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Originally published online as doi:10.1189/jlb.0905505 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:867-875.)
© 2006 by Society for Leukocyte Biology

Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation

Chong Hui Li*, Jiang Huai Wang*,1 and H. Paul Redmond*

* Department of Academic Surgery, National University of Ireland/University College Cork, Cork University Hospital, Ireland

1Correspondence: Department of Academic Surgery, National University of Ireland (NUI)/University College Cork, Cork University Hospital, Cork, Ireland. E-mail: jh.wang{at}ucc.ie


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ABSTRACT
 
Tolerance to bacterial cell-wall components may represent an essential regulatory mechanism during bacterial infection. We have demonstrated previously that the inhibition of nuclear factor (NF)-{kappa}B and mitogen-activated protein kinase activation was present in bacterial lipoprotein (BLP) self-tolerance and its cross-tolerance to lipopolysaccharide (LPS). In this study, the effect of BLP-induced tolerance on the myeloid differentiation factor 88 (MyD88)-dependent upstream signaling pathway for NF-{kappa}B activation in vitro was examined further. When compared with nontolerant human monocytic THP-1 cells, BLP-tolerant cells had a significant reduction in tumor necrosis factor {alpha} (TNF-{alpha}) production in response to a high-dose BLP (86±12 vs. 6042±245 ng/ml, P<0.01) or LPS (341±36 vs. 7882±318 ng/ml, P<0.01) stimulation. The expression of Toll-like receptor 2 (TLR2) protein was down-regulated in BLP-tolerant cells, whereas no significant differences in TLR4, MyD88, interleukin-1 receptor-associated kinase 4 (IRAK-4), and TNF receptor-associated factor 6 expression were observed between nontolerant and BLP-tolerant cells, as confirmed by Western blot analysis. The IRAK-1 protein was reduced markedly in BLP-tolerant cells, although IRAK-1 mRNA expression remained unchanged as revealed by real-time reverse transcriptase-polymerase chain reaction analysis. Furthermore, decreased MyD88-IRAK immunocomplex formation, as demonstrated by immunoprecipitation, was observed in BLP-tolerant cells following a second BLP or LPS stimulation. BLP pretreatment also resulted in a marked inhibition in total and phosphorylated inhibitor of {kappa}B-{alpha} (I{kappa}B-{alpha}) expression, which was not up-regulated by subsequent BLP or LPS stimulation. These results demonstrate that in addition to the down-regulation of TLR2 expression, BLP tolerance is associated with a reduction in IRAK-1 expression, MyD88-IRAK association, and I{kappa}B-{alpha} phosphorylation. These findings further elucidate the molecular mechanisms underlying bacterial peptide tolerance.

Key Words: monocytes/macrophages • signal transduction pathway • tolerance • human


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INTRODUCTION
 
Many bacterial cell-wall components are able to activate monocytes/macrophages to produce inflammatory cytokines including tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1, and IL-6. Among these bacterial cell-wall components, lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) are known as the most important components for gram-negative and gram-positive bacteria in eliciting the systemic inflammatory response associated with infection. Endotoxin or LPS tolerance is defined as a reduced capacity of the host (in vivo) or of cultured monocytes/macrophages (in vitro) to respond to LPS activation following a first exposure to this stimulus. The phenomenon of endotoxin tolerance has been investigated widely [1 ], but to date, the molecular mechanisms of endotoxin tolerance remain to be resolved clearly. Conversely, studies in our [2 ] and other [3 ] laboratories have shown that BLP can not only activate host inflammatory cells but also induce self-tolerance and cross-tolerance to LPS. Our further in vivo work [4 , 5 ] has demonstrated that induction of BLP tolerance, in addition to protection against a lethal BLP challenge, also protects against endotoxic shock through a cross-tolerance to LPS. It is more important that induction of BLP tolerance confers protection against lethal microbial sepsis induced by live bacteria or cecal ligation and puncture in wild-type [4 ] and Toll-like receptor 4 (TLR4)-deficient [5 ] mice via enhanced bacterial clearance.

Families of TLRs have been shown to be involved in the recognition of bacterial components. At least 10 TLRs have been cloned, and most have been functionally characterized with respect to ligand recognition [6 ]. TLR4 is established as a signal-transducing receptor for LPS. TLR2 has been shown to signal the presence of BLP, peptidoglycan, lipoteichoic acid, and mycoplasmal lipopeptides [macrophage-activating lipopeptide-2 (MALP-2)]. Engagement of TLRs with their ligands leads to the production of various proinflammatory cytokines, chemokines, and effector molecules. TLRs and interleukin-1 receptor (IL-1R) share several signaling molecules including adaptor protein myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNF receptor-activated factor 6 (TRAF6). Upon activation, MyD88 is recruited to TLR4 or TLR2, and then IRAK-1 is recruited to the receptor through MyD88. IRAK-1 becomes highly phosphorylated and then relays the signal downstream by interacting with TRAF6. This in turn leads to the activation of the inhibitor of {kappa}B (I{kappa}B) kinase complex and the phosphorylation of I{kappa}B. Degradation of phosphorylated I{kappa}B by the proteasome then allows nuclear factor (NF)-{kappa}B to translocate into the nucleus. Target genes containing NF-{kappa}B-binding sites are then transcribed and expressed [7 , 8 ]. Therefore, the identification and characterization of molecules in BLP and LPS signaling pathways responsible for BLP tolerance and its cross-tolerance to LPS are vital if we are to extend our understanding of molecular events involved during bacterial infection and associated septic shock.

