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. 2007 Jun;148(3):529–536. doi: 10.1111/j.1365-2249.2007.03346.x

Toll-like receptor 4-dependent recognition of structurally different forms of chemically synthesized lipid As of Porphyromonas gingivalis

N Sawada 1, T Ogawa 1, Y Asai 1, Y Makimura 1, A Sugiyama 1
PMCID: PMC1941937  PMID: 17335558

Abstract

Porphyromonas gingivalis is a Gram-negative anaerobic oral black-pigmented bacterium closely associated with chronic periodontitis. Lipopolysaccharide (LPS) derived from P. gingivalis is shown to be unusual because the LPS contains a greater number of lipid A species, such as tri-, tetra-, and/or penta-acylated lipid As. In this study, a lipid A possessing penta-fatty acyl chains derived from P. gingivalis strain 381 (compound PG-381–5FA) was synthesized, and examined for its immunobiological activities, compared with a tri-acylated lipid A (compound PG-381–3FA) synthesized previously. Compound PG-381-5FA, similar to compound PG-381-3FA, demonstrated weaker activity in a Limulus test as compared with Escherichia coli-type synthetic lipid A (compound 506). Compound PG-381-5FA, followed by compound PG-381-3FA, induced KC, interleukin-6, and tumour necrosis factor-α production in peritoneal macrophages from LPS-responsive C3H/HeN mice, but not in those from LPS-hyporesponsive C3H/HeJ mice. Furthermore, compound PG-381-5FA, as well as compound PG-381-3FA, activated nuclear factor-κB via Toll-like receptor (TLR)4/mD-2, but not TLR2, in a manner similar to compound 506, and worked as an antagonist for compound 506-induced cell activation. In the case of human peripheral blood mononuclear cells, compound PG-381-5FA showed much stronger IL-6-inducing activity than compound PG-381-3FA. The present results demonstrate that the chemical synthesis of a penta-acylated lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, is attributable to immune cell activation through TLR4, similar to that of compound 506.

Keywords: lipid A, lipopolysaccharide, Porphyromonas gingivalis, synthetic compound, Toll-like receptor

Introduction

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and exhibits a variety of biological activities with host cells. Lipid A, the lipophilic portion and active centre of LPS, has been chemically synthesized and shown to have immunobiological activities [1,2]. These chemically well-defined synthetic lipid A compounds have been investigated extensively with regard to the structure-function relationships of lipid As.

Porphyromonas gingivalis, a Gram-negative, anaerobic oral black-pigmented rod, is suspected to be a periodontopathic bacterium and has been frequently isolated from the periodontal pockets of patients with chronic periodontal diseases [3]. The chemical and biological properties of P. gingivalis LPS and its lipid A are different from those of enterobacterial LPSs and their lipid As [4,5]. It was also shown that P. gingivalis LPS is heterogeneous, with a greater number of lipid A species, such as tri-, tetra-, and/or penta-acylated lipid As [4,6].

Furthermore, P. gingivalis LPS and its lipid A were reported to activate cells from LPS-hyporesponsive C3H/HeJ mice, as well as those from LPS-responsive C3H/HeN mice [7,8], which was thought to be due to its unique structure. To confirm this structure-activity relationship, we synthesized a counterpart of P. gingivalis strain 381-type tri-acylated lipid A, compound PG-381–3FA, and found that it induced interleukin (IL)-6 and tumour necrosis factor (TNF)-α production in peritoneal macrophages from C3H/HeN mice, but not C3H/HeJ mice [9]. In addition, cell activation by the purified natural lipid A of P. gingivalis and compound PG-381–3FA did not include activation of cells from Toll-like receptor (TLR) 4 knockout mice or C3H/HeJ mice [9,10].

