Up-Regulation of IRAK-M is Essential for Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccharide in Kupffer Cells

Journal of Surgical Research 150, 34 –39 (2008) doi:10.1016/j.jss.2007.12.759

Up-Regulation of IRAK-M is Essential for Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccharide in Kupffer Cells Zuo-Jin Liu, Ph.D.,*,†,1 Lu-Nan Yan, Ph.D.,† Xu-Hong Li, Ph.D.,* Fa-Liang Xu, Ph.D.,* Xian-Feng Chen, M.D.,* Hai-Bo You, M.D.,* and Jian-Ping Gong, Ph.D.* *Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China; and †Center of Liver Transplantation, West China Hospital, Sichuan University, Chengdu, China Submitted for publication August 5, 2007

Western blot, the activities of NF-␬B were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzymelinked immunosorbent assay. Results. The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-␬B activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01). Conclusions. Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-␬B mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis. © 2008

Background. Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptorassociated kinase M (IRAK-M) expression. The aim of this study is to clarify weather or not IRAK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-␬B) mediated pathway. Materials and methods. KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 ⴛ 10 6 cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-Mshort hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco’s modified Eagle’s medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined by reverse transcription-polymerase chain reaction and

Elsevier Inc. All rights reserved.

Key Words: endotoxin tolerance; IRAK-M; RNA interference; Kupffer cells; LPS. INTRODUCTION

Endotoxin or lipopolysaccharide (LPS), a glycolipid of the cell membrane of Gram-negative bacteria, is one of the most potent inducer of inflammation that acts via stimulating macrophages and other immune cells through toll-like receptors (TLRs) by pathogen-associated molecu-

1

To whom correspondence and reprint requests should be addressed at Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing University of Medical Sciences, 74 Linjiang Road, Chongqing 400010, PR China. E-mail: gongjianping11@ 126.com.

0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.

34

LIU ET AL.: UP-REGULATION OF IRAK-M

lar patterns (PAMPs), to produce various proinflammatory cytokines including tumor necrosis factor-alpha (TNF-␣) [1– 4]. Although these proinflammatory molecules are indispensable for counteracting the growth and dissemination of bacteria, excessive and prolonged activation of innate immunity is harmful to the host, which is characterized by fever, acute respiratory failure, and in a large number of cases, death [5]. Interestingly, a prior exposure to a low level of LPS induces a transient state of refractoriness to subsequent LPS exposure, a phenomenon known as endotoxin tolerance (ET). It has been well recognized that induction of ET protects the host from cellular damage caused by hyperactivation of macrophages and likely represents a mechanism of immune cell adaptation to a persistent bacterial infection [6 – 8]. While many investigators are actively studying this phenomenon, the mechanism underlying ET has not been fully elucidated. Changes of cell surface molecules, signaling proteins, proinflammatory and anti-inflammatory cytokines, and other mediators have been examined. Recently, transgenic mice lacking Interleukin-1 receptor-associated kinase M (IRAK-M) exhibited enhanced inflammatory responses induced by TLR ligands, indicating that IRAK-M negatively regulates TLRs signaling and may be involved in the mechanisms of ET [9]. Liver macrophages, Kupffer cells (KCs), which preside in a strategic position within liver sinusoids, are continually exposed to various PAMPs, including LPS; therefore, ET is an important mechanism to maintain the homeostasis of KCs [10]. It has been reported that LPS-induced excessive release of cytokines from KCs contributes to organ damage during sepsis [11–14]. However, no studies about alteration of IRAK-M during ET development in KCs have been performed. In this study, we used a vector-based siRNA expression system, which overcomes the limitations of transience and high cost of synthetic siRNAs, to specifically inhibit IRAK-M expression. By assessing the nuclear factor-kappa B (NF-␬B) activation and TNF-␣ protein production in isolated C57BL/6J mice KCs, We have demonstrated that silencing of IRAK-M obviously dampened ET induced by a low dose of LPS (10 ng/mL) pretreatment prior to stimulation with 100 ng/mL LPS, supporting our view that IRAK-M negatively regulates TLRs signaling and participates in the mechanisms of ET in KCs through dampening NF-␬B mediated pathway. MATERIALS AND METHODS Experimental Animals Male C57BL/6J mice, 8 to 10 wk old weighing between 19 and 21 g were purchased from the Chongqing experimental animal center of Chongqing University of Medical Sciences. They were kept at 24°C, 55% humidity, 12 h day-night rhythm. All animals received humane

35

care in accordance with the National Institutes of Health guidelines and the legal requirements in China.

