An anti-inflammatory role of A20 zinc finger protein during trauma combined with endotoxin challenge

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An anti-inflammatory role of A20 zinc finger protein during trauma combined with endotoxin challenge Bo Liu, MD,a Dianming Jiang, MD,a Yunsheng Ou, MD,a Zhenming Hu, MD,a Jianxin Jiang, MD,b,* and Xia Lei, MDc,** a

Department of Orthopaedic Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, China State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China c Department of Dermatology, Daping Hospital, Third Military Medical University, Chongqing, China b

article info

abstract

Article history:

Objective: To investigate the anti-inflammatory role of A20 zinc finger protein during

Received 6 March 2013

trauma combined with bacterial endotoxin challenge and explore the molecular mecha-

Received in revised form

nism underlying this process.

27 May 2013

Methods: Traumatic bone impact injury was induced in the hind limbs of mice. One hour

Accepted 13 June 2013

after injury, mice were challenged with purified gram-negative bacterial endotoxins,

Available online 5 July 2013

lipopolysaccharides (LPSs), by tail vein injection. Effects on A20 messenger RNA and protein expressions were assessed by reverse transcriptionepolymerase chain reaction

Keywords:

and Western blotting, respectively. A20 recombinant adenoviruses, full-length (pAdA20 1-

Trauma

775) and N-terminal mutant (pAdA20 1-367), were constructed and used to infect RAW264.7

Lipopolysaccharide

macrophage cells or mice. Responses in the tumor necrosis factor a (TNF-a)enuclear factor

A20 zinc finger protein

kB (NF-kB) signaling pathway were evaluated by enzyme-linked immunosorbent assay (for

Anti-inflammatory

TNF-a) and electrophoretic mobility shift assay (for NF-kB). Results: Trauma combined with LPS challenge and LPS challenge alone dramatically promoted A20 expression in mouse liver tissues. LPS challenge increased A20 messenger RNA levels appreciably in RAW264.7 cells within 1 h. Full-length A20 recombinant adenoviruses (pAdA20 1-775) suppressed NF-kB activity and TNF-a expression and protected against liver damage and animal death otherwise induced by trauma combined with LPS challenge. Conclusions: A20 zinc finger protein plays an anti-inflammatory role and protects against liver injury associated with trauma combined with LPS challenge. ª 2013 Published by Elsevier Inc.

1.

Introduction

Severe traumatic injury can trigger a pathophysiologic response leading to systemic inflammation, organ dysfunction, and death. Concomitant infection exacerbates the

systemic inflammatory response and accelerates the development of life-threatening complications, such as sepsis, acute respiratory distress syndrome, and multiple organ dysfunction syndrome [1]. The gram-negative bacterial endotoxin, lipopolysaccharide (LPS), has been shown to

* Corresponding author. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China. Tel./fax: þ86 23 68757401. ** Corresponding author. Department of Dermatology, Daping Hospital, Third Military Medical University, Chongqing 400042, China. Tel.: þ86 23 68757595; fax: þ86 13228688156. E-mail addresses: [email protected] (J. Jiang), [email protected] (X. Lei). 0022-4804/$ e see front matter ª 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2013.06.031

