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(Journal of Leukocyte Biology. 2008;83:672-679.)
© 2008 by Society for Leukocyte Biology

Early and preferential induction of IL-1 receptor-associated kinase-M in THP-1 cells by LPS derived from Porphyromonas gingivalis

Hisanori Domon*,{dagger},{ddagger},1, Tomoyuki Honda*,{dagger},{ddagger},1, Taro Oda{dagger}, Hiromasa Yoshie{dagger} and Kazuhisa Yamazaki*,{ddagger},2

* Laboratory of Periodontology and Immunology, Department of Oral Health and Welfare, Niigata University Faculty of Dentistry,
{dagger} Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical Dental Sciences, and
{ddagger} Center for Transdisciplinary Research, Niigata University, Niigata, Japan

2Correspondence: Laboratory of Periodontology and Immunology, Department of Oral Health and Welfare, Niigata University Faculty of Dentistry, 5274 Gakkocho 2-ban-cho, Chu-o-ku, Niigata 951-8514, Japan. E-mail: kaz{at}dent.niigata-u.ac.jp


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ABSTRACT
 
LPS of Porphyromonas gingivalis (P. gingivalis) is suggested to be a virulence factor in periodontitis, stimulating host cells to produce proinflammatory mediators. However, P. gingivalis LPS has been reported to show lower biological activity compared with Escherichia coli (E. coli) LPS. Although differences in the chemical structure of lipid A and the receptor conferring LPS signaling are thought to account for these characteristics, the precise reason is unknown. Here, we demonstrate that P. gingivalis LPS up-regulates IL-1R-associated kinase (IRAK)-M, a negative regulator of the TLR signaling pathway, in a THP-1-derived macrophage more robustly than E. coli LPS. Although down-regulation of IRAK-M by small interfering (si)RNA augmented transcription and translation of TNF-{alpha}, IL-6, and IL-12 p40 in LPS-stimulated macrophages, the effect of siRNA was more prominent in P. gingivalis LPS-stimulated cells. Degradation of IRAK-1 was more obvious in E. coli LPS-stimulated macrophages than the cells stimulated with P. gingivalis LPS, suggesting that P. gingivalis LPS-induced IRAK-M suppressed dissociation of IRAK-1 from the receptor complex, resulting in escape from subsequent degradation. This activity may be involved in the chronic infection of this bacterium in periodontal tissue by serving as an escape mechanism from immune surveillance.

Key Words: human • monocytes/macrophages • signal transduction


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INTRODUCTION
 
A major bioactive substance of Gram-negative bacteria is LPS, which constitutes the outer membrane of the bacteria and is considered to be a potent inducer of proinflammatory cytokine production by neutrophils, monocytes, macrophages, and fibroblasts [1 ]. LPS-induced proinflammatory cytokines are indispensable for counteracting the growth and dissemination of gram-negative bacteria, and they often also mediate immune pathology themselves, such as the connective destruction and alveolar bone resorption seen in the periodontal diseases. However, an inflammatory response is resolved by the release of endogenous anti-inflammatory mediators, and the recent discoveries of IL-1R-associated kinase (IRAK)-M- and suppressor of cytokine signaling (SOCS)-1-dependent negative regulatory mechanisms in TLR-signaling pathways suggest distinct types of safety mechanisms for controlling the inflammatory response.

IRAK-M is highly restricted to monocytes/macrophages. It is induced upon TLR stimulation and prevents dissociation of IRAK-1 and IRAK-4 from MyD88 and the formation of IRAK-TNFR-associated factor 6 (TRAF6) complexes [2 ]. SOCS-1 expression is promptly induced in macrophages upon LPS stimulation and inhibits LPS-induced NF-{kappa}B and STAT-1 activation in macrophages [3 , 4 ]. In addition, recent studies have shown that Src homology 2 domain-containing inositol phosphatase (SHIP) [5 ] and PI3K [6 ] are involved in the negative regulation of the LPS-induced inflammatory response. As a homeostatic mechanism, the role of negative regulators is reasonable to suppress excess damage of host tissues.

