Anti-inflammatory and Protective Properties of Daphnetin in Endotoxin

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The Anti-inflammatory and Protective Property of Daphnetin in the Endotoxin-induced Lung Injury Wen-wen Yu, Zhe Lu, Hang Zhang, Yan-hua Kang, Yun Mao, Huan-huan Wang, Wei-hong Ge, and Li-yun Shi J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on November 30, 2014

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Journal of Agricultural and Food Chemistry

The Anti-inflammatory and Protective Property of Daphnetin in the Endotoxin-induced Lung Injury

Wen-wen Yu†,‡, Zhe Lu†, Hang Zhang†, Yan-hua Kang†, Yun Mao†, Huan-huan Wang†, Wei-hong Ge‡, Li-yun Shi*,† † Key Lab of Inflammation and Immunoregulation, School of Medicine, Hangzhou Normal

University, Hangzhou, Zhejiang 310036, China ‡ College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou,

Zhejiang 310053, China * To whom correspondence should be addressed: Liyun Shi, Key Lab of Inflammation and Immunoregulation, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, P. R. China. Tel: +86-571-28865632; Fax: +86-571-28865632. E-mail: [email protected]

Key words: inflammation, lung injury, ubiquitination

Running Title: Daphnetin Suppresses Acute Lung Injury via TNFAIP3

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ABSTRACT

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Uncontrolled inflammatory responses cause tissue injury and severe immunopathology.

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Pharmacological interference of intracellular proinflammatory signaling may confer a therapeutic

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benefit under these conditions. Daphnetin, a natural coumarin derivative, has been used to treat

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inflammatory diseases including bronchitis. However, the protective effect of daphnetin in

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inflammatory airway disorders has yet to be determined, and the molecular basis for its anti-

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inflammatory properties is unknown. We show here that daphnetin treatment conferred

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substantial protection from endotoxin-induced acute lung injury (ALI), in parallel with reductions

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in production of inflammatory mediators, symptoms of airway response, and infiltration of

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inflammatory cells. Further studies indicated that activation of macrophage and human alveolar

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epithelial cells in response to lipopolysaccharide (LPS) was remarkably suppressed by

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daphnetin, which was related to the down-regulation of NF-κB-dependent signaling events.

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Importantly, we demonstrate that TNF-α-induced protein 3 (TNFAIP3), also known as A20, was

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significantly induced by daphnetin, which appeared to be largely responsible for the down-

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regulation of NF-κB activity through modulation of non-degradative TRAF6 ubiquitination.

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Accordingly, the deletion of TNFAIP3 in primary macrophages reversed daphnetin-elicited

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inhibition of immune response, and the beneficial effect of daphnetin in the pathogenesis of ALI

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was, partially at least, abrogated by TNFAIP3 knockdown. These findings demonstrate the anti-

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inflammatory and protective function of daphnetin in endotoxin-induced lung inflammation and

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injury, and also reveal the key mechanism underlying its action in vitro as well as in vivo.

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INTRODUCTION

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Acute lung injury (ALI) is characterized by overwhelming production of proinflammatory

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mediators, accumulation of inflammatory cells and deposition of fibrin and edema in the alveolar

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space (1). An unchecked inflammatory response causes considerable tissue injury and

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progressive deterioration of lung function. Little is known regarding the cause of ALI and its


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complications; therefore, treatment is currently limited to supportive therapy, leading to a

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mortality rate approaching 40% (2).

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It has been recognized that persistent activation of nuclear factor-κB（NF-κB）is central to

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the pathogenesis of many inflammatory lung disorders. NF-κB is a ubiquitous transcription

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factor that dictates the expression of a variety of proinflammatory genes and is therefore

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regarded as a major driving force underlying inflammation and injury (3). The canonical NF-κB

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signaling pathway is induced by the stimulation of specific receptors such as toll-like receptor

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(TLR), interleukin-1 receptor (IL-1R) and tumor necrosis factor receptor (TNFR), leading to the

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recruitment of adaptor proteins such as myeloid differentiation factor88 (MyD88) or Toll/IL-1R

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homology domain-containing adaptorinducing interferon-β (TRIF) to the intracellular domain and

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activation of the downstream mediators. As a key signaling molecule in this cascade, TNF

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receptor-associated factor (TRAF) 6 serves to deliver the signaling by exerting its ubiquity ligase

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activity. It can link polyubiquitin chains to its lysine (K63) residue and other target proteins such

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as TGF-β-activated kinase 1 (TAK1), which in turn signals to the downstream mitogen activated

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protein kinase (MAPK) and/or inhibitor-κB (IκB) kinase (IKK). The activated IKK causes the

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phosphorylation and proteasome-mediated degradation of IκB, thereby facilitating the release of

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NF-κB and its entry into the nucleus, where NF-κB interacts with target motifs and initiates

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expression of proinflammatory genes (4, 5).