KCs Preparation and Culture KCs were isolated from mouse liver by collagenase digestion and differential centrifugation using Percoll as described elsewhere [12]. Briefly, the liver was excised after perfusion via the portal vein with Ca 2⫹ and Mg 2⫹ free Hanks’ balanced salt solution containing 0.05% collagenase IV (Sigma, St. Louis, MO) at 37°C and cut into small pieces in collagenase buffer. The suspension was filtered through nylon gauze, and the filtrate was centrifuged at 450 g for 10 min at 4°C. Cell pellets were resuspended in buffer, parenchymal cells were removed by centrifugation at 50 g for 3 min, and the nonparenchymal cells were centrifuged on a 70%:30% Percoll gradient (Sigma) at 600 g for 10min. KCs concentrated at the interface of the 30% and 70% were collected and cultured in 24-well culture plates (Sarstedt, Newton, NC) at a density of 1 ⫻ 10 6 cells/well in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL of penicillin G and 100 mg/mL of streptomycin sulfate) at 37°C in the presence of 5% CO 2. Nonadherent cells were removed after 1 h by replacing the buffer. All adherent cells phagocytized latex beads, indicating that they were KCs. Viability of KCs determined by trypan blue exclusion was ⬎90%.

Plasmid Constructs for IRAK-M Short Hairpin RNAs (shRNA) A vector-based shRNA expression system was used to endogenously express shRNA in mammalian cells. We selected the target regions in the exons 285 and 303 bp boundary of IRAK-M cDNA according to Tushul ’s principle and designed DNA oligonucleotides for in vitro transcription. The target sequences for murine IRAK-M were 5=-TCA ACG AGC TAT CCA CTT A-3= (S1) and 5=-TAA GTG GAT AGC TCG TTG A-3= (S2). The DNA sequences were cloned into the BamHI/HindIII restriction site of the pGenesil vector (Genesil Biotechnology Co., Wuhan, China), containing the cDNA of GFP and karamycin resistance gene. The control RNAi vector (Genesil Biotechnology Co.) was constructed by insertion of a scrambled sequence that did not show significant sequence homology to rat, mouse, or human gene sequences. All of the inserted sequences were identified with restriction-endonuclease digestion and sequencing. The doublestranded siRNA molecules were prepared by brief boiling and slow cooling, and stored at ⫺80°C until they were used for transfections.

KCs Transfection and Treatment All plasmids were transfected with Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. For RNAi, 1 ␮g shRNA expression plasmid was used. To select karamycin-resistant cells, 2.5 ␮g/mL G418 (Sigma) was applied and isolated the resistant colonies. All transfected experiments were done in triplicate and repeated at least twice with different DNA isolates. The efficiency of transfection was observed with fluorescent microscope. KCs, seeded in 24-well plates at 1 ⫻ 10 6 cells/well and grown overnight prior to transfection, were randomly divided into two groups: the pIRAK-MshRNA group (transfected IRAK-M shRNA) and the control group (transfected control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group incubation in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and endotoxin pretreatment group incubation in the same medium containing 10 ng/mL LPS, named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h, because IRAK-M gave an intermediate rate of induction and peaked at 3 h after LPS stimulation as described elsewhere [15].

