In this study, the effect of Lactobacillus plantarum lipoteichoic acid (pLTA) on LPS-induced MAPK activation, NF-κB activation, and the expression of TNF-α and IL-1R-associated kinase M (IRAK-M) was examined. The expression of the pattern recognition receptor and the survival rate of mice were also examined. pLTA pretreatment inhibited the phosphorylation of ERK, JNK, and p38 kinase. It also inhibited the degradation of IκBα and IκBβ, as well as the activation of the LPS-induced TNF-α factor in response to subsequent LPS stimulation. These changes were accompanied by the suppression of the LPS-induced expression of TLR4, NOD1, and NOD2, and the induction of IRAK-M, with a concurrent reduction of TNF-α secretion. Furthermore, the overexpression of pattern recognition receptors such as TLR4, NOD1, and NOD2 and the degradation of IRAK-M by transient transfection were found to reinstate the production of TNF-α after LPS restimulation. In addition, the i.p. injection of pLTA suppressed fatality, and decreased the level of TNF-α in the blood, in LPS-induced endotoxin shock mice. In conclusion, these data extend our understanding of the pLTA tolerance mechanism, which is related to the inhibition of LPS-induced endotoxin shock, and suggest that pLTA may have promise as a new therapeutic agent for LPS-induced septic shock.

The lipoteichoic acid (LTA)3 of Gram-positive bacteria is considered to be analogous to the LPS of Gram-negative bacteria because LTA shares many of its biochemical and physiological properties (1). Like LPS, LTA is an amphiphile that is formed by linking a hydrophilic polyphosphate polymer to a glycolipid (2). Several previous investigations have shown that LTA is a ligand against TLR2 and TLR4. Recent studies, however, have clearly shown that repurified LTA from Staphylococcus aureus induces TNF-α secretion through the TLR2-signaling pathway (3, 4). These findings support the conclusion that the LTA preparations used in earlier studies were contaminated by endotoxin. Another problem associated with earlier LTA preparations is a loss of biological activity in purified LTA. The purification methods included a phenol extraction step that contributed to the decomposition of LTA as a result of the loss of its alanine substituents (5). Recently, a novel purification technique using butanol extraction provided a highly purified biologically active LTA preparation.

The number of reported cases of sepsis and septic shock caused by Gram-negative and -positive bacteria, fungi, viruses, and parasites continues to grow every year (6). According to some reports, the proportion of sepsis due to Gram-negative bacteria varied between 30 and 80%, and that of sepsis due to Gram-positive bacteria varied between 6 and 24%. However, the contribution of Gram-positive bacteria to sepsis has increased in recent years. The mortality rates in patients with septic shock vary from 20 to 80% (7, 8). The production of cytokines, such as TNF-α and IL-1, initiated by bacterial components such as LPS, LTA, and peptidoglycan (PGN), can lead to the development of systemic inflammatory response syndrome and multiorgan system dysfunction. Therefore, many studies have attempted to elucidate the molecular pathways leading to an inflammatory response to abrogate them during clinical sepsis. Recent reports have described a general pathway involving the interaction between LPS and TLR: following release into the bloodstream, LPS from bacteria combines with the LPS-binding protein, and the resulting complex binds to CD14. CD14 recruitment, in turn, results in the release of inflammatory cytokines associated with sepsis (9, 10).

Recently, pathogen-induced tolerances using LPS, LTA, and PGN have been introduced to protect against the overproduction of proinflammatory cytokines associated with sepsis. However, the application of pathogen-induced tolerance to human disease entails some risks because the formulation of pathogenic fractions can induce septic shock. In this study, therefore, we isolated highly purified LTA from Lactobacillus plantarum (pLTA), known as a human health-promoting effector, and show that pLTA is more effective at inhibiting LPS-induced TNF-α production than pathogen-induced tolerance. We also identify a variation of signal transduction and demonstrate the regulation of pattern recognition receptors (PRRs), which occurred in our study through pLTA tolerance.