Porphyromonas gingivalis is the predominant gram-negative bacteria in the periodontal pockets of patients with periodontitis and is considered to be one of the major pathogens associated with various forms of periodontal diseases [7 ]. The LPS of P. gingivalis has been suggested as a possible virulence factor, stimulating host cells to produce proinflammatory mediators [8 , 9 ]. P. gingivalis LPS possess unique chemical and biological properties different from those of enterobacterial LPS.

Lipid A, the active center of the LPS from P. gingivalis, possesses structurally and functionally unique characteristics such as resistance to polymyxin B [10 ] and much less toxicity than Escherichia coli LPS in vivo [11 ]. Compared with E. coli LPS, P. gingivalis LPS has been reported to have low biological activity, such as the induction of cytokine production [12 ]. The major species of P. gingivalis lipid A is composed of unique branched fatty acids, with longer carbon chains than in enterobacterial lipid A, the absence of a phosphoryl group at position 4' of the nonreducing glucosamine, and certain other structural differences [13 ]. In addition to the uniqueness of the chemical structure, this lower biological activity of P. gingivalis LPS has been considered to be attributable to signaling through TLR2 but not TLR4 [14 ]. However, it is now clear that responsiveness to LPS from P. gingivalis can be conferred by TLR4 as well as TLR2 [15 ]. This indicates the difference of the activation pathway may not be the sole reason for the lower biological activity of P. gingivalis LPS compared with enterobacterial LPS.

Therefore, in the present study, we investigated the effect of P. gingivalis LPS on the expression of negative regulators of the TLR signaling pathway, particularly IRAK-M.


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MATERIALS AND METHODS
 
Reagents and antibodies
PMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS from E. coli O111:B4 was purchased from Sigma-Aldrich, and LPS from P. gingivalis 381 was kindly provided by Hidefumi Kumada and Toshio Umemoto (Department of Microbiology, Kanagawa Dental University, Yokosuka, Japan). Recombinant human TNF-{alpha} was obtained from R & D Sytems Inc. (Minneapolis, MN, USA).

Rabbit anti-human IRAK-M (Chemicon International, Temecula, CA, USA), rabbit anti-human SOCS-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human SHIP (Upstate Biotechnology, Inc., Lake Placid, NY, USA), mouse anti-human IRAK-1 (Santa Cruz Biotechnology), mouse anti-human GAPDH (Abcam, Cambridge, UK), and ECL Plus Western blotting reagent pack (Amersham Biosciences, Buckinghamshire, UK) were used for Western blotting.

Cell preparation and culture
The monocytic cell line THP-1 was maintained in 25 mM Hepes-buffered RPMI 1640, supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin, hereafter referred to as medium. All incubations were carried out at 37°C in an atmosphere of 5% CO2 in air.

For the experiments, the cells were incubated in a 24-well culture plate (TPP, Trasadingen, Switzerland) at a concentration of 2 x 105 cells/ml in the medium supplemented with 200 nM PMA to induce differentiation into macrophage-like cells, hereafter referred to as macrophages. After 48 h of incubation, the cells were washed extensively with RPMI 1640 and cultured further in the medium without FCS for 12 h, and the medium was changed to remove the cytokines induced by cell adherence. Then, various doses of LPS were added to the culture, and the cells were stimulated for various periods of time (1, 3, 6, 9, 12, 15, 18, and 24 h).

Gene expression analysis
Total RNA was isolated from unstimulated and stimulated macrophage using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, and treated with RNase-free DNase I (Invitrogen). The RNA was then reverse-transcribed to cDNA using a random primer (Takara Bio Inc., Shiga, Japan) and Moloney murin leukemia virus RT (Invitrogen).