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Given the central role for NF-κB signaling in driving and propagating inflammatory responses,

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it is important to control the key signaling events which is critically involved in the inflammation

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and the related immunopathology. Protein ubiquitination is known to play a central role in

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regulating NF-κB-dependent signaling and inflammatory responses. It is a dynamic process

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mediated by both E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) (6). The E3

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ubiquitin ligases promote the attachment of ubiquitin to a specific lysine in the target substrate,

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forming a polyubiquitin chain with other ubiquitins or linear polyubiquitination by binding to a



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methionine residue. The type of polyubiquitin chain determines the fate of the conjugated

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substrate. A polyubiquitin chain formed through the K48 of ubiquitin

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proteasomal degradation of the modified protein, whereas the K63 polyubiquitin chain or linear

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polyubiquitination can promote the protein-protein interactions and signal transduction (7). On

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the other hand, the ubiquitination process can be reversed by DUB enzyme, which, through

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cleaving the ubiquitin chains from their substrates, leads to the termination of NF-κB signaling

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(8).

tends to cause

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Among the known DUBs, TNF-α-induced protein 3 (TNFAIP3), also known as A20, has been

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recognized as a potent modulator of NF-κB signaling. TNFAIP3 has typical deubiquitinating

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activity with its ovarian tumor (OTU) domain at its amino terminus. Also, a carboxy-terminal zinc

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finger (ZnF) domain endows it with E3 ubiquitin ligase activity (9) . In most cell types, TNFAIP3

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serves as a negative feedback regulator of the NF-κB-driven response, either by directly

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removing ubiquitin moieties from the signaling molecule of TRAF6 or through binding to TRAF6

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to prevent its interactions with Ubc13, an E2-conjugating enzyme (10, 11). Consequently,

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TNFAIP3-null cells appear to be highly activated and secrete excessive proinflammatory

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cytokines. TNFAIP3-deficient mice die prematurely from multi-organ inflammation. Moreover,

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the clinical relevance of TNFAIP3 is underscored by the fact that a loss-of-function mutation

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within the TNFAIP3 gene is closely associated with certain inflammatory and autoimmune

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diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory

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bowel disease (IBD), psoriasis and type I diabetes (12, 13).

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In the respiratory system, maintaining TNFAIP3 levels is essential for tissue homeostasis.

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TNFAIP3 acts as a checkpoint to prevent the production of the proinflammatory cytokine

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interleukin-8 (IL-8) and down-regulate TLR2- and TLR4-induced inflammation in healthy human

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bronchial airway epithelial cells (14). Loss of TNFAIP3 resulted in the amplification of pro-

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inflammatory responses and exaggerated lung inflammation and injury, and its levels showed to

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be negatively associated with the number of inflammatory cells infiltrated and the levels of 4 ACS Paragon Plus Environment

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proinflammtory cytokines (15). Recently, a report showed that TNFAIP3 levels were

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substantially lower in patients with cystic fibrosis (CF), a type of lung disease with intrinsically

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dysregulated inflammation, and that TNFAIP3 levels were proportional to forced expiratory

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volume in 1s (FEV1), indicative of lung function. Additionally, a non-functional form of TNFAIP3

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has been found in inflamed airway epithelial cells, which was unable to colocalize with TRAF6

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and fail to prevent or diminish NF-κB activation (16). Consistent with these findings, the clinic

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data critically implicated aberrant expression or dysfunction of TNFAIP3 in the pathogenesis of

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inflammatory lung disorders such as asthma, CF and chronic obstructive pulmonary disease

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(COPD) (17). Thus, TNFAIP3 is emerging as a potent diagnostic marker as well as therapeutic

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target in these diseases and the endeavor to search for the novel TNFAIP3-targed treatment is

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currently under the way.

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Daphnetin is a natural product extracted from plants of the genus Daphne and belongs to the

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coumarin family. It has been shown to possess a variety of biological properties, including anti-

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inflammation, anti-hypoxia, anti-microbial and anti-cancer properties (18,19). The traditional

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Chinese medicine Zushima, primarily composed of daphnetin, has been used to treat

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inflammatory diseases in clinic. It can alleviate collagen-induced arthritis as revealed by the

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suppressed synovial hyperplasia, joint destruction and chondrocyte degeneration, and

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accompanying this, the infiltration of inflammatory cells and production of interleurin 1β (IL-1β),

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tumor necrosis factor α (TNF-α) and macrophage migration inhibitory factor (MIF) were

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significantly repressed by daphnetin treatment (20). In addition, daphnetin exhibited the ability to

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blunt the differentiation of inflammatory Th17 and Th1 cells and promote the development of

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regulatory T (Treg) cells, thus alleviating T cell-mediated immunopathology in the inflammatory

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autoimmune disorders (21). It is noted that daphnetin has been used to treat inflammatory

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airway diseases such as bronchitis and demonstrate to be a promising alternative for these

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disorders with little side effect (18). However, the protective efficacy of daphnetin in

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inflammatory pulmonary disorders has not yet been fully explored, and particularly, the ability of 5 ACS Paragon Plus Environment


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daphnetin to antagonize inflammatory signaling and the molecular mechanism underlying its

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action remain to be determined.

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In the present study, we, for the first time, revealed the significant protective effect of

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daphnetin against lung inflammation and injury. By impairing the key signaling events involved

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in NF-κB activity, daphnetin negatively regulated the TLR-triggered inflammatory response.

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Moreover, TNFAIP3 was identified as a target of daphnetin to modulate the K63-linked

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ubiquitination of TRAF6 and thereby NF-κB-driven proinflammatory signaling. This finding

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provides insight into the action mode of daphnetin in the inflammatory context and has profound

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implications for its use as a protective or even therapeutic agent in ALI and related compliance.

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MATERIALS AND METHODS

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Reagents. Daphnetin (7, 8-dihydroxycoumarin), with a purity greater than 99.4%, was obtained

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from Tauto Biotech Co., Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO), LPS (055:B5), 3-(4,

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5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Griess reagent and 4′, 6-

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diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, USA). All

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antibodies, unless otherwise indicated, were obtained from Cell Signaling. Antibodies against β-

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actin were obtained from Sigma-Aldrich, and the pRL-TK plasmids were purchased from

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Promega. All cell culture reagents and media were obtained from Life Technologies.

