD14 (D) levels on the cells. The

mpared to control; nnnPo0.001 compared to LPS alone. PMB: an inhibitor of LPS, as


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allowed to clot at room temperature. Serum was separated bycentrifugation, and stored at �80 1C until analysis. For the lethalendotoxin shock model, mice were randomly divided into4 groups (8 mice per group). Group 1 mice were injectedi.p. with DMSO in saline; group 2 mice were injected i.p. withbis-N-norgliovictin (30 mg/kg of body); group 3 mice wereinjected i.p. of LPS (25 mg/kg); group 4 mice were injected i.p.with bis-N-norgliovictin (30 mg/kg) 1 h before administration ofLPS (25 mg/kg). The survival of mice was monitored over 7 days.Liver and lung samples were obtained 12 h after LPS challenge(mice were treated as in the survival study, n¼6, tissues werecollected after mice were sacrificed) and fixed for hematoxylinand eosin staining or stored at �80 1C until use.

For preparing of liver single-cell suspension, a piece of liverissue was excised and minced in PBS, filtered the tissue suspen-sion through a 200-mesh filter and then centrifuged at 1500 rpmfor 5 min. Cells were incubated with Fc blocker prior to stainingwith PE-F4/80, FITC-CD11c and Alex Fluor 647-CD206 antibodiesfor 30 min at 4 1C followed by 3 washes in PBS. 20000 cells werecollected and analyzed by flow cytometry.

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2.14. Statistical analysis

All data were expressed as the mean7S.E.M unless otherwiseindicated. Differences between groups were compared by analysis

Fig. 4. Inhibitory effect of bis-N-norgliovictin on LPS-induced NF-kB signal activation in

norgliovictin (2 mg/ml) for 1 h before LPS (100 ng/ml) stimulation for another 15 min. Th

Bands were quantified with a densitometer. (C) RAW264.7 cells were treated with DMS

with bis-N-norgliovictin (2 mg/ml f or 1 h) and then exposed to LPS (100 ng/ml for 1

antibody against p65 (Methods). (D) RAW 264.7 cells were transfected with NF-kB lucif

1 mg/ml, 2 mg/ml) for 1 h before stimulated with LPS (100 ng/ml) for additional 5 h. Rela

at least three independent experiments. #Po0.05 compared to control, nPo0.05, nnP

vehicle plasmid. RLA, relative luciferase activity.


of variance followed by post hoc Tukey’s test to correct formultiple comparisons. Differences were considered to be statisti-cally significant at Po0.05. Each experiment was repeated atleast three times. All calculations were performed using the Prism5 software (GraphPad, San Diego, CA).

3. Results

3.1. Bis-N-norgliovictin inhibits LPS-induced production of

proinflammatory mediators in macrophages

We first examined the effects of bis-N-norgliovictin on cellviability of macrophages by using the CCK-8 assay. As in Fig. 1B,results showed that bis-N-norgliovictin (up to 16 mg/ml) treat-ment for indicated times did not induce any cellular toxicity inRAW264.7 cells. We got the similar results in thioglycolate-elicited peritoneal macrophages (Supplemental Fig. S1).

Subsequently, to determine the anti-inflammatory effect ofbis-N-norgliovictin, multiple proinflammatory mediators inducedby LPS were monitored in both macrophage cell lines and primarymacrophages. We first exposed RAW264.7 cells with variousconcentrations of bis-N-norgliovictin for 1 h, and then stimulatedwith LPS (100 ng/ml) for another 6 h or 20 h. Dexamethasone(Dex, 10 mg/ml) was used as a positive control in this experiment.Intracellular expression of IFN-b (LPS stimulation for 6 h) was

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RAW264.7 cells. ((A) and (B)) RAW264.7 cells were treated with or without bis-N-

e phosphorylation or degregation of IkBa were evaluated by western blot analysis.

O (Con, the top row) or LPS alone (100 ng/ml for 1 h, the middle row) or pretreated

h, the third row) were then processed for immunofluorescence staining with an

erase reporter plasmid. Cells were pre-treated with bis-N-norgliovictin (0.5 mg/ml,

tive luciferase activity (RLA) was determined. Data were shown as mean7S.E.M of

o0.01 and nnnPo0.001 compared to LPS alone. Veh, cells were transfected with


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measured by intracellular staining and FACS analysis. Resultsshowed that pretreatment of bis-N-norgliovictin (Z1 mg/ml)significantly inhibited LPS-induced intracellular IFN-b production(Fig. 2B). The amounts of TNF-a, IL-6 and MCP-1 (LPS stimulationfor 20 h) produced in the supernatants were measured by ELISA.Results showed that bis-N-norgliovictin decreased LPS-inducedTNF-a, IL-6 and MCP-1productions in a concentration-dependentmanner (Fig. 2B). However, bis-N-norgliovictin did not alterproduction of these proinflammatory mediators without LPSstimulation. In primary peritoneal macrophage cells, we deter-mined that bis-N-norgliovictin had the same inhibitory effect(Supplemental Fig. S2).