In a recent report, innate host responses to multiple lipid A species obtained from P. gingivalis LPS were found to be unusual, as they were able to function as an agonist for TLR2 and also as an antagonist or agonist for TLR4 [11,12]. However, we previously showed that a synthetic lipid A compound with tri-fatty acyl chains of P. gingivalis exhibited cell activation through TLR4, but not TLR2 [10]. In the present study, we investigated the receptor utilized by P. gingivalis lipid A for cell activation by using synthetic lipid As with a different chemical structure.

Materials and methods

Animals

C3H/HeN and C3H/HeJ mice (male, 9-week-old) were obtained from Japan SLC, Hamamatsu, Japan. The animals received humane care in accordance with our institutional guidelines and the legal requirements of Japan.

Synthesis of P. gingivalis strain 381 lipid A possessing penta-fatty acyl chains (compound PG-381-5FA)

The P. gingivalis strain 381-derived lipid A possessing penta-fatty acyl chains is shown in Figs 1a and 2 [4,6]. GlcNTroc (Fig. 1b) was prepared according to a method described previously [13]. The fatty acid moiety, 3-(R)-p-methoxybenzyloxy-13-methyltetradecanoic acid (Fig. 1c), prepared using a method similar to one described previously [9], was introduced into GlcNTroc (Fig. 1b) by acylation of the 3-hydroxyl group to obtain 3-fatty acyl GlcNTroc (Fig. 1d). The 0.allyl glycoside of 3-fatty acyl GlcNTroc (Fig. 1d) was removed using [Ir(COD)(Ph2MeP)2]PF6 (Wako Pure Chemical, Osaka, Japan), H2 gas, H2O, and I2 (Wako Pure Chemical), then converted to the trichloroacetimidate group using CCl3CN (Wako Pure Chemical) and 1,8-diazabicyclo[5·4·0] undec-7-ene (DBU, Wako Pure Chemical) to obtain 1-trichloroacetimidate 3-fatty acyl GlcNTroc (Fig. 1e). 3,6-dihydroxy sugar (Fig. 1f) was prepared according to a method described previously [13]. The fatty acid moiety, 3-(R)-p-methoxybenzyloxy-15-methylhexadecanoic acid (Fig. 1g), prepared as described previously [9], was introduced into GlcNTroc (Fig. 1f) by deprotection and acylation of the protected N-amino group to obtain N-fatty acyl 3,6-dihydroxy sugar (Fig. 1h). 1-Trichloroacetimidate 3-fatty acyl GlcNTroc (Fig. 1e) was condensed with N-fatty acyl 3,6-dihydroxy sugar (Fig. 1h), with Sc(OTf)3 (Sigma-Aldrich Co., St. Louis, MO, USA) in CH2Cl2 at − 20°C used to generate the disaccharide (Fig. 1i). The fatty acid moiety, 3-(R)-p-methoxybenzyloxy-palmitic acid (Fig. 1j), was prepared according to a method previously reported [14] by acylation of a 3-hydroxy group to obtain a tri-fatty acyl disaccharide (Fig. 1k). The fatty acid moiety, 3-(R)-palmitoyloxy-15-methylhexadecanoic acid (Fig. 1L), prepared as described previously [9], was introduced into the tri-fatty acyl disaccharide (Fig. 1k) by deprotection and acylation of the protected N-amino group to obtain penta-fatty acly disaccharide (Fig. 1m). The allyl glycoside of the penta-fatty acyl disaccharide (Fig. 1m) was removed in the same manner as that shown in Fig. 1(d–e) and converted to a 1-phosphate derivative using n-butyl lithium-dibenzyl phosphorochloridate [15], Thereafter, all of the protecting groups were reductively removed to generate synthetic lipid A of P. gingivalis 381 (Fig. 1a; compound PG-381–5FA) by treatment with Pd(OH)2/C (Sigma-Aldrich Co.). The reactant was purified by liquid-liquid partition chromatography on a Sephadex LH-20 column (CHCl3:MeOH:H2O:Et3N = 5:8:5:0·005) to produce the desired compound.

Fig. 1.