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Cell Lines and Generation of Peritoneal Macrophages. The A549 and RAW264.7 cell lines

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were obtained from the American Type Culture Collection and grown in RPMI 1640 medium

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containing 10% (v/v) heat-inactivated fetal bovine serum. To prepare murine peritoneal

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macrophages, 8-10-week-old mice were injected intraperitoneally (i.p.) with 3% thioglycolate

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broth. After 72 h, the peritoneal cells were harvested, and macrophages were enriched by quick

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adhesion. All cells were grown at 37°C in a humidified atmosphere in the presence of 5% CO2.

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Cell Viability Assay. Cell viability was determined using an MTT reduction assay. In brief,

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RAW264.7 cells were treated with various concentrations of daphnetin or vehicle for 24 or 48 h.


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Then the culture supernatants were replaced with MTT (0.5 mg/ml) and the resulting dark blue

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crystals were dissolved with DMSO. The absorbance values were read at 550 nm and obtained

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by replication in at least three independent experiments.

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RNA Isolation and Quantitative RT-PCR. Total RNA was isolated using TRIzol reagent

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(Takara) following the manufacturer’s protocol. SYBR Green PCR Master Mix (Bio-Rad) was

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used to detect mRNA levels, and relative expression levels were determined by applying the

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∆∆Ct method using β-actin as the endogenous control. The following primers were used: TNF-α,

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forward 5’-AAGGCCGGGGTGTCCTGGAG-3’ and reverse 5’-AGGCCAGGTGGGGACAGCTC-

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3’;

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AGTGCATCATCGTTGTTCATAC-3’; IL-1β, forward 5’-AACCTCACCTACAGGGCGGACTTCA-

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3’ and reverse 5’-TGTAATGAAAGACGGCACACC-3’; and inducible nitric oxide synthase

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(iNOS),

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GGCTGTCAGAGCCTCGTGGCTTTGG-3’.

IL-6,

forward

forward

5’-CCACTTCACAAGTCGGAGGCTTA-3’

5’-CCCTTCCGAAGTTTCTGGCAGCAGCG-3’

and

and

reverse

reverse

5’-

5’-

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Bronchoalveolar Lavage, Differential Cell Counts and Histological Analysis. Briefly, the

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trachea was exposed through a midline incision and cannulated with a sterile 22-gauge needle.

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Bronchoalveolar lavage fluid (BALF) was obtained by flushing 3 times with 1 ml of 0.5 mM

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EDTA/PBS. After centrifugation, the supernatants were stored at -80°C until use. The total cell

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numbers in the BALF were counted with a hemocytometer, and differential cell counts were

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determined on cytospin preparations with Diff-Quick staining (IMEB). Alternatively, neutrophils

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and macrophages in the BALF were assessed through immunostaining and subsequent flow

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cytometry. For histological analyses, mouse lung samples were washed thoroughly in PBS,

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fixed in 4% (wt/vol) formalin and embedded in paraffin; 5 µM sections were then stained with

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hematoxylin and eosin (H&E) using standard procedures (22).

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Plasmid Transfection and Luciferase Reporter Assays. To investigate NF-κB reporter

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activity, the mouse PGL3-NF-κB reporter plasmid was transfected into peritoneal macrophages



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or RAW264.7 cells using Jet-ENDO transfection reagents (Polyplus). After 24 h, the cells were

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treated with DMSO or daphnetin at different concentrations (20, 40, 60, 80, and 100 µM

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respectively) followed by stimulation with LPS (100 ng/ml) for 6h. The cells were then collected

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and lysed for dual luciferase assays (Promega).

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RNA Interference. Small Interference RNA (siRNA) targeting TNFAIP3 and scramble siRNA

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were synthesized by GIMA Co. (Shanghai, China). siRNA duplexes were transfected into

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macrophages using INTERFERin-HTS according to standard protocols (Polyplus). For the in

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vivo knockdown of TNFAIP3, 60 µg of siRNA per mouse was administered intratracheally (23).

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The knockdown efficacy was determined through RT-PCR or Western blotting.

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Animal Experiments. All procedures were conducted in accordance with university

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guidelines and approved by the ethical committee for Animal Care and the Use of Laboratory

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Animals, Hangzhou Normal University. The mice were anesthetized i.p. with ketamine

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hydrochloride (100 mg/kg) and xylazine (10 mg/kg). Daphnetin was injected at 5 mg/kg (i.p.) for

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a total of 100 µl into each mouse, and 1 h later, a total volume of 50µl PBS or LPS (1 mg/kg)

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was instilled intratracheally (24). For mortality studies, C57BL/6 mice were injected with

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daphnetin (5 or 10 mg/kg, i.p.) or DMSO 1 h before LPS injection (25 mg/kg, i.p.). Survival was

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monitored twice daily for up to 6 d.

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Determination of Cytokines and MPO Levels. The levels of TNF-α, IL-6 and IL-1β were

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measured in the culture supernatants or BALF by ELISA (R&D Systems). Lung

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(Myeloperoxidase) MPO levels were determined using mouse MPO ELISA, (Hycult Biotech)

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following the manufacturers’ instructions (25).