36

JOURNAL OF SURGICAL RESEARCH: VOL. 150, NO. 1, NOVEMBER 2008

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) The mRNA expressive level of IRAK-M in KCs was detected by RT-PCR analysis respectively. Briefly, total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions and quantified with the ratio of absorption values of RNA samples at 260 and 280 nm. Each total RNA sample was reverse-transcribed to complementary DNA (cDNA) using RT-PCR kit (Roche, Basel, Switzerland) and stored at ⫺70°C until PCR. ␤-Actin was used as an internal positive control. Specific primer sequences of IRAK-M and ␤-actin were as follows and the sizes of production were 500 and 300 bp, respectively: IRAK-M: 5=-GAA AAC GAC CCT GGA CCT CT-3= and 5=-CAC GGC TGA GTG TTG TGC AA3=. ␤-Actin: 5=-CAT TGT GAT GGA CTC CGG AG -3= and 5=ATA GTG ATG ACC TGG CCG TC -3=. All PCR products were electrophoresed on 2% agarose gels. The relative expression of mRNAs were assessed by taking the ratio of the intensity of the DNA bands of IRAK-M to ␤-actin using the Bio-Image analysis system (Gel Doc 2000) and expressed as arbitrary units.

Western Blotting Analysis The expression of IRAK-M protein in KCs was detected by Western blotting analysis. Protein extracts were obtained by homogenizing samples in a cell lysis buffer containing 20 mmol HEPES (pH 7.9), 25% glycerol, 0.42 mmol NaCl, 15 mmol MgCl 2, 0.2 mmol ethylenediamine tetraacetic acid, 0.5 mmol phenylmethylsulfonyl fluoride, and 0.5 mmol dithiothreitol, then by two cycles of centrifugation at 12,000 g for 15 min. Protein concentration was determined by Bradford assay kit (Bio-Rad, Hercules, CA). Extracted protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Dupont, Wilmington, DE). Membrane was washed with 0.1% Tween20-phosphate-buffered saline and incubated with 5% dry nonfat skimmed milk powder in 0.1% Tween20-phosphate-buffered saline, pH 7.4 for 1 h, then with rabbit anti-mouse IRAK-M polyclonal antibody (diluted 1:200, I5157, Sigma) and goat anti-rabbit IgG for 1 h. Finally, the immune complexes were developed with an enhanced chemiluminescence detection kit (Pierce, Waltham, MA) and the membranes were then immediately exposed to autoradiographic film (Eastman Kodak, Borhern, Belgium). The relative amount of SOCS-1 protein was quantified from relative absorbance of the band by image analysis system (Bio-Rad Gel Doc 2000).

shift assay (EMSA) was performed for analysis of NF-␬B transcriptional activity as described in a previous study [16]. The double stranded consensus-binding sequences for the EMSA comprised the oligonucleotide 5=-AGTTGAGGGGACTTTCCCAGG-3= for NF-␬B and for the mutant NF-␬B 10 ␮g of nuclear extract was incubated with an end-labeled, double-stranded, NF-␬B oligonucleotide probe. The reaction was performed in a total of 20 ␮L binding buffer (5 mmol/L HEPES pH 7.8, 50 mmol/L KCl, 0.5 mmol/L dithiothreitol and 10% glycerol) for 20 min at room temperature. After incubation, samples were fractionated on a 5% polyacrylamide gel and complex formation was visualized by autoradiography film (Eastman Kodak). In parallel, NF-␬B DNA-binding activity in nuclear extracts was measured using the Trans-a.m. NF-␬B p65 enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Rixensart, Belgium) according to the manufacturer’s instructions [17]. Briefly, 5 ␮g nuclear extract added to a 96-well plate to which oligonucleotide containing NF-␬B consensus-binding site had been immobilized. The NF-␬B complex bound to the oligonucleotide was detected by adding a specific mAb for p65 subunit. A secondary horseradish peroxidaseconjugated mAb was added and developed with tetramethylbenzidine substrate. After an optimal development time, the reaction was stopped using H 2SO4 0.5 mol/L, and absorbance was measured at 450 nm.

ELISA TNF-␣ level in supernatant was determined by ELISA with specific Ab purchased from BD PharMingen (Hamburg, Germany). ELISA plates were read using the Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA) and TNF-␣ levels quantified against a standard curve from 0 to 4,000 pg/mL plotted with a four-parameter curve. The detection limits were 5 pg/mL.

Data Analysis Results were expressed as mean ⫾ SD. All statistical analyses were performed using SPSS 10.0 software (SPSS, Inc., Chicago, IL). Statistical analyses were performed by analysis of variance. Differences were considered significant at the level of P ⬍ 0.05.