THP-1 and U-937, human monocyte-like cells, were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. THP-1 cells were seeded onto 96- or 24-well plates. After incubation for 24 h, the THP-1 cells were used for stimulation with pLTA and/or LPS (Escherichia coli 055:B5; Sigma-Aldrich). Bone marrow-derived macrophages (BMM) were isolated from wild-type and TLR2-deficient BALB/c mice, which were provided by S. Akira (Osaka University, Osaka, Japan). Briefly, femurs and tibiae were taken out, and surrounding muscle was removed. The marrow was flushed out with medium using a syringe with a 0.45-mm (26 G) diameter needle, and then cells were washed with PBS; RBC were disrupted using 1 ml of RBC lysis buffer for 10 min and washed with PBS two times. To differentiate BMM, they were plated in 24-well plates at 1 × 106 cell/ml in DMEM complete medium (1% penicillin/streptomycin, 10% FBS) adding 10% L-929-conditioned medium. After 3 days, medium was exchanged without L-929-conditioned medium in DMEM for 12 h and then stimulated with LPS and/or LTA.

Highly purified LTA was isolated from L. plantarum K8 (KCTC10887BP) by n-butanol extraction, as previously described (11). The purity of the purified LTA was determined by measuring the protein and endotoxin contents through the conventional silver staining after PAGE and through the Limulus amebocyte lysate assay (BioWhittaker), respectively. DNA or RNA contamination was assessed by measuring UV absorption at 260 and 280 nm (12). The modification of LTA from L. plantarum was performed as previously described with minor modifications (5, 13). The dealanylation of LTA was achieved by increasing the pH of the water phase after butanol extraction, and stirring at pH≈8.5 with a Tris buffer at room temperature (21°C) for 24 h. The deacylation of LTA was performed at 37°C for 24 h with 0.1 M NaOH. Both modified LTA fractions were dialyzed against water, and the d-alanine content of LTA was examined through the indirect ELISA method, using a rabbit polyclonal anti-d-alanine Ab (Abcam).

To quantify the mRNA expression, real-time PCR amplification was conducted using the ABI Prism 7000 Sequence Detection System (Applied Biosystems), and the PCR products were detected with SYBR Green. The following sequences of the forward and reverse primer pairs were used: 5′-GGACCTGACTGACTACCTCA-3′ and 5′-GCACAGCTTCTCCTTAATGT-3′ for β-actin; 5′-AATGGTGCCATTATGAACTC-3′ and 5′-GCTTGCTCTGTCAGCTTAAT-3′ for TLR1; 5′-ACCCTAGGGGAAACATCTCT-3′ and 5′-AGCTCTGTAGATCTGAAGCATC-3′ for TLR2; 5′-TGAAGAATTCCGATTAGCAT-3′ and 5′-AATAGTCACACTCACCAGGG-3′ for TLR4; 5′-AGACCTGTCTGACAATCCTG-3′ and 5′-GACAGATTGAGGGAGTTCAG-3′ for CD14; 5′-ATCCAGAATCAGATCACAGC-3′ and 5′-AACAGATAATCCGCTTCTCA-3′ for NOD1; 5′-CTAGAAGAACTCTGCCTGGA-3′ and 5′-AGGTGATGCAGTTATTGGAC-3′ for NOD2; and 5′-CACCTTCATCATGGAACTCT-3′ and 5′-CAACAAGCTGTGTCTCTGAA-3′ for LPS-induced TNF-α factor (LITAF). The expression of mRNA was normalized with β-actin.

After the cells were stimulated with LTA and/or LPS, cell supernatants were collected and assayed for cytokine production by standard sandwich ELISA. The TNF-α production was determined using the monoclonal anti-human (mouse IgG1, clone 28401) and biotinylated anti-mouse TNF-α-specific polyclonal Ab (goat IgG) for human TNF-α detection, and monoclonal anti-mouse (goat IgG) and biotinylated anti-human TNF-α-specific polyclonal Ab (goat IgG) for mouse TNF-α detection (R&D Systems), according to the manufacturer’s instructions.

TLR or NOD expression vectors (Invivogen) were transiently transfected into THP-1 cells using WelFec-EX (JBI), according to the manufacturer’s manual with small modifications. Briefly, THP-1 cells were plated into 24-well plates at 1 × 105 cells/well in RPMI 1640 (1% antibiotics and 10% FBS), grown overnight, and transfected for 6 h with the diverse concentrations of receptor expression and pUNO-empty vector by using 3 μg/well WelFec-Ex transfection reagent. After 24 h, transfectants were used for the examination of TNF-α production by LPS and/or pLTA treatment. U937 or HEK293 cells were cotransfected with the pNF-κB-Luc vector and the pRL-SV40 vector as described above. The transfected cell lines were prestimulated with pLTA for 18 h followed by restimulation with LPS. After incubation for an additional 18 h, cells were harvested, lysed, and the cell lysates were assayed for firefly and Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega) on an el 800 (Bio-Tek Instruments). The firefly luciferase activity of individual transfections was normalized against the Renilla luciferase activity.