Recent studies reported that modulation of the signaling pathway via TLR4 is involved in the development of LPS tolerance. A potential mechanism for tolerance to LPS is the down-regulation of its receptor molecules. The surface expression of CD14 has been excluded as an underlying mechanism for LPS tolerance because of up-regulated [9 , 10 ] or unchanged [11 ] CD14 expression observed in LPS-tolerant cells. However, down-regulated cell-surface TLR4 expression was correlated with LPS tolerance in mouse peritoneal macrophages [11 , 12 ]. Proximal post-receptor signaling proteins, which are altered in LPS tolerance, include reduced protein levels of IRAK-1 [13 , 14 ] and decreased formation of the TLR4-MyD88 [14 ] and MyD88-IRAK [13 , 15 ] complex. Down-regulation of NF-{kappa}B DNA-binding activity [14 15 16 ] and mitogen-activated protein kinase (MAPK) phosphorylation [14 , 16 ] is considered a significant marker for LPS tolerance, which has also been shown to be associated with decreased degradation and inhibited phosphorylation of I{kappa}B-{alpha} and I{kappa}B-ß [16 , 17 ].

Compared with LPS tolerance, however, the molecular mechanisms involved in BLP tolerance and its cross-tolerance to LPS remain largely unexplored. Our previous work has shown that suppressed TLR2 expression and inhibited MAPK phosphorylation and NF-{kappa}B activation are associated with BLP tolerance [2 ]. In this study, we further investigated the regulation of a TLR2-mediated upstream signal transduction pathway in BLP-tolerant human monocytic THP-1 cells.


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MATERIALS AND METHODS
 
Reagents and antibodies
BLP, a synthetic BLP (Pam3-Cys-Ser-Lys4-OH) derived from the immunologically active NH2 terminus of BLP, was obtained from Roche Molecular Biochemicals (Indianapolis, IN), which was endotoxin-free as confirmed by the Limulus amebocyte lysate assay (Charles River Endosafe, Charleston, SC). LPS from Escherichia coli serotype O55:B5 was purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal antibodies (pAb) for TLR4, IRAK-1, TRAF6, and ß-actin were purchased from Santa Cruz Biotechnology (CA). Rabbit anti-TLR2 and anti-MyD88 pAb were obtained from Abcam (Cambridge, MA). Rabbit pAb against IRAK-4 was obtained from Upstate Biotechnology (Waltham, MA). Rabbit pAb, which recognize I{kappa}B-{alpha} and phosphorylated I{kappa}B-{alpha} (Ser32), were purchased from Cell Signaling Technology (Beverly, MA). All culture medium and reagents used for cell culture were purchased from Invitrogen Life Technologies (Paisley, Scotland, UK). All other chemicals, unless indicated, were from Sigma-Aldrich.

Cell culture
Human monocytic THP-1 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin sulfate (100 µg/ml), and glutamine (2 mM) at 37°C in a humidified 5% CO2 atmosphere.

Induction of BLP tolerance and its cross-tolerance to LPS
THP-1 cells (1x106 cells/ml) in six-well plates (Falcon, Lincoln Park, NJ) were preincubated with culture medium (nontolerant) or 100 ng/ml BLP (BLP-tolerant) for 24 h, washed twice with phosphate-buffered saline (PBS), and incubated in fresh culture medium for 2 h. Thereafter, nontolerant cells were stimulated with 1000 ng/ml BLP, and BLP-tolerant cells were restimulated with 1000 ng/ml BLP or 1000 ng/ml LPS for the indicated time periods.