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Immunofluorescence Staining and Confocal Microscopy. RAW264.7 cells, seeded on

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slides at 30% confluence, were pretreated with daphnetin (160 µM) or DMSO 30min prior to

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LPS stimulation. After the indicated time, the cells were collected, fixed with 100% methanol,

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washed, and permeabilized in 0.2% saponin. Upon blocking with 5% bovine serum, the cells

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were stained with primary rabbit anti-p65 overnight at 4°C and then stained with goat anti-rabbit 8 ACS Paragon Plus Environment

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IgG conjugated to Texas Red (Invitrogen). The nuclei were labeled with DAPI (Invitrogen). The

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cells were finally mounted in Vectashield and detected through fluorescence confocal

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microscopy (LSM confocal microscope, Carl Zeiss, Inc.) (26).

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Immunoprecipitation and Immunoblotting. Briefly, cell lysates were prepared, and the

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protein concentrations were determined using a Bicinchoninic acid (BCA) protein assay

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(Thermo Fisher Scientific). For the immunoprecipitation of polyubiquitinated proteins with

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linkage-specific Abs, the cells were lysed at room temperature in buffer containing 1% Triton-X-

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100, 6 M urea, and 2 mM N-ethylmaleimide (NEM). Cell lysates were precleared and then

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incubated overnight with a specific primary antibody, followed by the incubation of protein A/G

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plus agarose beads. After extensive washing, the bead-bound complexes and cell lysates were

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resolved on 10% SDS-PAGE gels and finally immunoblotted with the appropriate monoclonal

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antibodies (27). Targeted proteins were visualized using an ECL Western blotting kit (Millipore).

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Statistical Analyses. The data are presented as the means ± SD of independent

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experiments. The statistical significance between two groups was analyzed using Student’s t-

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test. Survival data were analyzed with the Kaplan-Meier method and the log-rank test.

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Differences with a p value of 0.05 or less were considered significant.

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RESULTS

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Daphnetin Confers Protection Against LPS-induced Lung Inflammation and Injury.

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Daphnetin is a natural plant-derived product with the chemical structure of 7, 8-

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dihydroxycoumarin, which showed to have no notable effect on cell survival as revealed in our

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study (Fig. 1A, B). It has long been used in the treatment of inflammatory disorders and is

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therefore considered to have potential to regulate the immune response. To further understand

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the biological activity of daphnetin, we used a prototypical model of LPS-induced lung

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inflammation and injury, which has been widely used to investigate the mechanisms of ALI

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(28,29). Histopathological examination revealed that, compared with the control mice, the



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animals pretreated with daphnetin exhibited lessened alveolar damage and inflammation,

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characterized by reduced interstitial edema and debris deposit, less inflammatory cell recovery

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and decreased MPO activity in lung tissues (Fig. 2A-C). Moreover, protein leakage into the

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BALF and production of proinflammatory cytokines including IL-6, TNF-α and IL-1β were

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repressed by daphnetin (Fig. 2D and 2E). Based on these results, daphnetin appeared to

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protect against endotoxin-induced lung inflammation and injury.

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To further understand its protective role in the inflammatory setting, we evaluated the effect of

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daphnetin in sepsis-induced mortality. The mice were preinstilled with daphnetin (5 or 10 mg/kg,

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i.p.) or vehicle and then challenged with lethal doses of LPS. Animal survival was monitored for

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up to 6 days. As shown in Fig. 2F, the mice treated with daphnetin exhibited significantly

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prolonged survival compared with the animals pretreated with the vehicle, and a larger

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protection was conveyed by daphnetin at the higher dose relative to the lower dose.

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Daphnetin Negatively Regulates the Inflammatory Response. Given the importance of

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macrophages in the pathogenesis of lung inflammation and injury, we then assessed the effect

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of daphnetin in the LPS-stimulated macrophage response. As expected, LPS stimulation in

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macrophages caused a remarkable production of proinflammatory cytokines, including IL-6,

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TNF-α and IL-1β, at the mRNA and protein levels, which nevertheless was significantly

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repressed by daphnetin in a dose- and time-dependent manner (Fig. 3A and 3B). Because nitric

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oxide (NO) is a well-known mediator in ALI pathogenesis, we examined the expression of

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inducible nitric-oxide synthase (iNOS), the enzyme required for NO production. The results

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showed a profound reduction in iNOS expression in daphnetin-treated macrophages (Fig. 3C),

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suggesting a general effect of daphnetin on the production of the inflammatory mediators.

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Unexpectedly, expression of IL-10, an anti-inflammatory cytokine, was also found to be

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repressed (Fig. 3D). Furthermore, we extended the study into A549 cells, human alveolar

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epithelial cells critically involved in lung inflammation. The results showed that LPS-induced

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expression of IL-6 and TNF-α in A549 cells was also markedly suppressed by daphnetin (Fig. 10 ACS Paragon Plus Environment

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2E). Together, the data defined a crucial role for daphnetin in the negative regulation of TLR-

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triggered innate and inflammatory responses.

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Daphnetin Modulates NF-κB and MAPK Signaling. It is known that NF-κB and MAPK

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signaling are essential for the expression of proinflammatory genes in response to TLR

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stimulation. To investigate the molecular mechanism through which daphnetin controls

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inflammatory cytokine production, we analyzed the key signaling events involved. As shown in

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Fig. 4A, the phosphorylation of MAPKs, including p38, ERK and JNK kinases, was down-

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regulated by daphnetin. In addition, daphnetin suppressed LPS-stimulated NF-κB activation,

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and the critical signaling events including phosphorylation of RelA/NF-κB, phosphorylation and

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degradation of IκBα, and activation of the upstream kinase IKK were substantially impaired by

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daphnetin treatment in murine macrophages (Fig. 4B-D). Consistent with these results, NF-κB-

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driven transcriptional activity in RAW264.7 and A549 cells was also repressed by daphnetin in a

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dose-dependent manner (Fig. 4E). Additionally, considering that derepression of Rel/NF-κB

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from the restrain of IκBα and the translocation of RelA into the nuclear constitutes the key step

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for the proinflammatory gene transcription, we thus detected the impact of daphnetin on LPS-

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initiated nuclear translocation of RelA. The result clearly showed that a robust nuclear

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translocation of RelA induced by LPS was remarkably affected in daphnetin-treated

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macrophages, particularly at the early stage of activation (Fig. 5A and 5B). Together, daphnetin

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profoundly modulated the key signals required for the expression of proinflammatory genes,

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which might contribute to its anti-inflammatory property and protective effect in ALI.