We next used quantitative real-time PCR to examine themRNA expression of these mediators. As expected, LPS-inducedTNF-a, IL-6, MCP-1 and IFN-b mRNA expressions were increaseddramatically, and were clearly suppressed by pretreatment ofbis-N-norgliovictin in a dose-dependent manner (Fig. 2B).

According to previous cell viability assay, the concentration ofbis-N-norgliovictin used in the proinflammatory mediatorsexperiment was much less than 16 mg/ml. We reasoned that thebis-N-norgliovictin-induced inhibition of cytokine release was notrelated to its cytotoxicity.

3.2. Bis-N-norgliovictin does not alter LPS recognition or TLR4/MD-

2/CD14 expression

As LPS triggers the TLR4 signaling pathway, the observed anti-inflammatory effects appeared to be mediated by inhibition ofTLR4 signaling. Therefore, we first investigated whether itaffected TLR4 signaling from the origination. We conductedexperiments to evaluate the effect of bis-N-norgliovictin on LPSbinding to the macrophages. The cells were incubated with FITC-LPS, and the LPS binding was analyzed by flow cytometry. Results

Fig. 5. Bis-N-norgliovictin inhibited LPS-induced MAPKs activation in RAW264.7 cells.

phospho-JNK and phospho-p38 were evaluated by western blot. Bands were quantifie

reporter plasmid. Cells were treated as in Fig. 4D. Effect of bis-N-norgliovictin on LPS-s

were shown as mean7S.E.M of at least three independent experiments. #Po0.05 com

transfected with vehicle plasmid pGL6. RLA, relative luciferase activity.


in Fig. 3A and B showed that incubation of cells with Polymyxin B(PMB, an inhibitor of LPS) resulted in significant inhibition of thebinding of LPS to both RAW264.7 cells and thioglycolate-elicitedperitoneal macrophages; in contrast to PMB, bis-N-norgliovictindid not block the binding even at a concentration of 2 mg/ml.To examine whether bis-N-norgliovictin was able to modulateTLR4/MD-2 or CD14 expression in response to LPS exposure, flowcytometric analysis was performed. LPS exposure for 12 h and24 h could significantly increase TLR4/MD-2 and CD14 proteinlevels (Supplemental Fig. S3); but as shown in Fig. 3C and D, bis-N-norgliovictin pretreatment did not alter expression of TLR4/MD-2 or CD14 compared to LPS stimulated cells. These resultssuggested that bis-N-norgliovictin inhibited cytokine productionwithout antagonizing the binding of LPS to CD14/TLR4/MD-2complex or modulating their expression.

3.3. Bis-N-norgliovictin exerts anti-inflammatory effect through

TLR4-triggered MyD88 and TRIF-dependent pathways

The results described above suggest that bis-N-norgliovictinmight target the downstream event of TLR4 signaling. Therefore,we next investigated the effect of bis-N-norgliovictin on TLR4signaling pathways involving NF-kB, MAPKs and IRF3.

As the phosphorylation and degradation of IkBa proteins leadsto the translocation of NF-kB, so here, we examined the IkBaactivation by western blot assay. As determined in our previousstudy, LPS (100 ng/ml) induced significant phosphorylation anddegradation of IkBa at 15 min (Dou et al., 2011), but pretreatmentof bis-N-norgliovictin for 1 h inhibited these effects (Fig. 4A andB). The effect of bis-N-norgliovictin on LPS-induced nucleartranslocation of NF-kB was valued by immunofluorescence ana-lysis. As shown in Fig. 4C, under LPS (100 ng/ml) stimulation,the subunit of NF-kB p65 translocated from the cytosol to the

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((A) and (B)) RAW264.7 cells were treated as in Fig. 4A. Levels of phospho-ERK1/2,

d with a densitometer. (C) RAW264.7 cells were transfected with AP-1 luciferase

timulated AP-1 activity was determined by luciferase reporter gene analysis. Data

pared to control, nPo0.01 and nnnPo0.001 compared to LPS alone. Veh, cells were


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nucleus. While, the cells pretreated with 2 mg/ml bis-N-norglio-victin for 1 h followed by LPS treatment showed less p65translocation. Luciferase reporter gene assay also showed theinhibitory action of bis-N-norgliovictin on LPS-induced NF-kBactivation (Fig. 4D).