Western blot analysis and immunoprecipitation
Following BLP or LPS stimulation, nontolerant and BLP-tolerant THP-1 cells were collected at the different time-points and washed with ice-cold PBS. The cells were then lysed at 4°C in lysis buffer (Cell Signaling Technology) containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and proteinase inhibitor cocktail. The resultant lysates were centrifuged to remove particulate materials. Protein concentration was determined using a micro bicinchoninic acid protein assay (Pierce, Rockford, IL). Equal amounts of cytoplasmic protein extracts were denatured at 100°C for 10 min in loading buffer [60 mM Tris, 2.5% sodium dodecyl sulfate (SDS), 10% glycerol, 5% mercaptoethanol, 0.01% bromphenol blue]. Protein from each sample (40–60 µg) was separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). The membrane was blocked for 1 h at room temperature with PBS containing 0.05% Tween-20 and 5% nonfat milk and probed overnight at 4°C with primary pAb at conditions recommended by the manufacturers. Blots were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Dako, Cambridgeshire, UK) at room temperature for 1 h and developed with SuperSignal chemiluminescent substrate (Pierce) and exposed to Kodak X-Omat AR film (Sigma-Aldrich). For immunoprecipitation, equal amounts of extracted protein were incubated with 6 µg goat anti-MyD88 pAb (Santa Cruz Biotechnology) overnight at 4°C on a rotator. Thereafter, 50 µl of a 50% slurry of prewashed protein G-agarose beads (Upstate Biotechnology) was added to each sample and incubated at 4°C for an additional 2 h. The samples were spun briefly in a microcentrifuge and washed three times in the lysis buffer. Loading buffer (30 µl) was added to each sample and boiled for 10 min. The samples were then separated by SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with rabbit anti-IRAK-1 pAb and rabbit anti-MyD88 pAb, respectively.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA from nontolerant and BLP-tolerant THP-1 cells was extracted with a GenEluteTM mammalian total RNA purification kit (Sigma-Aldrich) and reverse-transcribed with the SuperScript first-strand synthesis system (Invitrogen). Quantitative real-time PCR was performed in a total reaction volume of 20 µl containing 10 µl 2x QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA), 1 µl each oligonucleotide primer (1.6 pmol), and 2 µl cDNA. Amplification was performed on a LightCycler system (Roche Molecular Biochemicals) with an initial step of denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 10 s, 55°C for 25 s, and 72°C for 20 s. After completion of the cycling process, melting curve analysis was performed from 65°C to 95°C at 2°C/s with continuous fluorescence monitoring. The size of the PCR product was identified by 2.5% agarose electrophoresis. The data were analyzed with Q-gene software [18 ]. The target gene mRNA expression was normalized with the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The following human gene-specific PCR primers were synthesized by MWG Biotech (Ebersberg, Germany) and used: TLR2 (sense-5'-GCCTCTCCAAGGAAGAATCC-3', and antisense-5'-TCCTGTTGTTGGACAGGTCA-3'); TLR4 (sense-5'-AAGCCGAAAGGTGATTGTTG-3', and antisense-5'-CTGAGCAGGGTCTTCTCCAC-3'); MyD88 (sense-5'-GCTGAGAAGCCTTTACAGGTG-3', and antisense-5'-CTGGGGCAATAGCAGATGAAG-3'); IRAK-1 (sense-5'-GGACACGGACACCTTCAGC-3', and antisense-5'-CAGCCTCCTCTTCCACCAG-3'); IRAK-4 (sense-5'-TGCTCCTGCGAGTCTTTTG-3', and antisense-5'-AGGTGGCATATAGCTTTGTTC-3'); TRAF6 (sense-5'-TTGATGGCATTACGAGAAGCAG-3', and antisense-5'-GCAAACAACCTTCATTTGGACAT-3'); GAPDH (sense-5'-GAAGGTGAAGGTCGGAGTC-3', and antisense-5'-GAAGATGGTGATGGGATTTC-3').

Cytokine measurements
THP-1 cells in 24-well plates (Falcon; 2x105 cells/well) were preincubated with culture medium or BLP at 10, 100, and 1000 ng/ml for 24 h. Nontolerant and BLP-tolerant cells were then stimulated with 10, 100, and 1000 ng/ml BLP or LPS for 6 h. Cell-free supernatants were collected by centrifugation, transferred to new tubes, and stored at –70°C until analysis. The levels of TNF-{alpha} in the supernatants were determined using BDTM cytometric bead array (CBA; BD Biosciences, San Jose, CA) on a FACScan flow cytometer (BD Biosciences).

Statistical analysis
All data are presented as the mean ± SD. Statistical analysis was performed using ANOVA. Differences were judged statistically significant when the P value was less than 0.05.


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RESULTS
 
BLP- and LPS-induced TNF-{alpha} production are suppressed in BLP-tolerant THP-1 cells
BLP and LPS possess the potential to activate human monocytic THP-1 cells and induce proinflammatory cytokine TNF-{alpha} production (Fig. 1A and 1B ). However, when THP-1 cells were pretreated with a low dose of BLP for 24 h and then subjected to a second stimulation with the same or higher doses of BLP, TNF-{alpha} production was reduced significantly (Fig. 1A) . This induction of BLP tolerance in THP-1 cells was BLP pretreatment dose-dependent, i.e., the higher the BLP dose used in the pretreatment, the greater the attenuation in TNF-{alpha} release. In this in vitro experimental system, BLP pretreatment at 100 ng/ml was capable of inhibiting TNF-{alpha} release completely. It is important that BLP-tolerant THP-1 cells also had an impaired ability to produce TNF-{alpha} in response to LPS stimulation (Fig. 1B) , indicating a cross-tolerance to LPS induced by BLP pretreatment. This cross-tolerance to LPS exhibited by BLP-tolerant cells was also BLP pretreatment dose-dependent (Fig. 1B) . There were no differences in viability between nontolerant and BLP-tolerant THP-1 cells before and after a second BLP or LPS stimulation (data not shown).