RESULTS

Analysis of NF-␬B Transcriptional Activity

The Influence of pIRAK-M-shRNA on IRAK-M Expression in KCs

Nuclear extracts of KCs were prepared and protein concentrations were determined by assay kit (Bio-Rad). Electrophoretic mobility

First, we successfully isolated KCs, constructed pIRAK-M-shRNA, and identified it by restriction-

FIG. 1. The influence of pIRAK-M-shRNA on IRAK-M gene expression (mRNA levels) in transfeced KCs at 3 h after LPS stimulation. (A) The mRNA isolated from the pIRAK-M-NEP, pIRAK-M-EP, pCV-NEP, and pCV-EP is shown in lanes 1– 4 at 3 h after stimulation with 100 ng/mL LPS, respectively. (B) Gels were photographed and digitized, and optical densities of IRAK-M mRNA bands were normalized using optical densities of the ␣-actin mRNA bands. *P ⬍ 0.05, pCV-EP versus pIRAK-M-EP and pCV-NEP; **P ⬎ 0.05, pIRAK-M-NEP versus pIRAK-M-EP. (Color version of figure is available online.)

LIU ET AL.: UP-REGULATION OF IRAK-M

37

FIG. 2. The influence of pIRAK-M-shRNA on IRAK-M protein expression in KCs at 3 h after LPS stimulation. (A) The IRAK-M protein isolated from the pCV-EP, pCV-NEP, pIRAK-M-EP, and pIRAK-M-NEP is shown in lanes 1– 4 at 3 h after LPS stimulation, respectively. (B) Densitometry of IRAK-M was performed as described in the legend to Figure 1. *P ⬍ 0.05, pCV-EP versus pIRAK-M-EP and pCV-NEP; **P ⬎ 0.05, pIRAK-M-NEP versus pIRAK-M-EP. (Color version of figure is available online.)

endonuclease digestion and sequencing. The efficiency of transfection with plasmid in KCs was about 85%, identified by significant expression of green fluorescent protein. IRAK-M expression changes in KCs were examined with RT-PCR and Western blot (Fig. 1 and Fig. 2). At 3 h after 100 ng/mL LPS stimulation, IRAK-M expression was significantly induced in pCV-EP than that in pCVNEP, supporting the hypothesis that IRAK-M is upregulated by a lower dose of LPS (10 ng/mL) pretreatment prior to stimulation with 100 ng/mL LPS, ensued by ET inducement. However, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, strongly indicating that pIRAK-M-shRNA specifically inhibited IRAK-M expression induced by LPS pretreatment. The Role of IRAK-M on NF-␬B Transcriptional Activity in KCs

To further examine the role of IRAK-M on LPS signal transduction in KCs, NF-␬B activation was first examined by ELISA. We observed that 10 ng/mL LPS pretreatment obviously attenuated KCs response to a

higher dose of LPS stimulation (100 ng/mL), and this response was more intense in groups without IRAK-M depression. As shown in Fig. 3A, pCV-EP and pIRAKM-EP, receiving 10 ng/mL LPS pretreatment, demonstrated lower NF-␬B p65 DNA-binding activity, which was just about 37% and 63% of pCV-NEP and pIRAKM-NEP, respectively. At the same time, pCV-NEP and pIRAK-M-NEP both induced a clear increase in NF-␬B p65 activation, and there was no significant difference between the two groups. This observation was further confirmed in EMSA experiments performed on nuclear extracts (Fig. 3B); we observed a lowest level of NF-␬B activation in pCV-EP and a dramatic enhancement after 3 h of LPS stimulation in the other three groups, indication that up-regulation of IRAK-M attenuated NF-␬B activation in KCs. The Influence of IRAK-M on TNF-␣ Expression in KCs

TNF-␣, which plays a pivotal role in the inflammatory cytokine cascade, is also involved in early LPS induced liver injury. As NF-␬B family members are known to

FIG. 3. The influence of pIRAK-M-shRNA on NF-êB activation in KCs at 3h after LPS stimulation. (A) Nuclear extract was obtained from pIRAK-M-NEP, pIRAK-M-EP, pCV-EP, and pCV-NEP is shown in lanes 1– 4 at 3 h after LPS stimulation, respectively. (B) Time course determination of NF-êB DNA-binding activity using ELISA. Optical density values (A450 nm) were corrected for background levels. *P ⬍ 0.05, pCV-NEP versus pIRAK-M-EP and pCV-EP, ** P ⬎ 0.05, pCV-NEP versus pIRAK-M-NEP. (Color version of figure is available online.)