Besides NF-kB, activator protein-1 (AP-1) is also required for LPS-induced induction of proinflammatory cytokines. Thus we nextexamined the effect of bis-N-norgliovictin on LPS-induced MAPK/AP-1 activation. Previously in our study, ERK1/2 and JNK phosphor-ylation were detected at 15 min and 30 min, while p38 phosphoryla-tion was detected from 15 min to 120 min in RAW264.7 cells (Douet al., 2011). We then stimulated cells with LPS (100 ng/ml) for15 min after pretreated with 2 mg/ml bis-N-norgliovictin for 1 h.Results showed that 2 mg/ml bis-N-norgliovictin could suppressphosphorylation of JNK and p38, though bis-N-norgliovictin itselfslightly stimulated their activation. But phosphorylation of ERK1/2was almost unaltered by bis-N-norgliovictin (Fig. 5A and B). Subse-quently, we monitored the effect of bis-N-norgliovictin on LPS-induced activation of AP-1. As expected, AP-1 luciferase reportergene assay confirmed that bis-N-norgliovictin impaired AP-1 activa-tion in LPS-stimulated RAW264.7 cells (Fig. 5C).

Given that bis-N-norgliovictin inhibited both MyD88-dependent genes (TNF-a and IL-6) and TRIF-dependent gene(IFN-b), we next sought to examined whether it affected phos-phorylation of IRF3, a pivotal transcriptional regulator down-stream of TRIF pathway. Time course experiments revealed thatphosphorylation of IRF3 was increased from 60 min to 120 minafter the LPS (100 ng/ml) stimulation (Fig. 6A and C). But thephosphorylation was almost abolished in bis-N-norgliovictinpretreated (2 mg/ml, for 1 h) and LPS-stimulated (for 60 min) cells(Fig. 6B and D).

Taken together, these results suggested that bis-N-norgliovic-tin inhibited LPS-induced inflammation through both MyD88 andTRIF-dependent pathways.

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3.4. Bis-N-norgliovictin protects mice against LPS-induced endotoxic

shock

To substantiate the physiological function of bis-N-norgliovic-tin in vivo, we examined the therapeutic effect of bis-N-

Fig. 6. Inhibitory effect of bis-N-norgliovictin on LPS induced phosphorylation of IRF3. ((

of IRF3 in RAW264.7 cells. ((B) and (D)) RAW264.7 cells were treated with or without b

60 min. Levels of phospho-IRF3 and IRF3 were evaluated by western blot. Bands w

independent experiments. #Po0.05 compared to control, nPo0.01 compared to LPS a


norgliovictin in a mouse model of sepsis. Preliminary, we foundthat serum level of TNF-a reach the peak at 1 h after LPS (10 mg/kg) challenge, and decreased after that. While serum levels of IL-6and MCP-1 increased from 1 h till 6 h. All of the three had a highlevel at 2 h after LPS injection (Supplemental Fig. S4). Based onthese, we chose 2 h as the LPS challenge time point. As expected,injection of LPS into mice for 2 h induced an increase in serumTNF-a, IL-6 and MCP-1 levels (Fig. 7A). Intraperitoneal adminis-tration of bis-N-norgliovictin 1 h before LPS challenge signifi-cantly inhibited TNF-a and MCP-1 production induced by LPS in adose-dependent manner. The high dose of bis-N-norgliovictin(30 mg/kg) pretreatment could also inhibit IL-6 productioninduced by LPS. Apart from proinflammatory cytokines, LPS alsoincreased anti-inflammatory cytokine IL-10 in serum. Bis-N-norgliovictin pretreatment also inhibited IL-10 level in a similardose-dependent manner.

The survival studies were conducted by a lethal dose of LPS(25 mg/kg) injections into mice and the status were monitoredover 7 days. Consistently, all the mice in group 3 (LPSþDMSO)died within 3 days after LPS administration; in contrast, bis-N-norgliovictin therapy resulted in a markedly improved survival at7 days, which showed that 62.5% of bis-N-norgliovictin pretreatedmice (LPSþBis) still survived (Fig. 7B).