Figure 1
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  		Figure 1. TNF-{alpha} production induced by BLP or LPS stimulation in naive and BLP-tolerant THP-1 cells. Human monocytic THP-1 cells were preincubated with culture medium (naive) or 10, 100, or 1000 ng/ml BLP (BLP-tolerant) for 24 h. After being washed twice with PBS and incubated in fresh medium for 2 h, these cells were stimulated with 10, 100, or 1000 ng/ml BLP (A) or LPS (B) for 6 h. BDTM CBA determined the concentrations of TNF-{alpha} in the culture supernatants. Data are expressed as the mean ± SD (n=5).

Reduced total and phosphorylated I{kappa}B-{alpha} expression in BLP-tolerant THP-1 cells in response to BLP or LPS stimulation
Before NF-{kappa}B translocation into the nucleus, I{kappa}B proteins undergo phosphorylation, ubiquitination, and proteosomal degradation, thus releasing NF-{kappa}B dimers and unmasking their nuclear localization and DNA-binding domains. Therefore, we first compared the kinetics of I{kappa}B-{alpha} degradation in naïve THP-1 cells in response to BLP stimulation or in BLP-tolerant cells in response to BLP stimulation or LPS stimulation. As shown in Figure 2 , lane C, following BLP stimulation, the levels of total I{kappa}B-{alpha} protein in naïve cells decreased at 10 min and almost totally disappeared at 30 min of BLP stimulation, suggesting that BLP stimulation induces I{kappa}B-{alpha} degradation. Meanwhile, the phosphorylation of Ser32 of I{kappa}B-{alpha} appeared as early as at 10 min after BLP stimulation and was degraded completely by 30 min (Fig. 2 , lane C), indicating the sequential occurrence of phosphorylation and degradation of I{kappa}B-{alpha} in naïve THP-1 cells in response to BLP stimulation. In contrast, markedly reduced, total I{kappa}B-{alpha} and phosphorylated I{kappa}B-{alpha} expression in BLP-tolerant THP-1 cells were observed at 24 h after BLP pretreatment and following a second BLP or LPS stimulation (Fig. 2 , lanes T and CT), indicating the existence of hyporesponsiveness in BLP-tolerant cells to a further BLP or LPS stimulation.


Figure 2
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  		Figure 2. Reduced total and phosphorylated I{kappa}B-{alpha} expression in BLP-tolerant THP-1 cells, which were preincubated with culture medium (naive) or 100 ng/ml BLP (BLP-tolerant) for 24 h. Naive cells were stimulated with 1000 ng/ml BLP for the indicated time-points (lane C), whereas BLP-tolerant cells were restimulated with 1000 ng/ml BLP (tolerance, lane T) or LPS (cross-tolerance, lane CT) for the indicated time-points. Cytoplasmic protein was extracted and analyzed for total and phosphorylated I{kappa}B-{alpha} (p-I{kappa}B-{alpha}) expression by Western blot analysis as described in Materials and Methods. Results shown represent one experiment from a total of three independent experiments.

Protein expression of proximal post-receptor signaling molecules in BLP-tolerant THP-1 cells
We next addressed the effect of pre-exposure to BLP on the expression of several components involved in the TLR-MyD88-dependent upstream signaling pathway in THP-1 cells. Following BLP stimulation, there were gradual reductions in IRAK-1 protein expression in naïve THP-1 cells (Fig. 3 , lane C), indicating that BLP stimulation induces degradation of IRAK-1 protein. Pretreatment with a low-dose BLP for 24 h resulted in a marked decrease in IRAK-1 protein level in BLP-tolerant cells when compared with nontolerant cells (Fig. 3) . The expression of IRAK-1 protein in BLP-tolerant cells remained at low levels after restimulation with BLP (Fig. 3 , lane T) or LPS (Fig. 3 , lane CT). In contrast, Western blot analysis demonstrated that there were no significant alterations in the protein levels of MyD88, IRAK-4, and TRAF6 in naïve and BLP-tolerant cells, before or after BLP or LPS stimulation (Fig. 3) .


Figure 3
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  		Figure 3. Suppressed IRAK-1 protein expression in BLP-tolerant THP-1 cells, which were preincubated with culture medium (naive) or 100 ng/ml BLP (BLP-tolerant) for 24 h. Naive cells were stimulated with 1000 ng/ml BLP for the indicated time-points (lane C), whereas BLP-tolerant cells were restimulated with 1000 ng/ml BLP (lane T) or LPS (lane CT) for the indicated time-points. Cytoplasmic protein was extracted, and IRAK-1, IRAK-4, MyD88, and TRAF6 protein expression was detected by Western blot analysis. Results shown represent one experiment from a total of three independent experiments.