For real-time PCR, primers and a probe specific for GAPDH were purchased from Applied Biosystems (Foster, CA, USA). Primers and probes specific for IRAK-M and IRAK-1 were designed using Primer Express, Version 2.0, software (Applied Biosystems). The sequences are shown in Table 1 . Reactions were conducted in a 25 µl reaction mixture on the ABI Prism 7900 HT sequence detection system (Applied Biosystems) by using TaqMan gene expression assays (Applied Biosystems) containing 900 nM primer and 250 nM probe and incubated for 10 min at 95°C, followed by 40 cycles of a two-step amplification procedure composed of annealing/extension at 60°C for 1 min and denaturation for 15 s at 95°C. ABI Prism SDS 2.0 software (Applied Biosystems) was used to analyze the standards and to carry out the quantifications. The relative quantity of each mRNA was normalized to the relative quantity of GAPDH mRNA.


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  		Table 1. Primer and Probe Sequences for Real-Time PCR

Western blotting
The cells were washed with ice-cold PBS twice and lysed on ice in 150 µl lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.1% Tween-20 containing 10% glycerol, 0.1 mM PMSF, 10 pg/ml leupeptin, 1 mg/ml aprotinin, 10 mM β-glycerophosphate, 1 mM NaF, and 1 mM sodium orthovanadate). Cell debris was pelletted by centrifugation for 20 min at 12,000 g at 4°C. The protein concentration in the supernatant was determined using a Bio-Rad protein assay kit, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).

Each sample (12 µg) was solublized by SDS sample buffer, separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore Co., Bedford, MA, USA), Western-blotted with each antibody, and determined with ECL. For reprobing of GAPDH, the membranes were washed three times with wash buffer (Tris-buffered saline containing 0.1% Tween-20 and 0.5% skim milk) and Western-blotted with anti-GAPDH antibody as described above.

The membrane was exposed to X-ray film (Fuji RX-U, Fuji Film Co., Minamiashigara, Kanagawa, Japan), processed, and photographed. The intensity of the signal was quantified using computer software (Scion Image 4.02, Scion Corp., Frederick, MD, USA). The intensity of each molecule was expressed after normalization with the GAPDH intensity.

Cytokine assay
The levels of TNF-{alpha}, IL-6, and IL-12 p40 in the supernatants of macrophage culture were determined by using commercially available ELISA kits (BioSource, Camarillo, CA, USA), according to the manufacturer’s instructions.

Construction and transfection of small interfering RNA (siRNA)
Stealth RNA interference (RNAi) against IRAK-M and Stealth RNAi-negative control were purchased from Invitrogen. The sequences of the siRNA are sense: 5'-CCUUGGCACAUUCGAAUCGGUAUAU-3'; antisense: 5'-AUAUACCGAUUCGAAUGUGCCAAGG-3'. The sequence for the Stealth RNAi-negative control has not been published. Macrophages were transfected with 40 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and they were treated 24 h post-transfection. The specific gene silencing was confirmed by real-time PCR and Western blotting.

Detection of NF-{kappa}B
Activation of the p50 and p65 subunits of NF-{kappa}B in macrophages was determined by means of NF-{kappa}B p50/p65 ELISA-based transcription factor assay kits (Active Motif, Carlsbad, CA, USA). The cells transfected with IRAK-M-specific siRNA were stimulated with LPS from P. gingivalis and E. coli for 9 h, washed with PBS/phosphatase inhibitors (Active Motif), and subjected to extraction of the nuclear fraction. Extract preparation and NF-{kappa}B ELISA were carried out according to the protocols supplied by the manufacturer. The amount of total protein (0.5 µg) used in the NF-{kappa}B ELISA was determined in preliminary experiments.


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RESULTS
 
Stimulatory effect of P. gingivalis-derived LPS is weaker than that of E. coli LPS
To compare the effect of LPS on the production of proinflammatory cytokines by THP-1-derived macrophages, the cells were stimulated with 0.01, 0.1, or 1 µg/ml LPS from E. coli and P. gingivalis for 24 h. Although the production of all the cytokines was significantly up-regulated compared with unstimulated control in a dose-dependent manner, the effect of P. gingivalis LPS on TNF-{alpha}, IL-6, and IL-12 p40 production was much lower than that of E. coli LPS (Fig. 1 ).