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TNFAIP3 is Targeted by Daphnetin During the Inflammatory Response. Next, we sought

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to obtain further insights into the mechanism used by daphnetin to regulate NF-κB and MAPK

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signaling. TAK1, a member of the MAP3 kinase family, is presumed to be a key intermediate

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that transmits signals to the downstream IKK and MAPKs (30). Our present study showed that

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TAK1 phosphorylation was remarkably attenuated in daphnetin-treated macrophages as

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compared with that in the control cells (Fig. 6A). We then questioned whether TRAF6, which is 11 ACS Paragon Plus Environment


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known to associate with TAK1 and has the potential to regulate its activity, was affected by

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daphnetin in LPS-stimulated macrophages. TRAF6 is a RING domain ubiquitin ligase that

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mediates the assembly of K63-linked poly-Ub chains required for TAK1 and the subsequent IKK

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activation. As depicted in Fig. 6B, our data showed that TRAF6 level was not altered upon

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daphnetin treatment. However, K63-linked ubiquitination of TRAF6 triggered by LPS was

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substantially suppressed in daphnetin- but not vehicle-treated cells. We next wondered what

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drives the daphnetin-induced modulation of TRAF6 ubiquitination. As protein ubiquitination is a

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dynamic process tightly regulated by ubiquitinating and deubiquitinating enzymes, our attention

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was focused on the impact of daphnetin on TNFAIP3, an ubiquitin-editing enzyme with the

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ability to cleave K63-linked polyubiquitination of TRAF6 and thereby counteract NF-κB signaling.

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Indeed, in a dose- and time-dependent manner, treating macrophages with daphnetin caused a

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significant increase in TNFAIP3 expression, either at mRNA or protein levels (Fig. 6C and 6D).

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The mice pretreated with daphnetin exhibited enhanced expression of TNFAIP3 in lung tissues

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following LPS challenge when compared with the control animals (Fig. 6E and 6F). The data

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thus implicated TNFAIP3 activity in daphnetin-mediated regulation on the inflammation and

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lung injury..

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TNFAIP3 is Required for Daphnetin-mediated Anti-inflammatory Activity. To further

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relate TNFAIP3 to the action mode of daphnetin, we knocked down TNFAIP3 in macrophages

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using specific siRNA. As expected, TNFAIP3 deficiency in macrophages led to enhanced IκBα

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phosphorylation and degradation (Fig. 7A), indicative of augmented activation of NF-κB activity.

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And NF-кB transcriptional activity in response to LPS was consistently strengthened upon

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TNFAIP3 loss. Strikingly, specific interference of TNFAIP3 in daphnetin-treated cells elevated

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NF-кB transcriptional activity to a level comparable to that in DMSO-treated cells, indicating that

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daphnetin-driven down-regulation of NF-кB activity was largely abrogated by TNFAIP3 deletion

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(Fig. 7B). Consistent with these data, defective production of IL-6, TNF-α and IL-1β upon

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daphnetin administration was, to varying degrees, restored by TNFAIP3 knockdown (Fig. 7C 12 ACS Paragon Plus Environment

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and 7D). The data thus established the importance of TNFAIP3 in the anti-inflammatory activity

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of daphnetin in macrophages. Furthermore, TNFAIP3 protein in murine lungs was knockdown

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through the intratracheal delivery of the specific siRNA prior to daphnetin treatment, and the

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response of the mice to the endotoxin was evaluated (23). As shown in Fig. 8A, there occurred

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about 80% less TNFAIP3 protein in the mice given with TNFAIP3-specific siRNA relative to

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control siRNA at 48 h post delivery. The symptoms of lung inflammation and injury induced by

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LPS in TNFAIP3 siRNA-treated mice were significantly greater than those of their counterparts

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instilled with non-specific siRNA (Fig. 8B). Also, the repressed activation of p65/NF-κB by

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daphnetin was elevated by TNFAIP3 siRNA treatment, and production of inflammatory

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cytokines, protein leakage and MPO activity showed to be exaggerated by TNFAIP3 silence in

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daphnetin-treated mice, as compared with the animals receiving non-specific siRNA (Fig. 8C-F).

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The data thus suggested that TNFAIP3-mediated negative regulation of proinflammatory

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signaling contributed substantially to the anti-inflammatory activity and protective effect of

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daphnetin in ALI.