Reduced association of IRAK with MyD88 in BLP-tolerant THP-1 cells
Activation of TLR2 by BLP or TLR4 by LPS triggers a common intracellular signaling pathway, which involves MyD88, IRAK-1, and TRAF6 and promotes these intracellular intermediates to form immunocomplexes that play a crucial role in BLP and LPS signal transduction. Several studies have shown that impaired MyD88-IRAK [13 , 15 ] or TLR4-MyD88 [14 ] complex formation is evident in LPS-tolerant cells. To determine the effect of BLP tolerance on the association of IRAK with MyD88, first, we analyzed the capacity of IRAK to associate with MyD88 upon BLP stimulation by immunoprecipitation experiments. As shown in Figure 4 , lane C, there was little IRAK coprecipitated with MyD88 in naive THP-1 cells. Following BLP stimulation, the MyD88-IRAK complex formation increased at 10 min and declined at 30 min, and no IRAK was coprecipitated further with MyD88 at 2 h after BLP stimulation. This suggests that BLP stimulation induces a transient association of IRAK with MyD88 in naïve THP-1 cells. In contrast, the MyD88-IRAK complex was barely detected in BLP-tolerant THP-1 cells, and IRAK no longer associated with MyD88 to form the immunocomplex in BLP-tolerant cells following a second BLP (Fig. 4 , lane T) or LPS (Fig. 4 , lane CT) stimulation, indicating an inability of BLP- or LPS-induced association of IRAK with MyD88 in BLP-tolerant cells.


Figure 4
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  		Figure 4. Disruption of the MyD88-IRAK complex formation in BLP-tolerant THP-1 cells, which were preincubated with culture medium (naive) or 100 ng/ml BLP (BLP-tolerant) for 24 h. Naive cells were stimulated with 1000 ng/ml BLP for the indicated time-points (lane C), whereas BLP-tolerant cells were restimulated with 1000 ng/ml BLP (lane T) or LPS (lane CT) for the indicated time-points. Cytoplasmic protein was extracted, immunoprecipitated (IP) with anti-MyD88 pAb, and analyzed by Western blot (IB) using anti-IRAK-1 and anti-MyD88 pAb, respectively. The results of a representative experiment are shown (n=3).

Expression of TLR2, but not TLR4, is down-regulated by BLP tolerance
As shown in Figure 5 , the constitutive expression of TLR2 protein in naïve THP-1 cells was up-regulated by BLP stimulation for 30 min (Fig. 5 , lane C). In contrast, there was a slight reduction of constitutive TLR2 expression in BLP-tolerant cells, and a further reduction was noted in BLP-tolerant cells following BLP restimulation (Fig. 5 , lane T), indicating that induction of BLP tolerance in THP-1 cells prevents BLP-induced up-regulation of TLR2 expression. These results are keeping with our previous finding in which an inhibition of BLP-induced TLR2 activation in BLP-tolerized THP-1 cells was also observed [2 ].


Figure 5
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  		Figure 5. TLR2 and TLR4 protein expression in naive and BLP-tolerant THP-1 cells, which were preincubated with culture medium (naive) or 100 ng/ml BLP (BLP-tolerant) for 24 h. Naive cells were stimulated with 1000 ng/ml BLP for the indicated time-points (lane C), whereas BLP-tolerant cells were restimulated with 1000 ng/ml BLP (lane T) or LPS (lane CT) for the indicated time-points. Cytoplasmic protein was extracted, and Western blot analysis was used to detect TLR2 and TLR4 protein expression. Results shown represent one experiment from a total of three independent experiments.

LPS tolerance has been associated with the down-regulation of TLR4 expression [11 ]. It was also reported that LPS stimulation induced an enhanced expression of TLR2 [16 , 19 ]. However, it is not clear whether BLP tolerance affects the expression of TLR4. We analyzed the protein level of TLR4 by Western blot analysis in nontolerant and BLP-tolerant THP-1 cells in response to BLP or LPS stimulation. Figure 5 shows BLP pretreatment did not alter TLR4 protein expression before and after a second BLP or LPS stimulation.

Regulation of mRNA expression of TLR2, TLR4, MyD88, IRAK-1, IRAK-4, and TRAF6 in BLP-tolerant THP-1 cells
Quantitative real-time RT-PCR analysis was used to detect the mRNA levels of the six signaling molecules in naïve and BLP-tolerant THP-1 cells. Figure 6 shows that TLR2 (Fig. 6A) , TLR4 (Fig. 6B) , MyD88 (Fig. 6C) , IRAK-1 (Fig. 6D) , IRAK-4 (Fig. 6E) , and TRAF6 (Fig. 6F) mRNA were constitutively expressed in THP-1 cells. The mRNA levels of TLR4 and MyD88 are relatively higher, and the mRNA levels of IRAK-4 and TRAF6 are relatively lower. However, the mRNA levels of these signaling molecules were not regulated significantly by BLP or LPS stimulation in naive and BLP-tolerant cells. Thus, induction of BLP tolerance did not significantly influence the mRNA levels of these signaling intermediates involved in the MyD88-dependent upstream signaling pathway.