Figure 1
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  		Figure 1. Stimulatory effect of P. gingivalis LPS on proinflammatory cytokines is weaker than that of E. coli LPS. Macrophages were unstimulated or stimulated with various doses of E. coli and P. gingivalis for 24 h, and human TNF-{alpha}, IL-6, and IL-12 p40 induction in culture supernatants was analyzed by ELISA. Results are shown as mean ± SD of three independent experiments. Significant differences among the different LPS stimulations are shown (*, P<0.05; **, P<0.005; ***, P<0.0001).

Up-regulation of IRAK-M in macrophage by P. gingivalis LPS stimulation
IRAK-M gene is constitutively expressed in the macrophage and was up-regulated by stimulation with the LPS preparations in a dose-dependent manner. The effect was the most prominent in the P. gingivalis LPS-stimulated cultures at a concentration of 1 µg/ml. At up to 3 h of stimulation, no difference of effect on IRAK-M expression was found among the different types of LPS and doses, but significant up-regulation was observed for P. gingivalis LPS-stimulated cultures at 6 h and 9 h. The rate of up-regulation by E. coli LPS was augmented between 9 h and 12 h for both concentrations, and the relative expression was similar to that stimulated by 1 µg/ml P. gingivalis LPS at 12 h (Fig. 2A ).


Figure 2
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  		Figure 2. Up-regulation of IRAK-M in THP-1-derived macrophages exhibits a distinctive pattern by which the LPS derived from various bacteria stimulated the cells. (A) The time course of IRAK-M expression in macrophage was compared among LPS preparations from E. coli and P. gingivalis. After stimulation with 0.1 µg/ml or 1 µg/ml LPS for the indicated time, total RNA was extracted from the cells, and IRAK-M gene expression was analyzed by real-time PCR. Data are expressed as mRNA expression relative to expression without stimulation. Results are shown as mean ± SE of three independent experiments. There is a significant difference of the IRAK-M level between P. gingivalis LPS stimulation and E. coli LPS stimulation at 6 h or at 9 h at both concentrations (P<0.05). (B) Macrophages were unstimulated or stimulated with 1 µg/ml E. coli and P. gingivalis for the indicated periods of time. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-IRAK-M antibody. Results are representative of those in four independent experiments. (C) The result of densitometric analysis of Western blotting. Results are shown as mean ± SE of four independent experiments. Significant differences between unstimulated control and the different LPS stimulations are indicated (*, P<0.05; **, P<0.005; ***, P<0.0001).

P. gingivalis LPS-induced IRAK-M up-regulation was confirmed further by Western blotting. The up-regulation of IRAK-M was observed at 9 h of stimulation and was still evident at 15 h (Fig. 2B) , whereas E. coli LPS stimulation did not exhibit the up-regulation at any time-point. IRAK-M expression appeared to be down-regulated in E. coli LPS- but not P. gingivalis LPS-stimulated cultures at 6 h (Fig. 2C) .

As the increased expression of IRAK-M was noted at 6–9 h of stimulation with P. gingivalis LPS, it is conceivable that the finding is attributable to an autocrine response by other gene products induced by LPS stimulation rather than a direct effect of LPS stimulation on IRAK-M expression. Although TNF-{alpha} is reported to up-regulate IRAK-M gene expression, no effect of TNF-{alpha} stimulation was observed at concentrations up to 30 ng/ml (data not shown).

Expression of SOCS-1 and SHIP by the LPS stimulation
SOCS-1 and SHIP were constitutively expressed in THP-1-derived macrophages. In contrast to the IRAK-M expression, no apparent change of protein expression of SOCS-1 or SHIP was found for any LPS stimulation (data not shown).

Specific suppression of IRAK-M by siRNA and concomitant up-regulation of TNF-{alpha} by P. gingivalis LPS stimulation
To confirm the specific suppression of IRAK-M by siRNA, specific siRNA and negative control siRNA were transfected to macrophages at a concentration of 40 nM, and the expression of IRAK-M was measured at 24 and 36 h. As shown in Figure 3A , a 60–70% knock-down effect was found at both time-points.