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modulate the tumor necrosis factor a (TNF-a)enuclear factor kB (NF-kB) signaling pathway, which regulates several of the molecular processes underlying systemic inflammation, including apoptotic cell death and proinflammatory cytokine expression and activation. In particular, LPS induces TNF-a gene transcription and NF-kB protein activation, both of which are key components of systemic inflammatory response syndrome, sepsis, and septic shock [2,3]. NF-kB is a major transcription factor that regulates genes responsible for both the innate and the adaptive immune responses. The target genes include multiple proinflammatory cytokines, such as TNF-a, TNF-b, interleukin (IL) 1b, IL-2, and IL-6 [3]. Constitutive activation of NF-kB has been clinically observed in many inflammatory diseases, such as inflammatory bowel disease, arthritis, sepsis, gastritis, and asthma [4]. Therefore, efficient inhibition of NF-kB activity and downstream inflammatory cytokine production is considered a particularly promising therapeutic approach to delay or suppress the progression of systemic inflammatory response syndrome after injury. A20, a cytoplasmic zinc finger protein, has been characterized for its ability to inhibit NF-kB activity and TNF-mediated programmed cell death. A20-deficient mice exhibited severe inflammation and cachexia and were hypersensitive to both TNF and LPS treatment, suggesting an important in vivo role for the A20 zinc finger in limiting inflammation by terminating TNF-induced NF-kB responses [5]. Thus, we hypothesized that the A20 zinc finger protein may functionally contribute to the pathophysiology of trauma combined with endotoxin challenge and suppress the detrimental pathogenic processes. This study was designed to investigate the potential protective abilities of A20 against liver damage induced by trauma combined with LPS challenge.

2.

Materials and methods

2.1.

Cell culture

The mouse macrophage cell line RAW264.7 and human embryonic kidney 293 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in Roswell Park Memorial Institute 1640 culture medium (Gibco; Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/ mL). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 C and passaged at 70%e80% confluence.

2.2.

Animal model

A total of 80 healthy Kunming mice with body weights of 18e24 g were obtained from the Laboratory Animal Center, Institute of Surgery Research, Third Military Medical University (Chongqing, China). The animals were given free access to water but restricted to food for 12 h before experimentation. Mice were randomly divided into four groups (n ¼ 20 each): control, trauma, LPS challenge, and trauma combined with LPS challenge (trauma þ LPS). Anesthesia was administered by intraperitoneal injection of 1% pentobarbital sodium

(30 mg/kg). Traumatic bone impact injury was induced in the hind limbs of mice using a minitype multifunctional bioimpactor (BM-III; Department 4, Institute of Surgery Research, Third Military Medical University, Chongqing, China) as described previously [6]. In the trauma group, animals received 0.5 mL of normal saline via tail vein injection 1 h after injury. For LPS challenge, mice were administered 5 mg/kg body weight of LPS (Escherichia coli 026:B6; Difco, Detroit, MI) by tail vein injection 1 h after injury. After treatment, animals were housed under standard conditions until use in examination. All efforts were made to minimize animal suffering and reduce the number of animals used. All experimental procedures were approved by the Animal Ethics Committee of the Third Military Medical University.

2.3.

Reverse transcriptionepolymerase chain reaction

The messenger RNA (mRNA) level of A20 was examined using reverse transcriptionepolymerase chain reaction (PCR). Total RNA was extracted from liver tissues or cells at various time points (0.5, 1, 2, 4, 7, or 10 h) after final treatment injection using the TriPure (Boehringer Mannheim, Mannheim, BW, Germany) or Trizol (Invitrogen) reagents, respectively. Total RNA was retro-transcribed into double-chain complementary DNA by incubating the reaction mix (total RNA, Thermo Moloney murine leukemia virus reverse transcriptase, oligo (dT)16, dNTPs, 5 Moloney murine leukemia virus reverse transcriptase buffer, and RNasin ribonuclease inhibitor) at 37 C for 2 h, followed by 95 C for 5 min. Gene-specific primers for the A20 zinc finger and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Sangon Biotech Co, Ltd (Shanghai, China) as follows: A20, forward 50 -CACCTGAT CAACGCTGCAAAAT-30 , reverse 50 -CTTCAGGGCCTAGCTTC GAGTT-30 ; GAPDH, forward 50 -ACCTCAACTACATGGTCTAC-30 , reverse 50 -TTGTCATTGAGAGCAATCC-30 . The thermocycling conditions for A20 (316 bp) and GAPDH (802 bp) amplification were as follows: initial denaturation at 94 C for 5 min; 30 amplification cycles of 94 C for 50 s, 60 C for 58 s, and 72 C for 60 s; and a final extension step at 72 C for 8 min. The PCR products were confirmed by agarose gel electrophoresis, and a UVP GDS 8000 gel analysis system (UVP Ltd, Cambridge, UK) was used for gray-scale analysis. The integrated optical density of the A20 zinc finger gene and GAPDH was recorded. Data were averaged from three independent experiments.