Fig. 1

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Synthetic scheme of lipid A derivative containing penta-fatty acyl chains from Porphyromonas gingivalis strain 381. The synthetic procedures are described in ‘Materials and methods’.

Fig. 2.

Fig. 2

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Proposed chemical structure of the lipid A components of P. gingivalis strain 381 (compounds PG-381-5FA and PG-381-3FA) [4,6] as compared with E. coli-type lipid A (compound 506) [1].

Bacterial compound

E. coli-type synthetic lipid A (compound 506) and P. gingivalis strain 381-type tri-acylated lipid A (compound PG-381–3FA) were synthesized (Fig. 2) as described previously [9,16] and used in the present study. These synthetic products were dissolved at a concentration of 1 mg/ml in a 0·1% triethylamine aqueous solution. Bacterial synthetic lipopeptide Pam3CSK4 was purchased from EMC Microcollections (Tuebingen, Germany) and dissolved at a concentration of 1 mg/ml in pyrogen-free distilled water. The stock solution was kept at 4 °C and appropriately diluted with phosphate-buffered saline (PBS) or cell culture medium before use in the assay.

Limulus test

A Limulus test was performed with Pre Gel, an amoebocyte lysate prepared from Tachypleus tridentatus (Seikagaku Kogyo, Tokyo, Japan), according to the instructions packaged with the reagent. The dose that caused a reaction definitely stronger than the increased viscosity was used as the minimum effective dose.

Murine cytokine assay

Peritoneal exudate cells, obtained from a peritoneal wash of C3H/HeN and C3H/HeJ mice that had received an intraperitoneal injection of 1 ml of 3% sterile thioglycollate broth (Nissui Pharmaceutical Co., Tokyo, Japan) 4 days prior, were distributed into each well of a 96-well microculture plate (BD Falcon 353072; Becton Dickinson, Lincoln Park, NJ, USA) to make a monolayer, at a cell density of 2 × 105 cells per 0·2 ml of RPMI 1640 medium (Nikken Biomedical Laboratories, Kyoto, Japan) containing penicillin G (100 U/ml), streptomycin (100 µg/ml), and l-glutamine (300 µg/ml), supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) [17]. After a 1-h incubation, non-adherent cells were removed by aspiration. The monolayers were washed three times with PBS, and the indicated doses of the test specimens in 0·2 ml of RPMI 1640 medium with 10% FBS were added and then cultured at 37°C for 18 h. Culture supernatants were collected and stored at − 80°C until the assay for cytokine production. The production levels of KC, interleukin (IL)-6, and tumour necrosis factor (TNF)-α were measured in the culture supernatants using a commercial ELISA kit system (R & D Systems, Minneapolis, MN, USA, for KC, and Endogen, Cambridge, MA, USA, for IL-6 and TNF-α). Each assay was performed according to the manufacturer's instructions and the results were determined using a standard curve prepared for each assay.

Human cytokine assay

This experiment was performed with healthy adult volunteers (males, age 36 years). The subjects were informed regarding the study and each signed an informed consent form approved by the Ethics Committee of Asahi University (reference number 15007). Heparinized venous blood was subjected to fractionation using a Histopaque-1077 to obtain human peripheral blood mononuclear cells (PBMC). The cells were distributed into each well of a 96-well microculture plate to make a monolayer, at a cell density of 2 × 105 cells per 0·2 ml of RPMI 1640 medium containing penicillin G (100 U/ml), streptomycin (100 µg/ml), and l-glutamine (300 µg/ml), supplemented with 10% FBS. These cells were stimulated with the indicated doses of the test specimens for 24 h at 37°C. Following incubation, the culture supernatants were collected and analysed for secreted IL-6 using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA, USA).