Figure 6
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  		Figure 6. Detection of TLR2, TLR4, MyD88, IRAK-1, IRAK-4, and TRAF6 mRNA expression in BLP-tolerant THP-1 cells, which were preincubated with culture medium (naive) or 100 ng/ml BLP (BLP-tolerant) for 24 h. Naive cells were stimulated with 1000 ng/ml BLP (open bars) for the indicated time-points, whereas BLP-tolerant cells were restimulated with 1000 ng/ml BLP (shaded bars) or LPS (solid bars) for the indicated time-points. Total RNA was extracted and reverse-transcribed. Quantitative real-time RT-PCR was performed for detecting mRNA expression of TLR2 (A), TLR4 (B), MyD88 (C), IRAK-1 (D), IRAK-4 (E), and TRAF6 (F) as described in Materials and Methods. Data are expressed as the mean ± SD (n=5).


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DISCUSSION
 
BLP is the most abundant protein in the outer membrane of gram-positive and gram-negative bacteria. Some TLR2 agonists, such as BLP [2 ], MALP-2 [3 ], and LPS from Porphyromonas gingivalis [20 ] are capable of inducing self-tolerance as well as cross-tolerance to LPS, which signals via TLR4. The mechanisms for cross-tolerance between different bacterial components may depend on their signaling pathways. It has been demonstrated that the post-receptor intracellular transduction pathways for LPS signaling or BLP signaling are dependent on MyD88, IRAK-1, IRAK-4, TRAF6, and I{kappa}B-{alpha}, which in turn, leads to activation of NF-{kappa}B. We have shown previously that activation of MAPKs (p38, extracellular signal-regulated kinase, Jun N-terminal kinase) and NF-{kappa}B was suppressed in BLP-tolerant THP-1 cells in response to a second stimulation of BLP or LPS [2 ]. In this study, we further examined the effect of BLP tolerance on the protein and mRNA expression of the signaling molecules upstream of NF-{kappa}B activation.

TLRs are pattern recognition receptors, which recognize distinct molecular patterns associated with microbial pathogens [6 ]. For example, TLR2 is involved in the response to gram-positive BLP stimulation, and TLR4 is required for gram-negative LPS signaling. Upon engagement by different microbial ligands, TLRs are activated and initiate a cascade of intracellular signal events that eventually result in inflammatory cytokine production. Our previous work has demonstrated that in addition to the inhibition of downstream NF-{kappa}B activation and MAPK phosphorylation, induction of BLP tolerance down-regulates TLR2 expression [2 ]. In contrast, LPS tolerance did not affect BLP-induced TLR2 activation. In the present study, we further confirmed the reduced protein level of TLR2 in BLP-tolerant cells, indicating that BLP-induced tolerance is associated with suppression of TLR2 expression. Although BLP-tolerant cells exhibited a strong inhibition in TLR2 protein expression, there was no significant reduction in TLR2 mRNA expression in BLP-tolerant cells, suggesting that BLP tolerance-induced down-regulation of TLR2 expression occurs at the translational level rather than at the transcriptional level.

Induction of LPS tolerance is associated with reduced TLR4 expression [11 ]. However, it is not clear whether BLP-induced cross-tolerance to LPS is a result of the down-regulation of TLR4 expression. In the present study, pretreatment of human THP-1 cells with BLP significantly attenuated LPS-induced TNF-{alpha} production; however, the levels of TLR4 protein and mRNA expression in BLP-tolerant cells were not affected, as revealed by Western blot and real-time RT-PCR analysis, indicating that BLP-induced cross-tolerance to LPS is not through an inhibition in TLR4 expression. These data are keeping with previous work done by Sato et al. [3 ], as they also found that MALP-2, a TLR2 agonist, induced cross-tolerance to LPS, which was independent of TLR4-myeloid differentiation protein-2 expression. Indeed, it is uncertain whether a down-regulated TLR4 expression is responsible for the induction of LPS tolerance. For example, LPS pretreatment resulted in a transient reduction in TLR4 mRNA and protein expression, but levels returned to normal at the time of the second challenge with LPS in RAW264.7 cells [21 ]. Furthermore, overexpression of TLR4 in human embryonic kidney 293T cells did not prevent LPS-induced tolerance, as these cells still exhibited decreased NF-{kappa}B and MAPK activation in response to a second LPS stimulation [22 ].

Following TLR activation, IRAK-1 is recruited to the TLR through the adaptor protein MyD88, which then relays signaling downstream to TRAF6, leading to the activation of NF-{kappa}B and MAPKs. Indeed, MyD88-deficient [23 ] and TRAF6-deficient [24 ] mice displayed hyporesponsiveness to LPS. MyD88-deficient mice also showed no inflammatory response to several other bacterial components, including peptidoglycan [25 ] and MALP-2 [26 ], which signal through TLR2. Thus, MyD88 is the adaptor molecule shared by TLR2- and TLR4-mediated signaling pathways. It has been observed in murine macrophages [3 , 21 ], human monocytic THP-1 cells [13 ], and human monocytes [14 ] that mRNA and protein expression of MyD88 and TRAF6 are unchanged following LPS stimulation. In this study, we found that the mRNA and protein expression of MyD88 and TRAF6 were constitutively expressed in THP-1 cells, but there were no significant differences between naïve and BLP-tolerant cells before and after a subsequent stimulation with BLP or LPS. Therefore, despite the critical functions of MyD88 and TRAF6 in transducing BLP and LPS signaling, it appears that neither of them is involved in the regulation of BLP-induced self-tolerance and cross-tolerance to LPS.