2.4.

Western blotting

Liver tissues or cells were collected at the indicated time points (0.5, 1, 2, 4, 7, or 10 h) after final injection. The samples were then incubated with radioimmunoprecipitation assay buffer. Protein lysates were centrifuged at 20,000g for 30 min at 4 C. The supernatant fraction of each sample was collected, and the protein concentration was determined by Bradford assay. Total protein was separated by sodium dodecyl sulfateepolyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA). The membranes were blocked for nonspecific binding with 5% nonfat milk and then incubated with mouse anti-A20 or b-actin primary antibodies (1:1000 dilution) followed by horseradish peroxidaseeconjugated anti-mouse

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secondary antibody. The bound antibody complexes were detected using an enhanced chemiluminescence reagent (Amersham ECL; GE Healthcare, Pittsburgh, PA). Band images were scanned with UVP BioImaging Systems (UVP Ltd, Cambridge, UK) and analyzed by LabWork 3.0 software (UVP ltd, Upland, CA). The intensities of the immunoreactive bands were quantified by densitometric analysis. b-actin was used as the internal control. The relative integral absorbance of A20 zinc finger protein was calculated by the following formula: (Integral absorbance of A20 zinc finger protein)/(Integral absorbance of b-actin).

2.5.

were detected by a plate reader at 492 nm absorbance. The results were compared with a standard curve prepared with different concentrations of the standard sample.

2.8.

All data are presented as mean  standard deviations from three independent experiments. Intergroup differences were analyzed by analysis of variance and Student t-test using SPSS 11.0 software (SPSS, Chicago, IL). P values <0.05 or <0.01 were considered statistically significant.

3.

Electrophoretic mobility shift assay

NF-kB activity in liver tissues or cells was evaluated by electrophoretic mobility shift assay. Nuclear extraction was carried out using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL), according to the manufacturer’s instructions, and nuclear protein concentration was determined using the Folin’s phenol reagent. The NF-kB consensus sequence was labeled with [g-32P]-adenosine triphosphate by application of an oligonucleotide probe (Promega Corp, Madison, WI) and T4 Polynucleotide Kinase (Boehringer Mannheim, Mannheim, BW, Germany). DNA/ protein complexes were analyzed on polyacrylamide gels and then electrotransferred to a positively charged nylon membrane and UV cross-linked. Intensities of the immunoreactive DNA/protein complexes were scanned with UVP BioImaging Systems and analyzed by LabWork 3.0 software.

2.7.

Statistical analysis

In vivo and in vitro virus infection

Two green fluorescent proteinetagged A20 recombinant adenoviral plasmids, full-length (pAdA20 1-775) and Nterminal mutant (pAdA20 1-367), were constructed by use of the pAdEasy system (a kind gift from Dr. Tongchuan He, University of Chicago) as previously described [7]. RAW264.7 cells were seeded onto culture dishes (100 mm in diameter) at a density of 5  105 cells/mL. Virus infection was performed when cells reached 60%e70% confluence. Twenty-four hours after infection, cells were challenged with 100 ng/mL of LPS. For virus infection in animals, mice were randomly divided into three groups (n ¼ 10 each): control, trauma combined with LPS challenge (trauma þ LPS), and pAdA20 (1-775) virus infection combined with trauma and LPS challenge (pAdA20 1-775 þ trauma þ LPS). For LPS challenge, mice were administered 5 mg/kg body weight of LPS by tail vein injection at 1 h after injury. For virus administration, pAdA20 (1-775) virus was injected into mice at 24 h before injury and then at 1 h after injury, the mice were administered 5 mg/kg body weight of LPS by tail vein injection.

2.6.