Luciferase assay

IL-3-dependent murine Ba/F3 pro-B cells stably expressing p55IgκLuc, a nuclear factor (NF)-κB/DNA binding activity-dependent luciferase reporter construct (Ba/κB), murine TLR2 and a p55IgκLuc reporter construct (Ba/mTLR2), and murine TLR4/mD-2 and a p55IgκLuc reporter construct (Ba/mTLR4/mMD-2), were kindly provided by Dr K. Miyake (Institute of Medical Science, University of Tokyo, Tokyo, Japan) and used to detect NF-κB-dependent luciferase activity, as described previously [18]. Briefly, the cells were inoculated onto 96-well plates with 1 × 105 per 100 µl of RPMI 1640 (Sigma-Aldrich Co.) supplemented with 5% FBS, then stimulated separately with the indicated doses of the test specimens. After 4 h of incubation at 37°C, 100 µl of Bright-GloTM luciferase assay reagent (Promega, Madison, WI, USA) was added to each well and luminescence was quantified with a luminometer (Turner Designs Luminometer Model TD-20/20; Promega). In some experiments, 1 µg/ml of compound PG-381-5FA or -3FA was added to the culture 30 min prior to stimulation with 1 ng/ml of compound 506.

Statistics

Cytokine production was analysed by one-way analysis of variance (anova), using the Bonferroni or Dunn method. The results are presented as the mean ± standard error of the mean (s.e.m).

Results

Limulus test

The results obtained by the conventional Pre Gel test indicated that synthetic compound PG-381–5FA exhibited the Limulus amoebocyte lysate (LAL) gelation activity at a dose of 10 ng, nearly the same as that of the tri-acylated synthetic lipid A, compound PG-381-3FA, and their activities were 10−3 of that of compound 506.

Cytokine-inducing activities in mouse macrophages

We examined cytokine production by peritoneal macrophages from C3H/HeN and C3H/HeJ mice after stimulation with compound PG-381-5FA (Fig. 3). Compound PG-381-5FA, followed by compound PG-381-3FA, definitely induced KC, IL-6 and TNF-α production in peritoneal macrophages from C3H/HeN mice in a dose-dependent manner, though the activity was weaker than that of compound 506. On the other hand, neither compound PG-381–5FA, PG-381–3FA nor compound 506 induced cytokine production in C3H/HeJ macrophages. Pam3CSK4, a ligand for TLR2 but not TLR4, exhibited these cytokine productions by both C3H/HeN and C3H/HeJ macrophages.

Fig. 3.

Fig. 3

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Cytokine production by peritoneal macrophages from C3H/HeN and C3H/HeJ mice in response to stimulation by compound PG-381-5FA. Cells were cultured at 37 °C for 24 h in RPMI 1640 medium containing 10% FBS, with or without the indicated doses of each test specimen. Following incubation, the supernatants were collected and cytokine production was determined by ELISA. The experiments were done at least three times and representative results are presented. Each assay was done in triplicate and the data are expressed as the mean ± s.e.m. Significant differences were found between the groups with and without the test specimens (*P< 0·05, **P< 0·01).

NF-κB activation in Ba/mTLR4/mMD-2 cells

Compound PG-381-5FA, as well as compound PG-381-3FA, activated NF-κB via TLR4/mD-2, but not TLR2, in a manner similar to that of compound 506 (Fig. 4a, b, d). With the addition of compound PG-381-3FA to the assay mixture of compound PG-381-5FA, the multiple lipid A species, mimicking the natural lipid A species of P. gingivalis LPS, also induced NF-κB activation via TLR4/mD-2 (Fig. 4c). Furthermore, NF-κB activation of compound PG-381-5FA, as well as compound PG-381-3FA, was weaker in comparison with that of compound 506. Pam3CSK4 was used as positive control stimulant of TLR2 and induced an NF-κB activation in Ba/mTLR2 (Fig. 4e). Using RT-PCR, Ba/F3 cells were reported to constitutively express TLR2 mRNA; however, TLR3, TLR4, and TLR5 mRNA were not detected by RT-PCR or Northern hybridization [19]. For this reason, Ba/κB and Ba/mTLR4/mMD-2 cells responded to Pam3CSK4 in the present assay.