Cellular extracts were prepared as described with minor modification (14). Twenty micrograms of total protein were added in a Laemmli buffer, boiled for 5 min, resolved by 12% SDS-PAGE in a Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and blotted onto nitrocellulose membranes (100 V, 1.5 h, 4°C). After blocking for 1 h in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBST and probed for 2 h with anti-phospho-MAPK Ab (R&D Systems), anti-IκBα or anti-IκBβ (Santa Cruz Biotechnology) in TBS-T/0.5% nonfat milk. After being washed three times in TBST, the membranes were incubated with secondary HRP-conjugated donkey anti-rabbit Ig or sheep anti-mouse Ig (GE Healthcare Biosciences) for 2 h and washed five times in TBST; bands were detected using ECL reagents (GE Healthcare Biosciences), according to the manufacturer’s description.

Male BALB/c mice (4 wk old, Samtako) were used. Murine endotoxic shock was induced through the i.p. injection of 200 μl of aqueous solution containing LPS (25–45 mg/kg). The survival rate of pLTA and LPS of endotoxin shock mice were estimated by the i.p. injection of pLTA (10–300 mg/kg) or 15 mg/kg LPS before the i.p. reinjection of 45 mg/kg LPS. The survival rate was monitored over the next 4 days. The TNF-α level from serum was assayed by ELISA, as described above. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and triacylglycerol in the mice were measured using Vital Scientific Selctra XL (Diamond Diagnostics). These studies have been approved by the Institutional Animal Care and Use Committee of Kyung Hee University.

All experiments were performed at least three times. The data shown are representative results of the means ± SD of triplicate experiments. Differences were judged to be statistically significant when the p value was <0.05.

The LPS-induced TNF-α production in pLTA-pretreated THP-1 cells was reduced in a dose-dependent manner from 1 to 200 μg/ml (Fig. 1,A). The inhibitory effects of pLTA were enhanced in proportion to pretreatment time (Fig. 1 B). The inhibition of LPS-induced TNF-α production, however, was not exhibited in the simultaneous treatment of pLTA and LPS or pLTA posttreatment following LPS. These results suggest that pLTA-induced tolerance is different from LPS- or S. aureus LTA (aLTA)-induced tolerance which results in bilateral hyporesponsiveness to subsequent stimulation with LPS or aLTA (15).

FIGURE 1.

Effect of pLTA pretreatment on LPS-induced TNF-α production. A, THP-1 cells were pretreated for 18 h with pLTA at the indicated concentrations, and restimulated with 0.5 μg/ml LPS for 4 h. B, Cells were pretreated with 100 μg/ml pLTA for the indicated period of time before restimulation using 0.5 μg/ml LPS for 4 h. C, THP-1 cells were pretreated with subfractions isolated from L. plantarum, such as 100 μg/ml of the CW, CX, PGN, LTA, TA, and genomic DNA (DNA) and 10 μl/ml of culture sup for 18 h. Each batch of cells was then restimulated with 0.5 μg/ml LPS for 4 h. D, THP-1 cells were pretreated with 100 μg/ml pLTA, aLTA isolated from S. aureus, lLTA isolated from Lactobacillus lactis, pPGN isolated from L. plantarum, fPGN isolated from Shigella flexneri, and aPGN isolated from S. aureus for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. In all the experiments, the TNF-α level in the culture supernatants was determined using ELISA. Data (mean ± SD) of three independent experiments are presented. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs LPS only (A), PBS (B–D). The cells treated with neither pLTA nor LPS are indicated as C.

FIGURE 1.