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Enzyme-linked immunosorbent assay

The levels of TNF-a in supernatants of cells or liver tissue samples were evaluated by enzyme-linked immunosorbent assay (ELISA; TNF-a ELISA kit; Hyclone [HyClone, Logan, UT]). The samples were prepared for ELISA analysis by the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockrord, IL), according to the manufacturer’s instructions. ELISA results

Results

3.1. Expression of A20 zinc finger protein in mouse liver tissue and RAW264.7 cells after trauma combined with LPS challenge As demonstrated in Figure 1A, very low levels of A20 zinc finger mRNA were detected in mouse liver tissues under normal conditions. Traumatic bone impact injury alone did not affect the expression of A20 mRNA (P > 0.05 versus control at each indicated time point). Notably, LPS challenge alone or trauma combined with LPS challenge dramatically promoted the mRNA level of A20 zinc finger starting at 30 min after final injection. The expression peaked at 2 h after injection and gradually decreased thereafter. Nevertheless, the upregulated mRNA expression induced by LPS alone or trauma plus LPS treatment was significantly higher than that in the trauma group at each time point evaluated. The treatment-induced changes in A20 protein expression in mouse liver tissues, as detected by Western blot, followed the same patterns as that in the mRNA, except that the peak value of A20 protein occurred at 7 h after final injection (Fig. 1B). As shown in Figure 1C, the level of A20 in RAW264.7 cells increased at 0.5 h after LPS challenge, peaked at 1 h, and gradually decreased thereafter.

3.2. cells

Recombinant adenovirus infection of RAW264.7

The recombinant adenoviral plasmids encoding the fulllength or mutant A20 zinc finger gene were constructed and expressed in human embryonic kidney 293 packaging cells, which produced a high titer of each (Fig. 2A). The adenoviruses were then used to infect mouse RAW264.7 cells as described in Materials and methods, and the effect of LPS challenge on A20 protein level was evaluated by Western blotting. As expected, enhanced expression was found in cells infected with fulllength and mutant A20-based recombinant adenovirus (P < 0.05 versus LPS challenge; Fig. 2B).

3.3. Effect of A20 zinc finger protein expression on the LPS-induced cellular inflammation To determine the influence of induced A20 zinc finger protein expression on NF-kB activity in cells challenged with LPS, electrophoretic mobility shift assay was performed. As shown in Figure 3A, LPS challenge induced strong NF-kB activity in RAW264.7 cells. Infection of the full-length A20-based

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Fig. 1 e Expression of A20 zinc finger protein in response to trauma combined with LPS challenge. (A) The A20 mRNA levels in liver tissues were evaluated by reverse transcriptionePCR at 0.5, 1, 2, 4, 7, and 10 h after mice received trauma, LPS, or trauma plus LPS treatment. The amount of A20 mRNA was quantified relative to the level of GAPDH normalization control. (B) The A20 protein levels in liver tissues were examined by Western blot, and the amount of A20 zinc finger protein was quantified relative to the level of b-actin internal control. (AeB) *P < 0.05, **P < 0.01 versus trauma group, +P < 0.05 versus LPS challenge group, #P < 0.05, and ##P < 0.01 versus control. (C) The A20 mRNA levels in RAW264.7 cells that had been challenged with LPS for 0.5, 1, 2, 4, or 7 h, as detected by reverse transcriptionePCR and normalized to GAPDH. Comparison of expression at various time points. *P < 0.05 versus 1 h LPS challenge.

recombinant adenovirus greatly suppressed the LPS-induced NF-kB activity. However, infection of mutant pAdA20 had no appreciable impact on the activity of NF-kB. Moreover, the secreted TNF-a level was evaluated by ELISA using the culture medium supernatant of RAW264.7 cells. Infection of fulllength A20-based recombinant adenovirus was found to have also dramatically reduced TNF-a level secreted by RAW264.7 cells, whereas infection of mutant pAdA20 had no effect on TNF-a secretion (Fig. 3B).