IRAK-1 plays a crucial role in LPS signaling downstream of TLR4. Subsequent to LPS stimulation, IRAK-1 is recruited to the receptor and becomes highly phosphorylated. This is degraded rapidly and then signals downstream to TRAF6 [27 ]. Currently, four members of the IRAK family, IRAK-1 (IRAK), IRAK-2, IRAK-M, and IRAK-4, have been discovered [28 ]. IRAK-1 and IRAK-4 have kinase activity and mediate LPS signaling. Mice with a deletion of IRAK-4 are phenotypically similar to mice lacking the adaptor protein MyD88 [28 ]. Animals with a deletion of IRAK-1 are partly resistant to LPS-induced lethality [29 ]. LPS-induced IRAK-1 kinase activity and protein content were decreased in LPS-tolerant THP-1 cells [13 ] and human peripheral blood mononuclear cells (PBMC) [15 ], whereas the IRAK mRNA expression was increased in LPS-tolerant, human PBMC [15 ]. In mouse RAW264.7 cells, IRAK mRNA and protein expression were decreased following the induction of LPS tolerance [21 ]. Pretreatment with interferon-{gamma} (IFN-{gamma}) or granulocyte macrophage-colony stimulating factor (GM-CSF) during the first LPS challenge prevented the development of LPS tolerance by enhancing IRAK-1 protein expression [15 ]. In the present study, we found that BLP stimulation induced IRAK-1 degradation in naïve THP-1 cells, and the obvious decreases in IRAK protein expression were seen at 2–6 h post-BLP stimulation. This is consistent with a study by Siedlar et al. [30 ], who reported a significant reduction in IRAK-1 protein after 4 h of BLP treatment when using the human monocytic cell line Mono Mac 6. To further clarify the effect of BLP tolerance on IRAK-1 expression, THP-1 cells were subjected to BLP pretreatment to induce BLP tolerance. Our data showed that pretreatment of THP-1 cells with BLP (0.1 µg/ml) for 24 h resulted in a marked decrease in IRAK-1 protein level when compared with untreated cells, and the reduced IRAK-1 protein expression in BLP-tolerant cells was not reversed by a high dose (1.0 µg/ml) of BLP or LPS restimulation. Conversely, despite the massive reduction in IRAK-1 protein levels, there were no changes in its mRNA expression observed following BLP tolerance. Consistent with our findings, Siedlar et al. [30 ] also reported that pretreatment with 1.0 µg/ml BLP for 24 h led to a complete depletion of IRAK-1 protein in Mono Mac 6 cells. They also found that the decreased IRAK-1 protein levels did not accompany a reduction in the respective mRNA. Thus, our data suggest that the reduced IRAK-1 protein levels in BLP-tolerant THP-1 cells, as a result of its reduced synthesis or increased degradation, may be involved in BLP-induced self-tolerance and cross-tolerance to LPS. IRAK-4 is an integral part of the IL-1R signaling cascade and is capable of transmitting signals dependent on and independent of its kinase activity [31 ]. Furthermore, IRAK-4 is indispensable in the BLP and LPS signaling pathways. In contrast to IRAK-1, however, our results failed to demonstrate that IRAK-4 is involved in BLP tolerance.

Intracellular signal transduction of gram-positive or gram-negative bacterial cell-wall components such as BLP or LPS after engagement with TLRs (TLR2 or TLR4) requires the adaptor protein MyD88, which recruits IRAK-1. The association and interaction between MyD88 and IRAK are critical for subsequent engagement of TRAF6 and MAPK kinase-1 [23 , 32 ], which activate NF-{kappa}B and MAPK, respectively, leading to induction of gene expression and production of inflammatory cytokines. A defect of association between MyD88 and IRAK, therefore, may impair downstream transduction of BLP or LPS signaling. Several studies have shown that induction of LPS tolerance is associated with an inhibition in MyD88-IRAK complex formation [13 , 15 ], and IFN-{gamma} and GM-CSF prevent LPS tolerance, at least in part, by promoting IRAK association with MyD88 [15 ]. In the present study, we hypothesized that induction of BLP tolerance results in disrupted MyD88-IRAK association. Our coimmunoprecipitation data showed that BLP stimulation induced a rapid and transient MyD88-IRAK complex formation in naïve THP-1 cells. In contrast, induction of BLP tolerance caused a defect of association between MyD88 and IRAK, as BLP-tolerant cells were unable to form the MyD88-IRAK immunocomplex in response to BLP or LPS restimulation. The impaired association of IRAK with MyD88 observed in BLP-tolerant THP-1 cells may be caused by reduced MyD88/IRAK protein expression and/or loss of interaction between these two proteins. It has been shown that LPS tolerance induces a defect of association between MyD88 and IRAK, which correlates with reduced protein levels of IRAK [13 , 15 ]. IFN-{gamma} and GM-CSF restore MyD88 and IRAK complex formation by up-regulating IRAK expression [15 ]. Conversely, a disrupted TLR4-MyD88 association as a result of failure of interaction between TLR4 and MyD88 proteins was also demonstrated in LPS-tolerant human monocytes, as LPS tolerance did not affect the expression levels of these two proteins [14 ]. In the present study, we have shown that induction of BLP tolerance induces a marked decrease in IRAK-1 expression. Thus, the observed MyD88-IRAK disassociation in BLP-tolerant THP-1 cells possibly results from the reduced IRAK-1 protein levels, although we cannot exclude that an impaired interaction between MyD88 and IRAK-1 in BLP-tolerant cells may also exist. To our best knowledge, this is the first report that has demonstrated a disturbed MyD88-IRAK complex formation in BLP-tolerant cells, which may be responsible for BLP-induced self-tolerance and cross-tolerance to LPS.