Histological examinations of liver and lung tissues wereconsistent with our prior findings. In group 3 (LPSþDMSO),significant damage was observed in the lungs and livers asevidenced by edema, moderate inflammatory cell infiltration,and severe hemorrhage. Treatment with bis-N-norgliovictin(LPSþBis) ameliorated these damages (Fig. 7C).

3.5. Bis-N-norgliovictin reverses the sepsis-induced macrophage

accumulation and alters macrophage polarization in liver

As F4/80 is a transmembrane protein present on the cell-surface of mouse macrophages and is associated with maturemacrophages, we used F4/80 as a single marker to identifymacrophages in liver. CD11cþCD206� cells in F4/80þ macro-phages are thought to be M1 macrophages, while F4/80þCD11c�CD206þ cells are thought to be M2 macrophages(Nishimura et al., 2009). Results of FACS analysis (Fig. 8A) showed

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A) and (C)) LPS (100 ng/ml, treated as the indicated time) induced phosphorylation

is-N-norgliovictin (2 mg/ml) for 1 h before LPS (100 ng/ml) stimulation for another

ere quantified with a densitometer. Data were representative of at least three

lone.


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Fig. 7. Bis-N-norgliovictin protected against endotoxic shock. (A) Bis-N-norgliovictin inhibited LPS-induced production of cytokines in mice. Bis-N-norgliovictin (3 mg/kg,

10 mg/kg, 30 mg/kg) intravenously administered 1 h before LPS (10 mg/kg i.p.) challenge. Serum samples were collected at 2 h after LPS challenge. Cytokine levels were

determined by specific ELISA. n¼6 mice per group. Data were shown as mean7S.E.M. #Po0.0001 compared to DMSO, nPo0.05, nnPo0.01 and nnnPo0.001 compared

to LPSþDMSO. (B) Protective effects of bis-N-norgliovictin on LPS-induced lethality in mice. bis-N-norgliovictin (30 mg/kg) was intravenously administered 1 h before LPS

(25 mg/kg i.p.) challenge. n¼8 mice per group. (C) Hematoxylin and eosin staining of lung and liver sections from mice treated as in (B), assessed 12 h after treatment. n¼6

mice per group. DMSO, normal control; Bis, treated with bis-N-norgliovictin alone; LPSþDMSO, pretreated with DMSO and challenged with LPS; LPSþBis, pretreated with

bis-N-norgliovictin and challenged with LPS.


that about 13% cells in liver of negative control mice (DMSO) were F4/80þ macrophages, LPS stimulation up-regulated macrophage popu-lation to approximately 19%. In contrast, bis-N-norgliovictin pretreat-ment significantly reduced the macrophage population. Intriguingly,there was variation in the ratio of M1 and M2 macrophages amongF4/80þ cells. LPS induced a dominant increase in CD11cþCD206�macrophages (M1) from 4.45%72.44% to 27.5876.86% of F4/80þcells (n¼6, Po0.001 versus DMSO). However, bis-N-norgliovictinpretreatment substantially reduced this increasing (7.3977.21% ofF4/80þ cells, Po0.001 versus LPSþDMSO). On the other hand, bis-N-norgliovictin pretreatment did not alter the amount ofCD11c�CD206þ macrophages (M2) very much (P¼0.187 versusLPSþDMSO).

Subsequently, we assessed expression of M1 and M2 markergenes in liver cells. In liver cells of LPS plus DMSO injected mice,mRNA levels of inflammatory genes (M1) iNOS, TNF-a and IL-6were high, and anti-inflammatory genes (M2) Arg-1 and IL-10were low. Bis-N-norgliovictin pretreatment significantly down-regulated iNOS, TNF-a and IL-6 mRNA levels (Fig. 8B) but did notunaltered Arg-1 and IL-10 expressions (Fig. 8C). However, in vitro,we found that pretreatment of bis-N-norgliovictin inhibited bothM1 (iNOS) and M2 markers (Arg-1) (Supplemental Fig. S5).

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4. Discussion

Recent work carried out in our laboratory on the screening of anti-inflammatory small-molecules from marine natural products has ledto the identification of a novel anti-inflammatory compound frommarine-derived endophytic fungus S3-1-c. This compound was iso-lated to 499% purity and chemically characterized as a diketopiper-azine. In 1988, it was first reported to be identified from Gliocladium

virens (Kirby et al., 1988). Although it has been reported to be isolatedfrom various species of fungus, no biological activity has been


reported yet except for its low cytotoxic activity (Isaka et al., 2005;Prachyawarakorn et al., 2008; Zhao et al., 2009). This study, to ourknowledge, provides the first evidence of bis-N-norgliovictin show-ing anti-inflammatory activity in LPS-induced macrophages andsepsis model.