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DISCUSSION

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The unrestrained activation of leukocytes and persistent lung inflammation are presumed to

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be the major reason for excessive lung injury or acute respiratory distress (ARDS). Despite

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pharmacological developments over the last several decades, inflammation-associated

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pulmonary diseases still result in high morbidity and mortality rates. Exploring effective

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treatments is therefore of critical importance. Using a prototypical model of LPS-induced

303

inflammation and lung injury, we for the first time have established the protective effect of

304

daphnetin in inflammatory airway disorders and suggest a novel promising therapy for these

305

conditions. In particular, we demonstrated that daphnetin alleviated the symptoms of lung injury,

306

blocked the infiltration of inflammatory cells and dampened the expression of proinflammatory

307

cytokines such as IL-6, TNF-α and IL-1β, which are highly expressed in patients with severe 13 ACS Paragon Plus Environment


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308

pulmonary inflammatory diseases (31). In addition, animals treated with daphnetin displayed a

309

remarkable survival advantage in endotoxin shock, highlighting its beneficial effect in the

310

inflammation-associated disorders. More importantly, we demonstrate that daphnetin targeted

311

the DUB TNFAIP3 and selectively reversed TRAF6 ubiquitination to limit the NF-κB-driven

312

proinflammatory signaling. The study thus revealed the action mode of daphnetin in

313

counteracting inflammatory response, providing insights into the biological properties and

314

protective applications of this naturally occurring coumarin compound.

315

The NF-κB signaling pathway is essential in the transcription of most pro-inflammatory

316

cytokines and other effector molecules, thereby contributing to an excessive inflammatory

317

response and tissue injury when uncontrollably activated. Enhanced and prolonged NF-κB

318

activation has been described in chronic inflammatory lung diseases (32, 33). In keeping with its

319

therapeutic effect in LPS-induced pulmonary inflammation and injury, daphnetin exhibited the

320

ability to potentially modulate NF-κB signaling mostly by disturbance of the major events in this

321

cascade. We have shown that daphnetin reduced the phosphorylation and degradation of IκBα,

322

retarding the release of NF-κB and the subsequent nuclear translocation, the presumed

323

determinant for the initiation of transcription of proinflammaotry genes. In addition, we revealed

324

a role for daphnetin in modulating the TAK1/IKK axis upon LPS stimulation. TAK1 is a master

325

kinase that mediates the activation of IKK and MAPKs (28-30). Upon stimulation, TAK1 forms

326

ternary complexes with its adaptor molecule TAK1-binding proteins (TABs), conveying the

327

signaling to IKK and promoting phosphorylation and proteasome-mediated degradation of IκBα

328

(34). TAK1 thus becomes a key point in TLR-triggered inflammatory signaling and is vulnerable

329

to multiple layer of modulation, particularly to the post-translational mechanism. For example, it

330

has been revealed that phosphorylation of Thr-184/187 in the activation loop of TAK1 correlates

331

with its kinase activity (35). Our data showed that daphnetin significantly repressed TAK1

332

phosphorylation at Thr-184/187, which might account for the down-regulation of IKK and

333

MAPKs by daphnetin. Additionally, TAK1 was previously reported to be regulated by 14 ACS Paragon Plus Environment

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334

ubiquitination, methylation or O-GlcNAcylation (36). The DUB cylindromatosis (CYLD) proved to

335

physically interact with TAK1 and inhibit its ubiquitination and the resultant catalytic activity (37).

336

In our present study, we demonstrated that modulation of TAK1 by daphnetin in LPS-stimulated

337

macrophages was most likely through down-regulation of TRAF6 activity. Despite the previous

338

propose that TAK1 exerted an effect on the activity JNK and p38 MAPKs downstream of MKK6

339

signaling, a recent investigation indicated that, TAK1 also modulated ERK activity via regulation

340

of IKK-β activity (38). This agrees with our finding that, along with the defective TAK1 activity in

341

daphnetin-treated macrophage, LPS-induced MAPK activation was also profoundly affected.

342

Together, our data indicated that daphnetin might act on TAK1 and/or the upstream kinase to

343

disrupt IKK and MAPKs for termination of the inflammatory signaling.

344

Protein ubiquitination is a process that has been suggested to play important roles in a variety

345

of cellular events, including DNA repair, signal transduction, and receptor endocytosis (39).

346

Modification of TRAF6 by addition of K-63-linked polyubiquitin (polyUb) chains is essential for

347

the TLR-induced inflammatory response. The RING type E3 ligase TRAF6 can promote K63-

348

linked ubiquitination within the kinase domain, facilitating TAK1 phosphorylation and hence IKK-

349

NF-κB activation and transcription of proinflammatory cytokine gene expression (40). We herein

350

revealed that daphnetin impaired K63-linked ubiquitination of TRAF6 in LPS-stimulated

351

macrophages without altering TRAF6 protein level, thereby implicating modulation of the

352

nondegradative polyubiquitination of TRAF6 in the anti-inflammatory action of daphnetin. Given

353

the key role for TRAF6 in NF-κB-driven signaling, it can be expected that aberrant expression or

354

dysfunction of TRAF6 might cause the inflammation-associated disorders (41), and

355

understanding of the mechanism responsible for TRAF modulation should be helpful in search

356

of novel treatments. One important finding in the present study is the identification of TNFAIP3

357

as a target by daphnetin in regulating the inflammatory response. TNFAIP3 has been

358

recognized as a key regulator that dampens NF-κB signaling mostly by removing K63-linked

359

polyubiquitin chains from TRAF6 (13, 42). Our study revealed that daphnetin enhanced 15 ACS Paragon Plus Environment


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360

TNFAIP3 expression in macrophages as well as in lung tissues following LPS challenge. Using

361

TNFAIP3-specific siRNA, we showed that TNFAIP3 deletion in macrophages overcame the

362

daphnetin-elicited suppression of IκBα degradation, and hence, NF-кB transcriptional activity.

363

Thus, the reduced expression of IL-6 and TNF-α caused by daphnetin was almost abolished.