Figure 3
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  		Figure 3. Specific suppression of IRAK-M by siRNA transfection and the effects on the expression of inflammatory cytokines. (A) Negative control siRNA and IRAK-M-specific siRNA were transfected into macrophages at a concentration of 40 nM, and the expression of IRAK-M was analyzed by Western blotting at 24 and 36 h. The upper row shows the result of densitometric analysis of Western blotting (n=3; mean±SE). (B) Macrophages were transfected with negative control siRNA or IRAK-M-specific siRNA and then unstimulated or stimulated with 1 µg/ml E. coli LPS and P. gingivalis LPS for 9 h. Total RNA was extracted from the cells, and the expression of human TNF-{alpha}, IL-6, and IL-12 p40 mRNA was analyzed by real-time PCR. Results are shown as mean ± SE of three independent experiments. Significant differences between E. coli LPS-stimulated and P. gingivalis LPS-stimulated cultures are shown (*, P<0.05). (C) Macrophages were transfected with negative control siRNA or IRAK-M-specific siRNA and then unstimulated or stimulated with 1 µg/ml E. coli LPS and P. gingivalis LPS for 24 h. TNF-{alpha}, IL-6, and IL-12 p40 in culture supernatants were analyzed by ELISA. Results are shown as mean ± SD of three independent experiments. The significant differences between the IRAK-M-specific, siRNA-transfected cutltures and control siRNA-transfected cultures in each stimulation are indicated (*, P<0.05; **, P<0.01).

The effect of IRAK-M suppression on cytokine mRNA expression was determined (Fig. 3B) . It was noted that IRAK-M-specific siRNA transfection showed little effect on TNF-{alpha} expression in the macrophages stimulated with E. coli LPS. In contrast to TNF-{alpha} expression, mRNA for IL-6 and IL-12 p40 were up-regulated in IRAK-M-specific siRNA-transfected macrophages. Unlike E. coli LPS stimulation, IRAK-M-specific siRNA transfection exhibited an up-regulation of mRNA expression for IL-6 and IL-12 p40 as well as TNF-{alpha} in P. gingivalis LPS-stimulated macrophages. The knock-down effect of siRNA for IRAK-M mRNA was confirmed further at the protein level. In P. gingivalis LPS-stimulated macrophage, transfection of siRNA for IRAK-M up-regulated the production of all cytokines examined. As with mRNA expression, TNF-{alpha} content in the culture supernatant was not different between the negative control siRNA-transfected and specific siRNA-transfected cultures when stimulated with E. coli LPS (Fig. 3C) . IL-6 and IL-12 p40 productions were up-regulated by siRNA-induced down-regulation of IRAK-M.

Lower degradation of IRAK-1 in P. gingivalis LPS-stimulated macrophage
IRAK-M is considered to suppress dissociation of IRAK-1 from the receptor complex, resulting in escape from subsequent degradation. Therefore, it is assumed that up-regulation of IRAK-M correlates with down-regulation of IRAK-1 degradation. As shown in Figure 4 , there was no difference of stimulatory effect on IRAK-1 expression between P. gingivalis LPS and E. coli LPS stimulations (A); however, degradation of IRAK-1 was more prominent in macrophages stimulated with E. coli LPS compared with P. gingivalis LPS. It was obvious that degradation of IRAK-1 was suppressed concomitantly with up-regulation of IRAK-M expression by stimulation with P. gingivalis LPS at 9–15 h (B). Suppression of IRAK-M expression by siRNA resulted in IRAK-1 degradation in P. gingivalis LPS-stimulated macrophages (C).