Fig. 4.

Fig. 4

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TLR4-dependent NF-κB activation of compound PG-381–5FA. Ba/κB cells (open triangle), Ba/mTLR2 cells (closed circle), and Ba/mTLR4/mMD-2 cells (open circle) were stimulated for 4 h with the indicated doses of compound PG-381-5FA (a), compound PG-381-3FA (b), compounds PG-381-5FA and PG-381 (equivalent weights) (c), compound 506 (d), and Pam3CSK4 (e). NF-κB activation was determined using a luciferase assay. The results are shown as relative luciferase activity, which was determined as the ratio of stimulated to nonstimulated activity. Each assay was done in triplicate and the data are expressed as the mean ± s.e.m. Significant differences were seen compared with Ba/κB cells in each dose of test specimen (**P< 0·01).

Antagonistic effects

P. gingivalis LPS was demonstrated to show antagonistic activity against E. coli LPS [12,20,21]. Therefore, we examined the effect of compound PG-381 on compound 506-induced cell activation using luciferase assay (Fig. 5). Pre-administration of 1 µg/ml of compounds PG-381-5FA and -3FA consequently exhibited 64·9% and 80·9% reductions of 1 ng/ml of compound 506-induced NF-κB activation in Ba/mTLR4/mMD-2 cells, respectively.

Fig. 5.

Fig. 5

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Antagonistic effects of compound PG-381. Ba/mTLR4/mMD-2 cells were incubated with 1 µg/ml of PG-381-5FA or PG-381-3FA for 30 min prior to stimulation with 1 ng/ml of compound 506 for 4 h. NF-κB activation was determined using a luciferase assay. The results are shown as relative luciferase activity, which was determined as the ratio of stimulated to non-stimulated activity. Each assay was done in triplicate and the data are expressed as the mean ± s.e.m. Significant differences were seen compared with compound 506 alone (**P< 0·01).

Cytokine-inducing activities in human PBMC

Compound PG-381-induced IL-6 production by human PBMC was examined (Fig. 6). In contrast to the mouse peritoneal macrophages (Fig. 3), compound PG-381-5FA showed much stronger IL-6-producing activity than compound PG-381-3FA in a dose-dependent manner (Fig. 6a). In addition, since the molecular weight of compounds PG-381-5FA and -3FA is 1690·38 and 1195·59, respectively, we next stimulated the cells using the equivalent molar concentrations. As shown in Fig. 6(b), the differences in the activities between compounds PG-381-5FA and -3FA were not changed as compared with the case of mass concentration.

Fig. 6.

Fig. 6

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IL-6 production by human PBMC in response to stimulation by compound PG-381-5FA. Cells were cultured at 37 °C for 24 h in RPMI 1640 medium containing 10% FBS, with or without the indicated doses ( (a) mass concentration (b) molar concentration) of PG-381-5FA (closed circle) or PG-381-3FA (open circle). Following incubation, the supernatants were collected and cytokine production was determined by ELISA. The experiments were done at least three times and representative results are presented. Each assay was done in triplicate and the data are expressed as the mean ± s.e.m. Significant differences were found between the groups with and without the test specimens (**P< 0·01).

Discussion

The present study found that a chemically synthetic compound of penta-acylated lipid A, mimicking the lipid A portion of LPS from P. gingivalis strain 381 (compound PG-381–5FA), exhibited an endotoxic activity, as demonstrated by Limulus test results (data not shown). The activity of compound PG-381-5FA, similar to tri-acylated compound PG-381-3FA, was weak but definite as compared with that of the E. coli-type synthetic lipid A, compound 506.