Effect of pLTA pretreatment on LPS-induced TNF-α production. A, THP-1 cells were pretreated for 18 h with pLTA at the indicated concentrations, and restimulated with 0.5 μg/ml LPS for 4 h. B, Cells were pretreated with 100 μg/ml pLTA for the indicated period of time before restimulation using 0.5 μg/ml LPS for 4 h. C, THP-1 cells were pretreated with subfractions isolated from L. plantarum, such as 100 μg/ml of the CW, CX, PGN, LTA, TA, and genomic DNA (DNA) and 10 μl/ml of culture sup for 18 h. Each batch of cells was then restimulated with 0.5 μg/ml LPS for 4 h. D, THP-1 cells were pretreated with 100 μg/ml pLTA, aLTA isolated from S. aureus, lLTA isolated from Lactobacillus lactis, pPGN isolated from L. plantarum, fPGN isolated from Shigella flexneri, and aPGN isolated from S. aureus for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. In all the experiments, the TNF-α level in the culture supernatants was determined using ELISA. Data (mean ± SD) of three independent experiments are presented. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs LPS only (A), PBS (B–D). The cells treated with neither pLTA nor LPS are indicated as C.

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Although pLTA significantly inhibited LPS-induced TNF-α production, other cell components, such as crude extract (CX), the cell wall (CW), PGN, teichoic acid (TA), genomic DNA, and the culture supernatant (sup), did not induce the inhibition of LPS-induced TNF-α production (Fig. 1,C). In the other experiments, pLTA-induced tolerance against LPS was more effective than other ligand-induced tolerance (Fig. 1 D). Although pLTA had a high inhibitory effect on LPS-induced TNF-α production, pLTA itself showed less immune activation than LPS in THP-1 cells (data not shown). Taken together, these results suggest that pLTA may be more effective, as well as safer for the prevention of proinflammatory diseases, without any concern about side effects such as synergic septic shock, which can occur by pathogenic ligands such as LPS, aLTA, and S. aureus PGN (aPGN) (16, 17, 18).

LPS induces septic shock through PRRs, especially TLR4 (10). Therefore, we examined the role of pLTA pretreatment on the expression of PRRs. The mRNAs of TLR4, NOD1, and NOD2 were down-regulated on the THP-1 cells which were pretreated with 100 μg/ml pLTA for 18 h followed by restimulation with 0.5 μg/ml LPS for 2 h (Table I). TLR4, NOD1, and NOD2 mRNAs were reduced by 63.5, 60.6, and 51.3%, respectively, in the pLTA-pretreated group as compared with LPS alone. In contrast, the mRNA of other TLRs, including CD14, increased in the pLTA-pretreated group. When compared with only LPS, the mRNA production of TLR2 and CD14 was increased to 4.3- and 10.5-fold, respectively, in the pLTA pretreatment. The confocal microscope data showed that the expression of TLR4, NOD1, and NOD2 was reduced, while the expression of TLR2 and CD14 was up-regulated in the THP-1 cells pretreated with pLTA followed by LPS restimulation (data not shown). Given that TLR4, NOD1, and NOD2 contribute to septic shock, the down-regulation of those receptors by pLTA pretreatment may be related to the inhibition of LPS-induced septic shock. In the next experiments, therefore, we examined the role of PRRs on TNF-α reduction.

Table I.

The regulation of PRR in pLTA- or LPS-tolerized THP-1 cellsa

Pretreatment/Challenge (μg/ml)bTLR1cTLR2cTLR4cNOD1cNOD2cCD14c
pLTA 0/LPS 0 508 ± 108 676 ± 38 905 ± 456 900 ± 6 275 ± 45 51 ± 2 
pLTA 100/LPS 0.5 1971 ± 431* 4757 ± 757* 989 ± 324** 1559 ± 356 2370 ± 684** 1643 ± 503** 
LPS 0.1/LPS 0.5 2028 ± 520 3084 ± 359* 1175 ± 439* 1436 ± 888* 1491 ± 709* 2127 ± 545 
pLTA 0/LPS 0.5 790 ± 306 1089 ± 342 1558 ± 181 3957 ± 1152 4870 ± 1113 156 ± 89 
Pretreatment/Challenge (μg/ml)bTLR1cTLR2cTLR4cNOD1cNOD2cCD14c
pLTA 0/LPS 0 508 ± 108 676 ± 38 905 ± 456 900 ± 6 275 ± 45 51 ± 2 
pLTA 100/LPS 0.5 1971 ± 431* 4757 ± 757* 989 ± 324** 1559 ± 356 2370 ± 684** 1643 ± 503** 
LPS 0.1/LPS 0.5 2028 ± 520 3084 ± 359* 1175 ± 439* 1436 ± 888* 1491 ± 709* 2127 ± 545 
pLTA 0/LPS 0.5 790 ± 306 1089 ± 342 1558 ± 181 3957 ± 1152 4870 ± 1113 156 ± 89 
a

Data from pLTA 100/LPS 0.5 and LPS 0.1/ LPS 0.5 were significantly lower (∗, p < 0.05; ∗∗, p < 0.01) than pLTA 0/LPS 0.5.

b

THP-1 cells were pretreated with 100 μg/ml pLTA or 0.1 μg/ml LPS for 18 h prior to challenge with 0.5 μg/ml LPS. Culture cells were harvested 4 h after challenge, and cDNAs were synthesized for real-time PCR. The amount of mRNA was normalized with β-actin.

c

Data are represented as the mean ± SEM from three separate experiments.