3.4. Protective effect of A20 zinc finger protein on mice after trauma combined with LPS challenge Twenty-four hours after mice were given tail vein injection of full-length A20-based recombinant adenovirus, traumatic bone impact injury was induced in the hind limbs of these animals. One hour after injury, mice were challenged with LPS, and A20 protein expression was detected 9 h later. Administration of the full-length or mutant A20 adenovirus induced significant upregulation of A20 compared with the A20 level

detected in the trauma plus LPS challenge group (Fig. 4A). Moreover, the trauma combined with LPS challenge group presented with significantly enhanced amounts of alanine aminotransferase (ALT) and total bilirubin (TBIL) in blood serum compared with those of the control (Fig. 4B and C). However, pAdA20 (1-775) infection of mice treated with trauma plus LPS produced significantly decreased ALT and TBIL expression (P < 0.05 versus trauma þ LPS or A20 mutant þ trauma þ LPS). In addition, full-length A20 adenovirus administration dramatically reduced the animal mortality rate after trauma plus LPS treatment. Twenty-four hours after LPS challenge, the mouse mortality rate was only 26.7% compared with 53.3% for the trauma plus LPS treatment group (P < 0.05; Fig. 4D).

3.5. The underlying mechanism of A20 protection in mice in response to trauma combined with LPS challenge To explore the underlying mechanism of A20 zinc finger proteineinduced protection of mice liver after trauma combined with LPS challenge, we examined the NF-kB activity and TNF-a

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Fig. 2 e Full-length A20 adenovirus infection elevated A20 protein expression in RAW264.7 cells. (A) Human embryonic kidney 293 packaging cells produced a high titer of recombinant full-length pAdA20 virus after plasmid transfection, as evidenced by green fluorescent proteinepositive cells under fluorescent microscope. (B) Twenty-four hours after virus infection, cells were challenged with LPS for 3 h, and the protein level of A20 was evaluated by Western blotting. The amount of A20 protein was quantified relative to b-actin. *P < 0.05 versus 3 h LPS challenge. (Color version of figure is available online.)

Fig. 3 e Effect of A20 zinc finger protein expression on LPS-induced cellular inflammation. RAW264.7 cells were infected with full-length or mutant pAdA20 virus. Twenty-four hours after infection, cells were then challenged with LPS for another 3 h. (A) The NF-kB activity was determined by electrophoretic mobility shift assay. Quantified immunoreactivity data are presented. (B) The secreted TNF-a level in the supernatant of culture medium was determined by ELISA. *P < 0.05.

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Fig. 4 e Protective effect of A20 zinc finger protein in mice after trauma combined with LPS challenge. Twenty-four hours after mice were given tail vein injection of full-length A20-based recombinant adenovirus, traumatic bone impact injury was induced in the hind limbs of these animals. One hour after injury, mice were challenged with LPS. (A) A20 protein expression was detected by Western blotting at 9 h after LPS treatment. The amount of A20 was quantified relative to bactin. *P < 0.05 versus trauma D LPS. The blood serum ALT (B) and TBIL (C) levels (mmol/L) were assessed by biochemical tests. *P < 0.05 versus A20 full-length D trauma D LPS. (D) Twenty-four hours after LPS challenge, the mortality rate was calculated. *P < 0.05 versus A20 full-length D trauma D LPS.

expression in liver tissues. Injection of full-length A20-based recombinant adenovirus significantly reduced both the NFkB activity and TNF-a expression in liver tissues of mice treated with trauma plus LPS compared with that of noninfected (no adenovirus) mice or mice injected with mutant A20 plus trauma and LPS (Fig. 5A and B).

4.