Our previous work has indicated that BLP-induced NF-{kappa}B DNA-binding activity was decreased significantly in THP-1 cells previously exposed to BLP [2 ]. As NF-{kappa}B activation and translocation into the nucleus require I{kappa}B protein phosphorylation and degradation to release NF-{kappa}B dimers, we compared the activation and degradation of I{kappa}B-{alpha} in naïve and BLP-tolerant THP-1 cells restimulated with BLP or LPS. Our data showed that in response to a second BLP or LPS stimulation, I{kappa}B-{alpha} phosphorylation in BLP-tolerant cells was markedly suppressed. It was also noted that total I{kappa}B-{alpha} protein expression was reduced greatly following BLP tolerization in THP-1 cells compared with naïve cells. However, an inhibition of BLP-induced degradation of total I{kappa}B-{alpha} protein in BLP-tolerant cells was still observed. These data indicate that induction of BLP tolerance leads to abrogated I{kappa}B-{alpha} phosphorylation, which may be another mechanism downstream of IRAK-1 for BLP-induced self-tolerance and cross-tolerance to LPS. In contrast to our findings of markedly reduced, total I{kappa}B-{alpha} protein expression in BLP-tolerant cells, other studies [16 , 33 ] reported that following the induction of LPS tolerance, I{kappa}B-{alpha} phosphorylation and degradation were inhibited, but the total I{kappa}B-{alpha} was not reduced significantly. One may argue that reduced, total I{kappa}B-{alpha} protein levels in BLP-tolerant cells may favor NF-{kappa}B activation, as the functional ability of I{kappa}B proteins is to sequester NF-{kappa}B in the cytoplasm. Unlike naïve cells (monocytes/macrophages), in which the inhibitory effect on NF-{kappa}B would be lost in the absence of I{kappa}B, the presence of I{kappa}B in LPS- or BLP-tolerant cells may not be essential for an inhibited NF-{kappa}B activation and subsequent repressed target-gene transcription. For example, in response to LPS restimulation, an accumulation of predominant NF-{kappa}B p50 homodimers in the nucleus was evident in LPS-tolerant cells [10 , 34 ]. A recent study, using chromatin immunoprecipitation, further demonstrated that in contrast to naïve THP-1 cells, LPS restimulation-induced accumulation of NF-{kappa}B p65 in LPS-tolerant cells did not bind at the IL-1ß promoter, despite cytosolic NF-{kappa}B activation and translocation of p65 into the nucleus [35 ]. Therefore, inhibition of NF-{kappa}B activation and NF-{kappa}B-dependent gene expression in LPS-tolerant cells and most likely in BLP-tolerant cells may be modulated at different levels by multiple factors. Finally, as I{kappa}B-{alpha} is one of the target genes of NF-{kappa}B, its protein synthesis and expression are regulated tightly by NF-{kappa}B activation [10 , 36 ]. The reduced total I{kappa}B-{alpha} protein observed in BLP-tolerant THP-1 cells in the present study is presumed to be a result of the profound inhibition of NF-{kappa}B activity in the BLP-tolerant state.

As TLR2 and TLR4 share the same MyD88-dependent, post-receptor, intracellular signaling pathway directed to NF-{kappa}B activation, the mechanisms underpinning BLP-induced self-tolerance and cross-tolerance to LPS may also involve multiple changes in TLR2-mediated signal transduction pathways. Our results have demonstrated that in addition to the down-regulated TLR2 protein expression, decreased IRAK-1 expression, reduced MyD88-IRAK complex formation, and suppressed I{kappa}B-{alpha} phosphorylation may be responsible for BLP tolerance and its cross-tolerance to LPS.


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ACKNOWLEDGEMENTS
 
This work was supported by the Basic Research Grants Scheme from The Irish Research Council for Science, Engineering and Technology (SC/2003/27, to J. H. W.)

Received September 9, 2005; revised November 11, 2005; accepted November 23, 2005.


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