Figure 4
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  		Figure 4. Up-regulation of IRAK-M inhibits IRAK-1 degradation. (A) The time course of IRAK-1 expression in macrophages was compared for E. coli LPS and P. gingivalis LPS. After stimulation with 1 µg/ml LPS for the indicated time, total RNA was extracted from the cells, and IRAK-1 gene expression was analyzed by real-time PCR. Data are expressed as mRNA expression relative to expression without stimulation and are shown as the mean ± SE of four independent experiments. (B) Macrophages were unstimulated or stimulated with 1 µg/ml E. coli LPS and P. gingivalis LPS for the indicated periods of time. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-IRAK-1 and anti-GAPDH antibodies. Results are representative of those in four independent experiments. (C) Macrophages were transfected with negative control siRNA (left column) or IRAK-M-specific siRNA (right column) and then unstimulated or stimulated with 1 µg/ml E. coli LPS and P. gingivalis LPS for 9 h. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-IRAK-1 and anti-GAPDH antibodies.

Suppression of P. gingivalis LPS-induced IRAK-M by siRNA up-regulates NF-{kappa}B activation
As down-regulation of IRAK-M coincided with an up-regulation of proinflammatory cytokine expression, the effect of suppression of IRAK-M on the NF-{kappa}B activation was analyzed in P. gingivalis LPS- or E. coli LPS-stimulated cultures by means of ELISA. As shown in Figure 5 , NF-{kappa}B p50 and p60 were up-regulated in IRAK-M-specific siRNA-transfected macrophages compared with control siRNA-transfected macrophages in P. gingivalis LPS-stimuilated cultures. However, these effects were not observed for E. coli LPS stimulation.


Figure 5
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  		Figure 5. NF-{kappa}B p50 and p65 activation in P. gingivalis LPS, with or without IRAK-M suppression. Macrophages were transfected with negative control siRNA or IRAK-M-specific siRNA and then were left unstimulated or stimulated with 1 µg/ml E. coli LPS or P. gingivalis LPS for 9 h. NF-{kappa}B activation in cellular extracts was analyzed using a NF-{kappa}B p50/p65 ELISA kit. Results are shown as mean ± SD of triplicate determinants. Significant differences were observed for P. gingivalis LPS stimulation between the control siRNA and specific siRNA transfections (*, P<0.05).


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DISCUSSION
 
Although TLR signaling-mediated cytokine production and the resultant inflammatory response are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is harmful to the host, such as connective tissue destruction and alveolar bone resorption seen in periodontal disease. To maintain immune-system integrity, there are negative-feedback regulators in the TLR signaling pathway, such as IRAK-M, SOCS-1, and SHIP. The most prominent finding reported here is that periodontopathic bacterium P. gingivalis-derived LPS up-regulates IRAK-M expression rapidly and preferentially compared with E. coli LPS. As LPS stimulation demonstrated no significant change of the protein production of SOCS-1 and SHIP, subsequent detailed analysis was focused on IRAK-M, which is reported to play an important role, together with other molecules such as SOCS-1 and PI3K, in endotoxin tolerance [16 , 17 ] and peptidoglycan-induced tolerance in macrophages [18 ].

Unique characteristics of P. gingivalis LPS have been demonstrated in the context of endotoxin tolerance. For example, although pretreatment of THP-1 cells with E. coli LPS resulted in a severe reduction in the level of IL-1β, IL-6, and TNF-{alpha} upon secondary stimulation, pretreatment with P. gingivalis LPS showed a similar effect only on IL-1β production [19 ]. In contrast to the cytokine production, Ig-like transcript 3 (ILT-3) and B7-H1 expression were significantly up-regulated upon secondary stimulation by P. gingivalis LPS. However, E. coli LPS had no effect on the expression of these molecules. ILT-3 and B7-H1 are considered to contribute to the immunosuppressive function of APCs. In addition to the lower stimulatory effect of P. gingivalis LPS on cytokine production compared with E. coli LPS, P. gingivalis LPS-induced desensitization of APCs may favor immune tolerance, a property not observed for E. coli LPS [20 ]. In addition, Dobrovolskaia et al. [21 ] demonstrated that whereas TNF-{alpha} production was abrogated completely in the homotolerance induced by P. gingivalis LPS, TNF-{alpha} secretion was not inhibited completely in the homotolerance by E. coli LPS. This may be a result of a more robust induction of negative regulators in P. gingivalis LPS stimulation.