364

Most strikingly, when TNFAIP3 was knocked down in the airway prior to endotoxin challenge,

365

the protective effect of daphnetin appeared to be, at least partially, abrogated. These results

366

thereby establish a critical role for TNFAIP3 in the protective effect of daphnetin and suggest a

367

promising treatment for the inflammatory lung disorders. Indeed, the beneficial effect of

368

TNFAIP3 in counteracting airway inflammation has been documented. It has been found that

369

TNFAIP3 expression in bronchial airway epithelial cells or other innate immune cells is required

370

for the restricted NF-κB activation upon engagement of TLRs, IL-1R, IL-33R and even high-

371

affinity IgE receptor（FcεRI）(43, 44). In addition, TNFAIP3 contributed substantially to the

372

relief of lung immunopathology caused by infection of Pseudomonas aeruginosa or influenza

373

virus (45, 46). Recently, a trial on TNFAIP3-targeted gene therapy was conducted in allergic

374

asthma mice via the adenoviral transfer method, which showed that, compared with controls,

375

the animals with enforced TNFAIP3 expression can decrease the production of mucin and

376

inflammatory cytokines and attenuate the airway hyperresponsiveness (47). These findings

377

highlight the potential of TNFAIP3-targeted treatment in inflammatory lung disorders that are

378

largely associated with exaggerated NF-κB activity. However, the strategy by gene transfer of

379

functional TNFAIP3 into the airway may be limited by ethical issues. Pharmaceutical induction

380

of TNFAIP3 or members of the TNFAIP3 ubiquitin-editing complex has yet to be developed and

381

will require a significant financial and time investment. In view of the established low toxicity,

382

relatively low cost, and availability in herbal plants, the natural compound daphnetin and other

383

similar plant derivatives might have implications in treating inflammatory respiratory diseases

384

(48, 49). Even though, we noted that daphnetin pretreatment, in the absence of TNFAIP3, did


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385

not fully abolish its protection of the mice from LPS-induced lung injury and inflammation,

386

suggesting that daphnetin might also act via other antiinflammatory mechanisms.

387

In conclusion, we provide the first evidence to establish a beneficial effect of daphnetin in

388

LPS-induced inflammation and pulmonary injury and elucidate a molecular mechanism through

389

which daphnetin dampens NF-κB-dependent inflammatory signaling. Our results might serve as

390

a basis for the development of new approaches to lessen the severity of inflammation through

391

use of the naturally occurring coumarin derivative.

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26. Kang, Y.; Wang, F.; Lu, Z.; Ying, H.; Zhang, H.; Ding, W.; Wang, C.; Shi, L. MAPK kinase 3 potentiates Chlamydia HSP60-induced inflammatory response through distinct activation of NF-kappaB. J Immunol 2013, 191(1), 386-394. 27. Yang, M.; Wang, C.; Zhu, X.; Tang, S.; Shi, L.; Cao, X.; Chen, T. E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC{zeta}. J. Exp. Med. 2011, 208(10), 2099-2112. 28. Do-Umehara, H. C.; Chen, C.; Urich, D.; Zhou, L.; Qiu, J.; Jang, S.; Zander, A.; Baker, M. A.; Eilers, M.; Sporn, P. H.; Ridge, K. M.; Sznajder, J. I.; Budinger, G. R.; Mutlu, G. M.; Lin, A.; Liu, J. Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ. Nat. Immunol. 2013,14(5):461-469 29. Islam, M. N.; Das, S. R.; Emin, M. T.; Wei, M.; Sun, L.; Westphalen, K.; Rowlands, D. J.; Quadri, S. K.; Bhattacharya, S.; Bhattacharya, J. Mitochondrial transfer from bone-marrowderived stromal cells to pulmonary alveoli protects against acute lung injury. Nat. Med. 2012, 18(5):759-765 30.Sato, S.; Sanjo, H.; Takeda, K.; Ninomiya-Tsuji, J.; Yamamoto, M.; Kawai, T.; Matsumoto, K.; Takeuchi, O.; Akira, S. Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat. Immunol. 2005, 6 (11), 1087-1095. 31. Park, W. Y.; Goodman, R. B.; Steinberg, K. P.; Ruzinski, J. T.; Radella, F. N.; Park, D. R.; Pugin, J.; Skerrett, S. J.; Hudson, L. D.; Martin, T. R. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 2001, 164(10 Pt 1), 1896-1903. 32. Lim, J. H.; Stirling, B.; Derry, J.; Koga, T.; Jono, H.; Woo, C. H.; Xu, H.; Bourne, P.; Ha, U. H.; Ishinaga, H.; Xu, H.; Andalibi, A.; Feng, X. H.; Zhu, H.; Huang, Y.; Zhang, W.; Weng, X.; Yan, C.; Yin, Z.; Briles, D. E.; Davis, R. J.; Flavell, R. A.; Li, J. D. Tumor suppressor CYLD regulates acute lung injury in lethal Streptococcus pneumoniae infections. Immunity 2007, 27(2), 349-360. 20 ACS Paragon Plus Environment