Discussion

Severe trauma is the “first hit” for the human body, whereas endotoxin-induced inflammation may induce a more severe “second hit” after trauma [1]. The uncontrolled inflammatory responses play a critical role in the development and progression of complications after severe trauma. LPS is widely used to challenge the uncontrolled inflammation and is recognized as the trigger during the activation of cascades. Inflammatory factors serve as essential regulators in the endotoxemia after traumatic injury. Besides, liver is the

primary site for the attack of inflammatory factors induced by endotoxin. The mechanism of trauma combined with endotoxin attack is still poorly known. In this study, we determined the protective role of A20 on the regulation of inflammatory factors induced by LPS after severe trauma. Despite previous studies indicating the anti-inflammatory role of A20, the mechanism of A20-mediated liver protection in trauma combined with LPS challenge remains unclear. LPS can cause overactivation of the NF-kBemediated inflammatory signaling pathway, thereby inducing robust production of proinflammatory cytokines in the host and causing ancillary tissue damage and inappropriate activation of the immune response, with systemic detriment. As such, efficient inhibition of NF-kB activity appears to be a promising treatment approach for delaying or suppressing the progression of inflammatory diseases. The zinc finger protein A20, having a molecular weight of 80 kDa, was identified in human umbilical vein endothelial cells and originally characterized as a TNF primary response

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Fig. 5 e The underlying molecular mechanism of A20 protection in mice after trauma combined with LPS challenge. Twentyfour hours after mice were given tail vein injection of full-length or mutant A20-based recombinant adenovirus, traumatic bone impact injury was induced in the hind limbs of these animals. One hour after injury, mice were challenged with LPS. (A) The NF-kB activity was determined by electrophoretic mobility shift assay at 9 h after LPS challenge. (B) The TNF-a level in the liver tissue was determined by ELISA and presented as nanograms in total protein (gram). *P < 0.05 versus A20 fulllength D trauma D LPS.

protein [8]. This protein consists of an N-terminal domain (amino acids 1e386) and a C-terminal zinc finger domain (amino acids 387e790) [9]. A20 is currently recognized as an essential negative regulator of inflammation [5]. Using embryonic fibroblast cultured from A20-deficient mice, Lee et al. [5] revealed that A20 is also required to prevent TNFaeinduced persistent IkB kinase activity. The A20 zinc finger protein has also been demonstrated to suppress NF-kB activity by disruption of upstream ubiquitin enzyme complexes [10]. Multiple stimuli, including LPS [11], IL-1b [12], and CD40 [13], are capable of inducing A20 expression, suggesting that A20 may be a component of a negative feedback loop involved in tempering proinflammatory signaling. In the present study, the expression of A20 zinc finger protein in LPS-challenged RAW264.7 macrophage cells and mice subjected to trauma combined with LPS challenge was investigated. Our results demonstrated that normal liver tissues express low levels of A20 mRNA and protein, and traumatic injury alone has little or no effect on A20 expression. However, LPS or trauma combined with LPS challenge dramatically increases A20 expression, especially in the early stage after challenge. Consistent with the findings from our in vivo study, enhanced A20 levels were also detected in LPSchallenged cultured RAW264.7 cells during the early stage of LPS administration, suggesting that LPS could induce the rapid upregulation of intracellular A20 zinc finger protein. A previous study using MCF7 breast carcinoma cells reported that A20 was able to suppress the activation of NF-kB and

activating protein-1 transcription factors and could inhibit the related signal transduction pathways induced by TNF and IL-1 [14]. Hence, A20 appears to play a role in suppressing general inflammatory signaling pathways mediated by TNF-a and IL-1 through blockage of NF-kB and activating protein-1 activity [15,16]. Song et al. [17] have demonstrated that the N-terminal region of A20 interacts with TNF receptoreassociated factor 1. Overexpression of A20 inhibited NF-kB activation by several stimuli and subsequently suppressed NF-kBemediated inflammatory response, further confirming a functional relationship between A20 and inflammation suppression [17]. The unique characteristics of A20-deficient mice, including robust inflammation and hypersensitivity to TNF and LPS, likewise implicated A20 as an important limiting factor of inflammation [5]. Together with our observations, these studies indicate that the A20 zinc finger protein, serving as an important endogenous antiinflammatory factor, is involved in the early response to inflammation after trauma combined with LPS challenge. Upregulated expression of A20 might be of great importance for blocking inflammation during trauma-associated infection. To determine the mechanism of A20-induced antiinflammatory activity, we infected RAW264.7 cells with fulllength (pAdA20 1-775) A20 recombinant adenoviral plasmid and evaluated the influence of A20 overexpression on LPSinduced NF-kB activity and TNF expression. Our results indicated that full-length A20 recombinant adenovirus infection dramatically suppressed NF-kB activity and TNF-a generation.