Because of the central role of TLR4 in inflammation responseand pathogenesis of sepsis, therapeutic targeting of TLR4 signal-ing is a new strategy to treat sepsis. Evidence has emerged thatvarious small-molecule compounds have TLR4 signaling inhibi-tion activities with different mechanisms. For instance, E5531, anLPS antagonist, inhibits the binding of LPS to TLR4-MD-2 (Akashiet al., 2003); TAK-242 inhibits TLR4 signaling by binding directlyto a specific amino acid in the intracellular domain of TLR4(Kawamoto et al., 2008; Takashima et al., 2009); RSCL-0520blocks signals generated by TLR4 activation by down-regulationof NF-kB-regulated inflammatory cytokines, and the inhibitoryeffect involves both MyD88-dependent and -independent signal-ing cascades (Datla et al., 2010). Our results clearly demonstratedthat bis-N-norgliovictin neither inhibit LPS recognition nor mod-ulate TLR4/MD-2 or CD14 expression (Fig. 3). In fact, bis-N-norgliovictin inhibited NF-kB activation via suppressing IkBaphosphorylation and degradation, which led to the suppressionof nuclear translocation of p65 (Fig. 4); Moreover, it suppressedMAPK/AP-1 cascade by selectively decreasing phosphorylation ofJNK and p38, but not ERK1/2 (Fig. 5); In addition, bis-N-norglio-victin inhibited TRIF/IRF3 signal by almost abolishing the phos-phorylation of IRF3 induced by LPS (Fig. 6). These might explainwhy bis-N-norgliovictin inhibited the production of inflammatorymarkers (TNF-a, IL-6 and MCP-1) as well as type I interferon (IFN-b). Taken together, bis-N-norgliovictin is not an LPS antagonist orCD14/TLR4/MD-2 inhibitor but can inhibit LPS-induced MyD88and TRIF-involved signaling cascades.

Sepsis is marked by a systemic inflammatory response. Over-production of multiple proinflammatory mediators (such as TNF-a


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Fig. 8. Bis-N-norgliovictin altered macrophage polarization in liver. (A) Analysis of liver cells for F4/80, CD11c and CD206. Liver cells from different treated mice as

indicated were stained with antibodies against F4/80, CD11c, CD206 and isotype controls followed by flow cytometric analysis. Samples were first assessed the

percentages of F4/80þ cells and then gated for F4/80þ cells and examined for expression of CD11c and CD206. Quantitative PCR of inflammatory gene expression (B) and

anti-inflammatory gene expression (C) in liver cells as in (A). Data were shown as mean7S.E.M. #Po0.05 compared to DMSO; nPo0.05 and nnPo0.01 compared to

LPSþDMSO.


and IL-6) leads to severe injury and increased mortality in sepsis(Netea et al., 2003). Persistent or inappropriate expression of chemo-kines (such as MCP-1) may results in more extensive tissue damage(Gerard and Rollins, 2001). IFN-b deficiency leads to resistance to LPS-induced endotoxin shock, which suggest the central role of IFN-b inLPS-induced lethality (Karaghiosoff et al., 2003). Thus, regulation ofmultiple mediators could be more beneficial than suppression ofsingle mediator. In fact, the clinical trials targeting single inflamma-tory cytokine have been proved ineffective in the treatment ofsepsis (Abraham et al., 1998; Qiu et al., 2011). In our presentstudy, we demonstrated that bis-N-norgliovictin significantly


inhibited LPS-induced production of TNF-a, IL-6, MCP-1 and IFN-bin vitro, and inhibited TNF-a, IL-6 and MCP-1 in vivo. Strikingly, it alsosuppressed the anti-inflammatory cytokine IL-10, as well as pro-inflammatory cytokines and chemokine (Fig. 7A). Actually, accordingto the cohort study of 1886 subjects hospitalized with community-acquired pneumonia (CAP), the highest risk of death was withcombined high levels of IL-6 and IL-10 cytokine activity in severesepsis; while the lowest risk of death was with combined low IL-6and low IL-10 (Kellum et al., 2007).