38

JOURNAL OF SURGICAL RESEARCH: VOL. 150, NO. 1, NOVEMBER 2008

FIG. 4. The influence of pIRAK-M-shRNA on TNF-␣ level in supernatant at 3 h after LPS stimulation. TNF level in supernatant was determined by ELISA, *P ⬍ 0.05, pCV-NEP versus pIRAK-M-EP and pCV-EP, **P ⬎ 0.05, pCV-NEP versus pIRAK-M-NEP. (Color version of figure is available online.)

control the expression of the TNF-␣, was also quantified using ELISA (Fig. 4). TNF-␣ levels in the supernatant are significantly lower in pCV-EP and pIRAK-M-EP, which was about 43% and 72% of pCV-NEP and pIRAKM-NEP, respectively. However, TNF-␣ expression was also much lower in pCV-EP compared with pIRAK-MNEP, accompanied with specifically IRAK-M depression by siRNA. DISCUSSION

KCs represent more than 80% of macrophage population and are the first line to clearing circulating LPS crossing the gut mucosal barrier and entering the portal circulation [18]. Large quantities of LPS are known to induce the overproduction of cytokines, causing septic shock, while suboptimal doses could induce tolerance to subsequent exposure to LPS. ET is an important mechanism for maintaining the homeostasis of KCs, because KCs is continually exposed to various PAMPs, including LPS. Indeed, activation of KCs by gut-derived LPS plays an important role in liver injury by activating NF-␬B and producing TNF-␣ [14, 19]; however, inducing ET in KCs could specifically decrease this injury [20 –23]. Although LPS-mediated ET has been most extensively studied, molecular mechanisms of ET have not been fully clarified. ET involves multiple changes in cell signal transduction pathways. The discovery of TLR4 as the major receptors for LPS has prompted a resurgence of interest in ET mechanisms [24, 25]. TLR4 belongs to the toll/IL-1 receptor family that shares a conserved cytoplasmic domain required for signal transduction. IL-1 receptor and TLR4 activate a common intracellular pathway composed of MyD88, IRAKs, and TNF receptor-associated factor 6. IRAKs consist of two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. Proximal postreceptor signaling proteins that are altered in ET include decreased MyD88 recruitment to TLR4 and IRAK/MyD88 association, which lead to decrease NF-␬B activation and TNF-␣ gene expression [26, 27]. Interestingly, not all signaling pathways are down-

regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including IRAK-M expression [28]. A recent study has found that the expression of IRAK-M is crucial to LPS-induced ET in biliary epithelial cells [15]. A notable feature of IRAK-M is that it lacks kinase activity and is restricted to macrophages. IRAK-M ⫺/⫺ macrophages stimulated with LPS displayed increased NF-␬B and mitogen-activated protein kinase activation compared with IRAK-M ⫺/⫺ macrophages, and ET was significantly attenuated in IRAK-M ⫺/⫺ cells. Activation of TLRs by PAMPs resulted in IRAK-1 and IRAK-4 activation and their subsequent phosphorylation, causing loss of affinity for the TLR signaling complex and allowing the stimulation of downstream signaling through association with signaling molecules such as TRAF6. IRAK-M inhibits this process by inhibiting dissociation of IRAK-1 and IRAK-4 from the TLR signaling complex by either inhibiting the phosphorylation of IRAK-1 and IRAK-4 or stabilizing the TLR/MyD88/IRAK(-4) complex [14, 28]. Therefore, IRAK-M may also involve in the induction of ET in KCs. To examine this, we first successfully constructed the recombinant plasmid of pIRAK-M-shRNA and it specifically inhibited IRAK-M expression after transfected into KCs. We next determined the role of IRAK-M in ET induction in KCs. In the present study, we found that pretreatment with 10 ng/mL of LPS (primary LPS stimulation) for 24 h significantly induced tolerance to a subsequent challenge with 100 ng/mL of LPS (second LPS stimulation) as assessed by the activation of NF-␬B and production of TNF-␣, demonstrating the presence of ET in KCs. However, although primary LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted by silence IRAK-M gene, indicating that the absence of IRAK-M caused inhibition of ET and consequent abnormal enhancement of inflammatory response. It remains to be determined if ET involves a single critical signaling pathway or sequential multiple changes in signaling events during tolerance induction [29]. We indeed found that although ET was significantly attenuated, but it was not completely abrogated by specific silence of IRAK-M gene using RNA interference. The likely reason is that up-regulation IRAK-M expression is not the unique mechanism and obviously there are other additional regulatory mechanisms to control the response to LPS. Suppressor of cytokine signaling-1 is another inducible negative regulator of TLR signaling, although its induction occurs only through TLR4. ET has also been shown to be associated with decreased Gi protein content and activity, decreased protein kinase C activity, reduction in mitogen activated protein kinase activity, and reduced activator protein-1 and NF-␬B induced gene transactivation [30 –32]. In summary, we show that ET could be induced in KCs

LIU ET AL.: UP-REGULATION OF IRAK-M

by pretreatment with a low dose of LPS accompanied by up-regulation of IRAK-M, and the silence of IRAK-M by RNA interference obviously attenuated the inhibitory response induced by low dose LPS pretreatment, which were indicated by suppressed the NF-␬B activation and TNF-␣ production. Therefore, IRAK-M may be a key component of this important control system and a new target for the clinical treatment of sepsis.

14.

ACKNOWLEDGMENTS

17.

We thank Miss Wahg Beihang for enlightening discussions. This study was supported by grants from the National Natural Science Foundation of China (30471696, 30500473, 30772098), the Ph.D. Programs Foundation of the Ministry of Education of China (20050631001), and the Natural Science Foundation of Chongqing (2005BB5242).

15.

16.

18.

19.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

Broad A, Jones DE, Kirby JA. Toll-like receptor (TLR) response tolerance: A key physiological “damage limitation” effect and an important potential opportunity for therapy. Curr Med Chem 2006;13:2487. Broad A, Kirby JA, Jones DE. Toll-like receptor interactions: Tolerance of MyD88-dependent cytokines but enhancement of MyD88-independent interferon-␤ production. Immunology 2007; 120:103. Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of lipopolysaccharide-induced signal transduction in endotoxintolerized mouse macrophages: Dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression. J Immunol 2000;164:5564. Makhlouf M, Zingarelli B, Halushka PV, et al. Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators. J Immunol 2001;166:5161. Dinarello CA. Infection, fever, and exogenous and endogenous pyrogens: Some concepts have changed. J Endotoxin Res 2004; 10:201. Medvedev AE, Sabroe I, Hasday JD, et al. Tolerance to microbial TLR ligands: Molecular mechanisms and relevance to disease. J Endotoxin Res 2006;12:133. Fujihara M, Muroi M, Tanamoto K, et al. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: Roles of the receptor complex. Pharmacol Ther 2003;100:171. Liu ZJ, Liu CA, Gong JP. IRAK-4: The key molecule of TLR/ IL-1R common signal transduction system. Prog Physiol Sci 2005;36:276. Kobayashi K, Hernandez LD, Galan JE, et al. IRAK-M is a negative regulator of toll-like receptor signaling. Cell 2002;110:191. Wang YY, Dahle MK, Agren J, et al. Activation of the liver X receptor protects against hepatic injury in endotoxemia by suppressing Kupffer cell activation. Shock 2006;25:141. Jacob A, Zhou M, Wu R, et al. Proinflammatory cytokines from Kupffer cells down-regulate hepatocyte expression of adrenomedullin binding protein-1. Biochim Biophys Acta 2007;1772:766. Tsukada S, Enomoto N, Takei Y, et al. Dalteparin sodium prevents liver injury due to lipopolysaccharide in rat through suppression of tumor necrosis factor-alpha production by Kupffer cells. Alcohol Clin Exp Res 2003;27:7S. Sturm E, Havinga R, Baller JF, et al. Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats. J Hepatol 2005;42:102.