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LPS is known to trigger the activation of mitogen-activated protein kinase signaling pathways, which subsequently activate nuclear translocation of NF-kB, ultimately promoting expression (and secretion) of several inflammatory molecules, including IL-2, IL-6, IL-8, TNF-a, TNF-b, granulocytemacrophage colony-stimulating factor, and interferon b [18,19]. Therefore, it is possible that in our study, LPS induced rapid intracellular expression of A20, which in turn suppressed the nuclear translocation of NF-kB and decreased generation of inflammatory molecules, such as TNF-a. Moreover, the promoter region of the A20 gene contains two kappa B elements through which its expression is mediated by NF-kB [20]. The A20-binding inhibitor of NF-kB activation interacts with the C-terminal zinc finger domain of A20, and induced expression of A20-binding inhibitor of NF-kB activation has been shown to inhibit TNF-induced NF-kB activation [21,22]. Considering that activated NF-kB regulates transcriptional expression of a diverse array of immunomodulatory genes, including E-selectin, tissue factor, vascular cell adhesion molecule 1, IL-6, IL-8, granulocyte colonystimulating factor (G-CSF), and c-myc [23], it is possible that trauma combined with LPS administration may induce NF-kB translocation, leading to either direct or indirect induction of A20 transcription. The elevated expression of A20 appeared to function within a yet undefined negative feedback loop that blocks NF-kB activation and contributes to relief of the inflammatory cascade reaction. Cytokine-mediated cell signaling plays an important role in uncontrolled inflammatory response after injury. Cytokines can alter intracellular function of the same cell (autocrine), adjacent cells (paracrine), or remote cells (endocrine), facilitating a robust power to induce a rapid and exponential systemic inflammatory response, as is seen in inflammatory diseases such as sepsis and septic shock [24,25]. TNF-a is capable of attracting macrophages, monocytes, and polymorphonuclear neutrophils to inflammatory regions, activating the p38 signaling pathway and NF-kB in these cells and stimulating further release of cytokines, which if left unchecked will damage organs [26,27]. Therefore, we investigated whether the anti-inflammatory A20 protein was upregulated in mice after trauma combined with LPS challenge. We found that when A20 was overexpressed, by full-length A20 adenovirus administration, animal mortality from this persistent inflammatory syndrome was dramatically reduced. pAdA20 (1-775) infection greatly reduced the ALT and TBIL levels induced by trauma plus LPS treatment and decreased both the NF-kB activity and the TNF-a expression in liver tissues of these animals. A20 was characterized as a protective factor against liver damage after trauma with LPS challenge, a finding that complements the severe inflammation phenotype of A20-deficient mice [5]. Together with our observations, this evidence indicates that the full-length A20 adenovirus may provide a valuable approach for preventing inflammation induced by trauma and LPS challenge. Collectively, the results from our study revealed that the A20 zinc finger protein suppresses activation of NF-kB and release of the inflammatory cytokine TNF-a, ultimately reducing liver damage and animal mortality associated with the robust systemic inflammatory response triggered by trauma with concomitant infection. Thus, A20 functions as an

important endogenous anti-inflammatory factor during the trauma-induced inflammation process. Future studies should explore the association between A20 and other upstream and downstream factors involved in LPS-triggered signal transduction. Generation of such a detailed profile of A20 signaling may identify targets for pharmacologic manipulation and effective treatment of this complex disease process.