M1 program induction is related to sepsis severity. In theLPS-induced sepsis model, we showed that bis-N-norgliovictin


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pretreatment diminished the population of total macrophages(F4/80þ) and M1 macrophages in the liver, but did not alter M2macrophages population. Gene detection also confirmed the phe-nomenon: bis-N-norgliovictin inhibited the expression of iNOS, TNF-a and IL-6, markers of classically activated macrophages, whereasdid not interfere with expression of Arg-1 and IL-10, markers ofalternative activation. These suggested that this compound inhibitedM1 polarization but did not alter M2 activation. This mightcontribute to the protection effect of bis-N-norgliovictin in sepsis.However, we noticed that the IL-10 level in liver tissue seemeddifferent from that in circulation: pretreatment of bis-N-norgliovic-tin remarkably suppressed IL-10 level in serum, but not in liver.There may be at least two reasons for this: first of all, sepsis isextremely complex and the environment between tissues andcirculation differs; second, we detected the whole liver cells butnot only macrophages in them, other cells might interfere with thegene detection. To elucidate this question, further studies areneeded to be conducted.

Collectively, these data extend our understanding of the mole-cular mechanisms underlying the biological activities and pharma-cological use of bis-N-norgliovictin. The ability to negatively regulateTLR4 signaling and modulate macrophage polarization may accountfor the anti-inflammatory properties of bis-N-norgliovictin in vitroand in vivo. Thus, bis-N-norgliovictin can be a useful therapeuticcandidate for the treatment of sepsis and other inflammatorydiseases.

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Acknowledgments

We would like to thank Dr. H. Fan and Dr. P. Li for their valuablediscussions, and Dr. Z. Yin and Dr. G. Zhao for their excellenttechnical assistance. This work was supported by the NationalNatural Science Foundation of China (90813036, 81121062).

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Appendix A. Supporting information

Supplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.ejphar.2013.02.008.

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References

Abraham, E., Anzueto, A., Gutierrez, G., Tessler, S., San Pedro, G., Wunderink, R.,Dal Nogare, A., Nasraway, S., Berman, S., Cooney, R., Levy, H., Baughman, R.,Rumbak, M., Light, R.B., Poole, L., Allred, R., Constant, J., Pennington, J., Porter,S., 1998. Double-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shock NORASEPT II StudyGroup. Lancet 351, 929–933.

Abraham, E., Singer, M., 2007. Mechanisms of sepsis-induced organ dysfunction.Crit. Care. Med. 35, 2408–2416.

Akashi, S., Saitoh, S., Wakabayashi, Y., Kikuchi, T., Takamura, N., Nagai, Y.,Kusumoto, Y., Fukase, K., Kusumoto, S., Adachi, Y., Kosugi, A., Miyake, K.,2003. Lipopolysaccharide interaction with cell surface Toll-like receptor4-MD-2: higher affinity than that with MD-2 or CD14. J. Exp. Med. 198,1035–1042.

Angus, D.C., Linde-Zwirble, W.T., Lidicker, J., Clermont, G., Carcillo, J., Pinsky, M.R.,2001. Epidemiology of severe sepsis in the United States: analysis of incidence,outcome, and associated costs of care. Crit. Care. Med. 29, 1303–1310.

Benoit, M., Desnues, B., Mege, J.L., 2008. Macrophage polarization in bacterialinfections. J. Immunol. 181, 3733–3739.

Bozza, F.A., Salluh, J.I., Japiassu, A.M., Soares, M., Assis, E.F., Gomes, R.N., Bozza, M.T.,Castro-Faria-Neto, H.C., Bozza, P.T., 2007. Cytokine profiles as markers of diseaseseverity in sepsis: a multiplex analysis. Crit. Care 11, R49.

Carracedo, J., Ramirez, R., Martin-Malo, A., Rodriguez, M., Aljama, P., 2002. Theeffect of LPS, uraemia, and haemodialysis membrane exposure on CD14expression in mononuclear cells and its relation to apoptosis. Nephrol. Dial.Transplant. 17, 428–434.

Cohen, J., 2002. The immunopathogenesis of sepsis. Nature 420, 885–891.Datla, P., Kalluri, M.D., Basha, K., Bellary, A., Kshirsagar, R., Kanekar, Y., Upadhyay,

S., Singh, S., Rajagopal, V., 2010. 9,10-Dihydro-2,5-dimethoxyphenanthrene-


1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling mediated byToll-like receptors. Br. J. Pharmacol. 160, 1158–1170.