In the tolerance induction model, although IRAK-M mRNA was expressed 6 h after stimulation with Salmonella LPS, the LPS pretreatment induced a more immediate up-regulation of IRAK-M gene expression [16 ]. The induction of IRAK-M by P. gingivalis LPS was evident 6 h and 9 h after stimulation, which was significantly higher than that by E. coli LPS. At 12 h of stimulation, IRAK-M mRNA expression was up-regulated further by all the LPS preparations, and the expression levels came to be similar. However, Western blot analysis clearly demonstrated that elevated IRAK-M expression in P. gingivalis LPS-stimulated cultures was observed at 9 h after stimulation and maintained until 15 h, suggesting that the up-regulation of IRAK-M gene expression by P. gingivalis LPS stimulation had a prolonged effect on protein synthesis. On the other hand, there was a discrepancy between gene expression and protein synthesis of IRAK-M by E. coli LPS stimulation. Although we have not examined the effect of different bacteria-derived LPS, it is speculated that the expression profile of IRAK-M by E. coli LPS but not by P. gingivalis LPS stimulation is rather typical. Furthermore, at the protein level, IRAK-M expression seemed to be down-regulated by E. coli LPS at 6 h. Although the reason for this curious finding is not known, it is possible to speculate that the inflammatory response to this bacterium may be up-regulated by transient down-regulation of a negative regulator. Other tolerogenic molecules such as SOCS-1 and SHIP were not affected by the primary stimulation with LPS. Therefore, at least for primary stimulation, P. gingivalis LPS-induced IRAK-M may be responsible for the down-regulation of cytokine synthesis.

Down-regulation of IL-6 and IL-12 p40 expression was rescued by suppression of IRAK-M gene transcription by siRNA when macrophages were stimulated with E. coli LPS. However, the effect of the siRNA for IRAK-M on TNF-{alpha} was negligible for the stimulation with E. coli LPS. These findings are in accordance with those obtained by using macrophages derived from the IRAK-M–/– mouse [2 ]. Synthesis of IL-12 p40 and IL-6 were higher in the IRAK-M–/– macrophages compared with wild-type macrophages when the cells were stimulated with Salmonella LPS. However, no difference was observed for TNF-{alpha} synthesis [2 ]. Therefore, up-regulation of TNF-{alpha} production in siRNA-transfected macrophage stimulated with P. gingivalis LPS could be a P. gingivalis LPS-specific phenomenon. Alternatively, this response pattern could be specific for TNF-{alpha}. In this respect, it is reported that the LPS-induced TNF-{alpha} factor (LITAF) specifically regulates TNF-{alpha} transcription and is an important mediator of the LPS-induced inflammatory response that can be distinguished from the NF-{kappa}B pathway [22 ]. The involvement of LITAF in the expression of TNF-{alpha} may account for the difference of the rescue by IRAK-M-specific siRNA between TNF-{alpha} and IL-6 or IL-12p40 synthase. Nevertheless, as with the absence of effect of LPS stimulation on TNF-{alpha} in the macrophages from IRAK-M–/– mice [2 ], the reason for the rescue of TNF-{alpha} production in siRNA for the IRAK-M-transfected macrophages stimulated with P. gingivalis LPS remains to be elucidated.