In a previous study, we and other researchers demonstrated that P. gingivalis lipid A showed heterogeneity and exhibited quite different phosphorylation and acylation patterns when compared with those of enterobacterial lipid A [4,6]. We also reported that P. gingivalis natural lipid A had marked differences as compared with compound 506 with regard to cytokine-inducing activities in splenic B cells from C3H/HeJ mice [22]. From those findings, we speculated that P. gingivalis lipid A possesses a unique chemical structure. In the present experiments, the number of fatty acid chains was different from tri-acylated compound PG-381-3FA. Thus, penta-acylated compound PG-381-5FA exhibited cytokine-inducing activities in peritoneal macrophages from C3H/HeN mice, but not C3H/HeJ mice, which have a point mutation of the TLR4 molecule. It was also found that an increasing number of fatty acyl chain of β(1–6)glucosamine disaccharide bone structure tends to increase cell activation. Furthermore, multiple lipid A species, that is a mixture of compound PG-381-5FA and compound PG-381-3FA, showed TLR4-dependent NF-κB signalling activation in a luciferase assay (Fig. 4c). It was previously shown that multiple structurally different forms of lipid A in P. ginigvalis LPS contributed to cell activation through both TLR2 and TLR4 [11]. We also demonstrated in a prior study that lipoprotein contained in P. gingivalis LPS preparation functionally interacts with only TLR2 [23]. Therefore, it was suggested that only trace molecule(s) such as lipoprotein and lipopeptide are contained in the natural LPS/lipid A of P. gingivalis, and that contaminant(s) might induce cell activation via TLR2.

It was shown that P. gingivalis LPS contains tetra-acylated, as well as tri- and penta-acylated lipid A structures [6,11], and tetra-acylated lipid A activated HEK 293 cells transiently transfected with human and mouse TLR2 or TLR4/mD-2 [11]. It was revealed that P. gingivalis LPS attenuated proinflammatory cytokine production by human cells stimulated with E. coli LPS [21]. More recently, Reife et al. reported that P. gingivalis LPS containing tetra-acylated lipid A structure exhibited antagonistic effects on E. coli LPS-induced E-selectin mRNA expression in human endothelial cells [12]. We demonstrated here that compounds PG-381-5FA and -3FA worked as an agonist for TLR4 but not TLR2, and also functioned as antagonists for potent TLR4 agonist compound 506 in agreement with previous reports (Figs 3–5). Therefore, it appears that P. gingivalis lipid A containing tetra-acylated lipid A is a TLR4 agonist and TLR2-induced cell activation by tetra-acylated lipid A seems to be caused by contaminated lipoprotein and lipopeptide, which are hard to remove. In the near future, P. gingivalis tetra-acylated lipid A will be synthesized and its immunobiological activities should be well defined.

Previous studies have indicated that human and mouse TLR4/mD-2 complex differently recognized LPS/lipid A [24,25]. In particular, Hajjar et al. showed that human but not mouse TLR4 can distinguish LPS that contains penta-acylated lipid A rather than hexa-acylated lipid A [25]. Similarly, we showed that human PBMC recognized the differences between compound PG-381-5FA and -3FA in contrast to mouse peritoneal macrophages (Figs 3 and 6). These results suggested that human TLR4 can differentiate lipid A possessing decreasing numbers of fatty acids effectively and penta- and tri-acylated lipid As were differently recognized by human and mouse TLR4/mD-2.

In conclusion, we found that chemically synthesized lipid As containing different numbers of acyl chains exhibited weak but definite endotoxicity, similar to that of E. coli-type synthetic lipid A. Furthermore, those lipid As demonstrated potent cell activation of cytokine-inducing activities in peritoneal macrophages from C3H/HeN mice, but not C3H/HeJ mice, and of NF-κB via TLR4/mD-2, but not TLR2. The present results suggest that these different forms of P. ginigvalis lipid A functionally interact with only TLR4.

Acknowledgments

This work was supported in part by a Miyata research financial incentive of Asahi University (A). We thank Ms Chieko Kanamori for her technical assistance, and Mr Mark Benton for his critical reading of the manuscript.

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