Dou, H., Song, Y., Liu, X., Gong, W., Li, E., Tan, R., Hou, Y., 2011. Chaetoglobosin Fexfrom the marine-derived endophytic fungus inhibits induction of inflamma-tory mediators via Toll-like receptor 4 signaling in macrophages. Biol. Pharm.Bull. 34, 1864–1873.

Gerard, C., Rollins, B.J., 2001. Chemokines and disease. Nat. Immunol. 2, 108–115.Gordon, S., Martinez, F.O., 2010. Alternative activation of macrophages: mechan-

ism and functions. Immunity 32, 593–604.Gordon, S., Taylor, P.R., 2005. Monocyte and macrophage heterogeneity. Nat. Rev.

Immunol. 5, 953–964.Hams, E., Saunders, S.P., Cummins, E.P., O’Connor, A., Tambuwala, M.T., Gallagher,

W.M., Byrne, A., Campos-Torres, A., Moynagh, P.M., Jobin, C., Taylor, C.T.,Fallon, P.G., 2011. The hydroxylase inhibitor dimethyloxallyl glycine attenu-ates endotoxic shock via alternative activation of macrophages and IL-10production by b1 cells. Shock 36, 295–302.

Isaka, M., Palasarn, S., Rachtawee, P., Vimuttipong, S., Kongsaeree, P., 2005. Uniquediketopiperazine dimers from the insect pathogenic fungus Verticilliumhemipterigenum BCC 1449. Org. Lett. 7, 2257–2260.

Karaghiosoff, M., Steinborn, R., Kovarik, P., Kriegshauser, G., Baccarini, M., Donabauer,B., Reichart, U., Kolbe, T., Bogdan, C., Leanderson, T., Levy, D., Decker, T., Muller, M.,2003. Central role for type I interferons and Tyk2 in lipopolysaccharide-inducedendotoxin shock. Nat. Immunol. 4, 471–477.

Kawamoto, T., Ii, M., Kitazaki, T., Iizawa, Y., Kimura, H., 2008. TAK-242 selectivelysuppresses Toll-like receptor 4-signaling mediated by the intracellulardomain. Eur. J. Pharmacol. 584, 40–48.

Kellum, J.A., Kong, L., Fink, M.P., Weissfeld, L.A., Yealy, D.M., Pinsky, M.R., Fine, J.,Krichevsky, A., Delude, R.L., Angus, D.C., 2007. Understanding the inflamma-tory cytokine response in pneumonia and sepsis: results of the genetic andinflammatory markers of sepsis (GenIMS) study. Arch. Intern. Med. 167,1655–1663.

Kirby, G.W., Rao, G.V., Robins, D.J., 1988. New co-metabolites of gliotoxin inGliocladium virens. J. Chem. Soc. Pak. 1, 301–304.

Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., Hoffmann, J.A., 1996. Thedorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potentantifungal response in Drosophila adults. Cell 86, 973–983.

Liao, X., Sharma, N., Kapadia, F., Zhou, G., Lu, Y., Hong, H., Paruchuri, K.,Mahabeleshwar, G.H., Dalmas, E., Venteclef, N., Flask, C.A., Kim, J., Doreian,B.W., Lu, K.Q., Kaestner, K.H., Hamik, A., Clement, K., Jain, M.K., 2011. Kruppel-like factor 4 regulates macrophage polarization. J. Clin. Invest. 121,2736–2749.

Liu, X.G., Yao, M., Li, N., Wang, C.M., Zheng, Y.Y., Cao, X., 2008. CaMKII promotesTLR-triggered proinflammatory cytokine and type I interferon production bydirectly binding and activating TAK1 and IRF3 in macrophages. Blood 112,4961–4970.

Lopez-Bojorquez, L.N., Dehesa, A.Z., Reyes-Teran, G., 2004. Molecular mechanismsinvolved in the pathogenesis of septic shock. Arch. Med. Res. 35, 465–479.

Martin, G.S., Mannino, D.M., Eaton, S., Moss, M., 2003. The epidemiology of sepsisin the United States from 1979 through 2000. N. Engl. J. Med. 348, 1546–1554.

Mehta, A., Brewington, R., Chatterji, M., Zoubine, M., Kinasewitz, G.T., Peer, G.T.,Chang, A.C., Taylor Jr., F.B., Shnyra, A., 2004. Infection-induced modulation ofm1 and m2 phenotypes in circulating monocytes: role in immune monitoringand early prognosis of sepsis. Shock 22, 423–430.