Acknowledgment This paper was supported by the funds of the National Natural Science Foundation of China (NSFC, 30500512).

references

[1] Lenz A, Franklin GA, Cheadle WG. Systemic inflammation after trauma. Injury 2007;38:1336. [2] Chandel NS, Trzyna WC, McClintock DS, Schumacker PT. Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin. J Immunol 2000;165:1013. [3] Castellheim A, Brekke OL, Espevik T, Harboe M, Mollnes TE. Innate immune responses to danger signals in systemic inflammatory response syndrome and sepsis. Scand J Immunol 2009;69:479. [4] Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest 2001;107:7. [5] Lee EG, Boone DL, Chai S, et al. Failure to regulate TNFinduced NF-kappaB and cell death responses in A20-deficient mice. Science 2000;289:2350. [6] Zhao Y, Yang C, Wang H, et al. Therapeutic effects of bone marrow-derived mesenchymal stem cells on pulmonary impact injury complicated with endotoxemia in rats. Int Immunopharmacol 2012;15:246. [7] He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509. [8] Dixit VM, Green S, Sarma V, et al. Tumor necrosis factoralpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin. J Biol Chem 1990;265:2973. [9] Tewari M, Wolf FW, Seldin MF, O’Shea KS, Dixit VM, Turka LA. Lymphoid expression and regulation of A20, an inhibitor of programmed cell death. J Immunol 1995;154:1699. [10] Shembade N, Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science 2010;327:1135. [11] Zen K, Karsan A, Eunson T, Yee E, Harlan JM. Lipopolysaccharide-induced NF-kappaB activation in human endothelial cells involves degradation of IkappaBalpha but not IkappaBbeta. Exp Cell Res 1998;243:425. [12] Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C. A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets. J Exp Med 1999; 190:1135. [13] Sarma V, Lin Z, Clark L, et al. Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J Biol Chem 1995;270:12343. [14] Jaattela M, Mouritzen H, Elling F, Bastholm L. A20 zinc finger protein inhibits TNF and IL-1 signaling. J Immunol 1996;156: 1166.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 5 ( 2 0 1 3 ) 7 1 7 e7 2 5

[15] Duwel M, Welteke V, Oeckinghaus A, et al. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains. J Immunol 2009;182:7718. [16] Klinkenberg M, Van Huffel S, Heyninck K, Beyaert R. Functional redundancy of the zinc fingers of A20 for inhibition of NF-kappaB activation and protein-protein interactions. FEBS Lett 2001;498:93. [17] Song HY, Rothe M, Goeddel DV. The tumor necrosis factorinducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation. Proc Natl Acad Sci U S A 1996;93:6721. [18] Lin WN, Luo SF, Lee CW, Wang CC, Wang JS, Yang CM. Involvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells. Cell Signal 2007;19:1258. [19] Christman JW, Lancaster LH, Blackwell TS. Nuclear factor kappa B: a pivotal role in the systemic inflammatory response syndrome and new target for therapy. Intensive Care Med 1998;24:1131. [20] Krikos A, Laherty CD, Dixit VM. Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein,

[21]

[22]

[23] [24] [25] [26]

[27]

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A20, is mediated by kappa B elements. J Biol Chem 1992;267: 17971. Heyninck K, Kreike MM, Beyaert R. Structure-function analysis of the A20-binding inhibitor of NF-kappa B activation, ABIN-1. FEBS Lett 2003;536:135. Verstrepen L, Carpentier I, Verhelst K, Beyaert R. ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling. Biochem Pharmacol 2009;78:105. Baldwin AS Jr. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 1996;14:649. Adrie C, Pinsky MR. The inflammatory balance in human sepsis. Intensive Care Med 2000;26:364. Goris RJ. Pathophysiology of shock in trauma. Eur J Surg 2000; 166:100. Rao P, Hayden MS, Long M, et al. IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response. Nature 2010;466:1115. Dong J, Jimi E, Zeiss C, Hayden MS, Ghosh S. Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized TNFalpha-independent inflammatory disease. Genes Dev 2010;24:1709.