P. gingivalis LPS-specific IRAK-M up-regulation in macrophages was further confirmed by a lower degradation of IRAK-1 compared with E. coli stimulation. LPS binding to TLR triggers the recruitment of MyD88 via a homophilic Toll/IL-1R (TIR)-TIR interaction. MyD88 in turn brings IRAK-4 into the receptor complex. In addition, preformed Toll-interacting protein/IRAK-1 complexes are recruited to the receptor, which allows IRAK-1 to bind MyD88 via its death domain. In this way, IRAK-1 and IRAK-4 come in close proximity, which allows IRAK-4 to phosphorylate IRAK-1 on critical residues that are necessary to trigger IRAK-1 [23 ]. TRAF6 is also recruited transiently to the receptor complex via interaction with phosphorylated IRAK-1 [24 ], which with TRAF6, dissociate from the receptor and interact at the membrane with a preformed complex consisting of TGF-β-activated protein kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and TAB2 [24 , 25 ]. This interaction induces phosphorylation of TAB2 and TAK1, which then translocate together with TRAF6 and TAB1 to the cytosol [24 25 26 ]. IRAK-1 remains at the membrane, where it gets degraded, presumably via a ubiquitin-dependent mechanism [24 ]. During the unfolding of this pathway, IRAK-M does not prevent IRAK-1 or IRAK-4 recruitment to the receptor complex but inhibits IRAK-1 dissociation from the receptor complex after its activation. It therefore blocks the LPS-induced IRAK-1–TRAF6 interaction and NF-{kappa}B activation, thus preventing IRAK-1 translocation to the membrane and subsequent degradation [2 ].

Therefore, if P. gingivalis LPS up-regulates functional IRAK-M, a greater amount of IRAK-1 would be left undegraded after stimulation with P. gingivalis LPS compared with E. coli LPS. As shown in Figure 4 , Western blot analysis clearly demonstrates that a higher level of IRAK-1 was detected in P. gingivalis LPS-stimulated cells compared with E. coli LPS-stimulated cells. However, IRAK-1 degradation became more obvious when specific siRNA-transfected macrophages were stimulated with P. gingivalis LPS compared with E. coli LPS, suggesting that P. gingivalis LPS specifically up-regulates IRAK-M and suppresses subsequent IRAK-1 translocation, resulting in depressed transcription and translation of proinflammatory cytokines. Although the IRAK-M level induced by P. gingivalis LPS and subsequent suppression of IRAK-1 degradation seemed to be relatively small, the effect was large enough to up-regulate the NF-{kappa}B activation, as shown in the IRAK-M suppression experiment, confirming our hypothesis further.

The mechanism by which P. gingivalis LPS rapidly induce IRAK-M is unknown. It is reported that the inhibiting of PI3K induces the early transcription of IRAK-M [16 ], suggesting a connection between these two events. As P. gingivalis LPS can activate the PI3-Akt pathway in monocytes [27 ], we speculate that the effect of P. gingivalis LPS on the PI-3-Akt pathway could be weaker than that of E. coli LPS. However, this remains to be determined. As the macrophages responded promptly to LPS stimulation in TNF-{alpha} expression as early as 3 h, one might speculate that this cytokine could have some effect on the IRAK-M expression by an autocrine mechanism. In support of this, del Fresno et al. [28 ] showed that IRAK-M was induced by TNF-{alpha}, and the expression was abrogated by anti-TNF-{alpha} antibody. However, no effect of TNF-{alpha} on the IRAK-M expression was observed, even at a concentration up to 30 ng/ml (data not shown). Therefore, the exact mechanism by which P. gingivalis LPS induce IRAK-M remains to be elucidated.

In conclusion, a periodontopathic bacterium, P. gingivalis-derived LPS, exhibits certain unique characteristics in the induction of IRAK-M in macrophages. P. gingivalis LPS is able to induce IRAK-M earlier and more potently than E. coli LPS, resulting in substantially lowered production of proinflammatory cytokines. This activity could very well be involved in the well-known phenomenon of chronic infection of this bacterium in periodontal tissue by acting as an escape mechanism from immune surveillance. Although further studies are clearly needed, elucidation of the regulatory mechanisms for IRAK-M expression may provide new insight into not only the mechanisms for chronic infection by P. gingivalis and other bacteria but also the development of new therapeutic modalities for chronic inflammatory diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (160209063, 17659655, and 19390536) and the Promotion of Niigata University Research Project. Pacific Edit reviewed the manuscript prior to submission. We thank H. Kumada and T. Umemoto (Department of Microbiology, Kanagawa Dental University, Yokosuka, Japan) for kindly providing P. gingivalis LPS.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received June 26, 2007; revised November 8, 2007; accepted November 20, 2007.


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