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33.Deng, J. C.; Cheng, G.; Newstead, M. W.; Zeng, X.; Kobayashi, K.; Flavell, R. A.; Standiford, T. J. Sepsis-induced suppression of lung innate immunity is mediated by IRAK-M. J. Clin. Invest. 2006, 116(9), 2532-2542. 34. Wang, C.; Deng, L.; Hong, M.; Akkaraju, G. R.; Inoue, J.; Chen, Z. J. TAK1 is a ubiquitindependent kinase of MKK and IKK. Nature 2001, 412(6844), 346-351. 35. Yu, Y.; Ge, N.; Xie, M.; Sun, W.; Burlingame, S.; Pass, A. K.; Nuchtern, J. G.; Zhang, D.; Fu, S.; Schneider, M. D.; Fan, J.; Yang, J. Phosphorylation of Thr-178 and Thr-184 in the TAK1 T-loop is required for interleukin (IL)-1-mediated optimal NFkappaB and AP-1 activation as well as IL-6 gene expression. J. Biol. Chem. 2008, 283(36), 24497-24505. 36. Sakurai, H. Targeting of TAK1 in inflammatory disorders and cancer. Trends Pharmacol. Sci. 2012, 33(10), 522-530. 37.Ahmed, N.; Zeng, M.; Sinha, I.; Polin, L.; Wei, W. Z.; Rathinam, C.; Flavell, R.; Massoumi, R.; Venuprasad, K. The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation. Nat. Immunol. 2011, 12(12), 1176-1183. 38. Bhattacharyya, S.; Ratajczak, C. K.; Vogt, S. K.; Kelley, C.; Colonna, M.; Schreiber, R. D.; Muglia, L. J. TAK1 targeting by glucocorticoids determines JNK and IkappaB regulation in Toll-like receptor-stimulated macrophages. Blood 2010, 115(10), 1921-1931. 39. Chen, B. B.; Coon, T. A.; Glasser, J. R.; McVerry, B. J.; Zhao, J.; Zhao, Y.; Zou, C.; Ellis, B.; Sciurba, F. C.; Zhang, Y.; Mallampalli, R. K. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation. Nat. Immunol. 2013, 14(5), 470479. 40. Deng, L.; Wang, C.; Spencer, E.; Yang, L.; Braun, A.; You, J.; Slaughter, C.; Pickart, C.; Chen, Z. J. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitinconjugating enzyme complex and a unique polyubiquitin chain. Cell 2000, 103(2), 351-361. 41. Song, Z.; Yao, C.; Yin, J.; Tong, C.; Zhu, D.; Sun, Z.; Jiang, J.; Shao, M.; Zhang, Y.; Deng, Z.; Tao, Z.; Sun, S.; Bai, C., Genetic variation in the TNF receptor-associated factor 6 gene 21 ACS Paragon Plus Environment


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Footnotes 1. Funding This work was supported from National Key Scientific Research Project (2012CB911200), National Natural Scientific Funds (81270066 and 30872262), the funds from the Ministry of Education (201070, NCET-08-0927) and from the Provincial and Municipal Funds (2011R10027, LY12H1007, 2012C37052 and 20100633B11). 2. Acknowledgments We thank Ms. Hongping Ying (Center of Medical Research, Hangzhou Normal University) for histological analysis of lung tissues. 3. Abbreviations: TNFAIP3, TNF-α-induced protein 3; ALI, acute lung injury; MAPK, mitogen-activated protein kinase; DAPH, daphnetin; DMSO, dimethyl sulphoxide; TRAF6, TNF receptor-associated factor; IκB, inhibitor of κB; NF-κB, nuclear factor-κB; iNOS, inducible NO synthase; DUB, deubiquitinating enzyme; TLR, Toll-like receptor; JNK, c-Jun NH2-terminal protein kinase; ERK, extracellular signal-regulated kinase; MPO, myeloperoxidase; WT, wild type; KO, knockout; PRR, pattern recognition receptor; BALF: bronchoalveolar lavage fluid; TNFR, tumor necrosis factor receptor; MyD88, myeloid differentiation factor88; 4. Conflict of Interests The authors have no conflicting financial interests.


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Figure Legends FIGURE 1. Chemical structure and effect of daphnetin on macrophage survival. (A) The chemical structure of daphnetin. (B) Viability of RAW264.7 cells, that were treated with daphnetin (DAPH) at the dose indicated for 48h, was analyzed by the MTT assay. The data are represented as the mean ± SD of three independent experiments. FIGURE 2. Daphnetin renders the mice resistant to LPS-induced inflammation and acute lung injury. (A-E) Age- and sex-matched C57BL/6 mice (5 mice/group) were pretreated with DAPH (5 mg/kg, i.p.) or DMSO as a vehicle and then intratracheally challenged with LPS (1 mg/kg) or PBS. 12 h later the animals were sacrificed for the analysis. (A) Reprehensive H&E staining of lung tissues; (B) BAL fluid cell recovery; (C) MPO activity of lung tissues; (D) protein and (E) cytokine levels in BAL fluid. Original magnification, x 200. All the results are expressed as the means ± SD. *P < 0.05, **P < 0.01 by student’s t-test. (F) Mice were injected with DAPH (5 or 10 mg/kg, i.p.) or DMSO 1 h before LPS administration (25 mg/kg, i.p.). Kaplan-Meier survival plots were depicted and comparisons were made with the log-rank statistic (n = 10 per group). FIGURE 3. Daphnetin inhibits LPS-triggered production of proinflammatory cytokines. (AD) RAW264.7 cells were pretreated with DMSO or DAPH for 30 min at the dose indicated, and followed by LPS (100 ng/ml) stimulation for 6 h (for the mRNA) or 24 h (for the protein). Expression of IL-6, TNF-α and IL-1β, at the mRNA level (A) and protein level (B) was assessed by quantitative real-time PCR and ELISA, respectively; (C, D) Time- and does-dependent inhibition of iNOS and IL-10 expression by daphnetin. (E) LPS-induced production of IL-6 and TNF-α in A549 cells with pretreatment of DAPH (160 µM) or DMSO. The data are presented as the means ± SD of three independent experiments. * p

Anti-inflammatory and Protective Properties of Daphnetin in Endotoxin

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