20.

21.

22.

23.

24. 25.

26.

27.

28. 29.

30.

31.

32.

39

Thobe BM, Frink M, Hildebrand F, et al. The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-TLR4-, and TLR9-mediated signaling following trauma-hemorrhage. J Cell Physiol 2007;210: 667. Harada K, Isse K, Sato Y, et al. Endotoxin tolerance in human intrahepatic biliary epithelial cells is induced by up-regulation of IRAK-M. Liver Int 2006;26:935. Liu ZJ, Yan LN, Gong JP, et al. Glycine blunts transplantative liver ischemia-reperfusion injury by down-regulating interleukin 1 receptor associated kinase-4. Acta Pharmacol Sin 2006;27:1479. Gelbmann CM, Leeb SN, Vogl D, et al. CD40 expression on human pancreatic duct cells: Role in nuclear factor-␬B activation and production of pro-inflammatory cytokines. Gut 2003; 52:1448. Seki S, Habu Y, Kawamura T, et al. The liver as a crucial organ in the first line of host defense: The roles of Kupffer cells, natural killer (NK) cells, and NK1.1 Ag⫹ T cells in T helper 1 immune responses. Immunol Rev 2000;174:35. Miyaso H, Morimoto Y, Ozaki M, et al. Protective effects of nafamostat mesylate on liver injury induced by lipopolysaccharide in rats: Possible involvement of CD14 and TLR-4 downregulation on Kupffer cells. Dig Dis Sci 2006;51:2007. Granado M, Martin AI, Priego T, et al. Inactivation of Kupffer cells by gadolinium administration prevents lipopolysaccharideinduced decrease in liver insulin-like growth factor-I and IGFbinding protein-3 gene expression. J Endocrinol 2006;188:503. Yamashina S, Wheeler MD, Rusyn I, et al. Tolerance and sensitization to endotoxin in Kupffer cells caused by acute ethanol involve interleukin-1 receptor associated kinase. Biochem Biophys Res Commun 2000;277:686. Deng JC, Cheng G, Newstead MW, et al. Sepsis-induced suppression of lung innate immunity is mediated by IRAK-M. J Clin Invest 2006;116:2532. Medvedev AE, Lentschat A, Wahl LM, et al. Dysregulation of LPS-induced toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells. J Immunol 2002;169:5209. Fan H, Cook JA. Molecular mechanisms of endotoxin tolerance. J Endotoxin Res 2004;10:71. Li CH, Wang JH, Redmond HP. Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation. J Leukoc Biol 2006;79:867. Janssens S, Beyaert R. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol Cell 2003;11:293. Tazi KA, Quioc JJ, Saada V, et al. Up-regulation of TNF-␣ production signaling pathways in monocytes from patients with advanced cirrhosis: Possible role of Akt and IRAK-M. J Hepatol 2006;45:280. Crispe IN, Giannandrea M, Klein I, et al. Cellular and molecular mechanisms of liver tolerance. Immunol Rev 2006;213:101. Lopez-Collazo E, Fuentes-Prior P, Arnalich F, et al. Pathophysiology of interleukin-1 receptor-associated kinase-M: Implications in refractory state. Curr Opin Infect Dis 2006;19:237. Ferlito M, Squadrito F, Halushka PV, et al. Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization. Shock 2001;15:291. Cuschieri J, Billigren J, Maier RV. Endotoxin tolerance attenuates LPS-induced TLR4 mobilization to lipid rafts: A condition reversed by PKC activation. J Leukoc Biol 2006;80:1289. Pocock J, Gomez-Guerrero C, Harendza S, et al. Differential activation of NF-␬B, AP-1, and C/EBP in endotoxin-tolerant rats: Mechanisms for in vivo regulation of glomerular RANTES/ CCL5 expression. J Immunol 2003;170:6280.