Mullbacher, A., Eichner, R.D., 1984. Immunosuppression in vitro by a metabolite ofa human pathogenic fungus. Proc. Nat. Acad. Sci. U.S.A. 81, 3835–3837.

Netea, M.G., van der Meer, J.W., van Deuren, M., Kullberg, B.J., 2003. Proinflam-matory cytokines and sepsis syndrome: not enough, or too much of a goodthing? Trends Immunol. 24, 254–258.

Nishimura, S., Manabe, I., Nagasaki, M., Eto, K., Yamashita, H., Ohsugi, M., Otsu, M.,Hara, K., Ueki, K., Sugiura, S., Yoshimura, K., Kadowaki, T., Nagai, R., 2009.CD8(þ) effector T cells contribute to macrophage recruitment and adiposetissue inflammation in obesity. Nat. Med. 15 914-U116.

O’Neill, L.A.J., Bryant, C.E., Doyle, S.L., 2009. Therapeutic targeting of Toll-likereceptors for infectious and inflammatory diseases and cancer. Pharmacol.Rev. 61, 177–197.

Pahl, H.L., Krauss, B., SchulzeOsthoff, K., Decker, T., Traenckner, E.B.M., Vogt, M.,Myers, C., Parks, T., Warring, P., Muhlbacher, A., Czernilofsky, A.P., Baeuerle, P.A.,1996. The immunosuppressive fungal metabolite gliotoxin specifically inhibitstranscription factor NF-kappa B. J. Exp. Med. 183, 1829–1840.

Porcheray, F., Viaud, S., Rimaniol, A.C., Leone, C., Samah, B., Dereuddre-Bosquet, N.,Dormont, D., Gras, G., 2005. Macrophage activation switching: an asset for theresolution of inflammation. Clin. Exp. Immunol. 142, 481–489.

Prachyawarakorn, V., Mahidol, C., Sureram, S., Sangpetsiripan, S., Wiyakrutta, S.,Ruchirawat, S., Kittakoop, P., 2008. Diketopiperazines and phthalides from amarine derived fungus of the order pleosporales. Planta Med. 74, 69–72.

Qiu, P., Cui, X., Barochia, A., Li, Y., Natanson, C., Eichacker, P.Q., 2011. The evolvingexperience with therapeutic TNF inhibition in sepsis: considering the potentialinfluence of risk of death. Expert Opin. Invest. Drugs 20, 1555–1564.

Rittirsch, D., Flierl, M.A., Ward, P.A., 2008. Harmful molecular mechanisms insepsis. Nat. Rev. Immunol. 8, 776–787.

Romagne, F., 2007. Current and future drugs targeting one class of innateimmunity receptors: the Toll-like receptors. Drug Discovery Today 12, 80–87.

Saturnino, S.F., Andrade, M.V., 2007. Toll-like receptors, new horizons in sepsis.Curr. Mol. Med. 7, 522–531.

Sica, A., Mantovani, A., 2012. Macrophage plasticity and polarization: in vivoveritas. J. Clin. Invest. 122, 787–795.


1234567

89

10111213


Stout, R.D., Suttles, J., 2004. Functional plasticity of macrophages: reversibleadaptation to changing microenvironments. J. Leukocyte Biol. 76, 509–513.

Sutton, P., Newcombe, N.R., Waring, P., Mullbacher, A., 1994. In-vivo immunosup-pressive activity of gliotoxin, a metabolite produced by huam pathogenicfungi. Infect. Immun. 62, 1192–1198.

Takashima, K., Matsunaga, N., Yoshimatsu, M., Hazeki, K., Kaisho, T., Uekata, M.,Hazeki, O., Akira, S., Iizawa, Y., Ii, M., 2009. Analysis of binding site for the


novel small-molecule TLR4 signal transduction inhibitor TAK-242 and itstherapeutic effect on mouse sepsis model. Br. J. Pharmacol. 157, 1250–1262.

Wittebole, X., Castanares-Zapatero, D., Laterre, P.F., 2010. Toll-like receptor 4modulation as a strategy to treat sepsis. Mediat. Inflamm..

Zhao, W.Y., Zhu, T.J., Han, X.X., Fan, G.T., Liu, H.B., Zhu, W.M., Gu, Q.Q., 2009. A newgliotoxin analogue from a marine-derived fungus Aspergillus fumigatus Fres.Nat. Prod. Res. 23, 203–207.


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