To determine the involvement of TLR2 in signaling on stimulation with pLTA, we compared TNF-α production by BMM from BALB/c mice lacking functional TLR2 and wild-type BALB/c mice. BMM from wild-type mice exhibited TNF-α release on pLTA stimulation with 10 μg/ml, whereas no TNF-α was released from cells of TLR2-deficient mice (Fig. 2,A). In contrast to stimulation with pLTA, BMM from TLR2-deficient mice were equally responsive as compared with wild-type mice in terms of LPS-induced TNF-α production (Fig. 2,B). These results suggest that pLTA requires TLR2-dependent signal transduction to activate macrophages as aLTA does (19). Prior exposure to pLTA rendered cells hyporesponsive to subsequent stimulation with LPS. We tested whether prior exposure of cells to pLTA induced refractoriness to subsequent stimulation with LPS in TLR2-deficient mice. Release of TNF-α in response to pLTA was decreased in a dose-dependent fashion when wild-type BMM were pretreated with different concentrations of pLTA for 18 h before restimulation with 0.5 μg/ml LPS. In contrast to wild-type cells, LPS-induced TNF-α production from TLR2-deficient BMM was not affected by pretreatment with pLTA (Fig. 2 C), confirming the requirement of TLR2-mediated signaling in tolerance induction by pLTA.

FIGURE 2.

The role of TLR2 in pLTA-mediated tolerance. BMM from wild type and TLR2-deficient mice (n = 2) were stimulated with pLTA or LPS at the indicated concentrations (A and B), or pre-exposed to increasing concentrations of pLTA for 18 h followed by restimulation with 0.5 μg/ml LPS for 4 h (C). In all the experiments, murine TNF-α production was examined by ELISA from culture supernatants, and data are presented as means ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with pLTA-untreated cells.

FIGURE 2.

The role of TLR2 in pLTA-mediated tolerance. BMM from wild type and TLR2-deficient mice (n = 2) were stimulated with pLTA or LPS at the indicated concentrations (A and B), or pre-exposed to increasing concentrations of pLTA for 18 h followed by restimulation with 0.5 μg/ml LPS for 4 h (C). In all the experiments, murine TNF-α production was examined by ELISA from culture supernatants, and data are presented as means ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with pLTA-untreated cells.

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We tried to verify whether overexpression of PRRs would alter pLTA-mediated tolerance. The expression of pUNO-hTLR4 (InvivoGen) was confirmed by RT-PCR in THP-1 cells, and by confocal microscope in HEK293 cells (data not shown). TNF-α production was inhibited in the control cells transiently transfected with an empty vector after stimulation with 0.5 μg/ml LPS following 100 μg/ml pLTA pretreatment, whereas TNF-α was reinstated in the cells transiently transfected with pUNO-hTLR4 in a dose-dependent manner (Fig. 3,A). In the next experiments, we examined the role of NOD1 and NOD2 in pLTA-mediated tolerance. The expression of NOD1 and NOD2 was confirmed after transient transfection of pUNO-hNOD1 and pUNO-hNOD2 into THP-1 cells for RT-PCR and HEK293 cells for confocal microscope (data not shown). Although TNF-α production was inhibited in the THP-1 cells transfected with the empty vector after 0.5 μg/ml LPS stimulation following 100 μg/ml pLTA pretreatment, LPS-induced TNF-α production was reinstated in NOD1 and NOD2 transfectants in a dose-dependent manner (Fig. 3,B). Similarly, NF-κB activation was inhibited in U937 cells cotransfected with the empty vector and the pNF-κB-luc vector after stimulation with 0.5 μg/ml LPS following 100 μg/ml pLTA pretreatment, whereas it was increased in U937 cells cotransfected with PRR and the pNF-κB-luc vector in a PRR dose-dependent manner (Fig. 3 C). These results may suggest that TLR4, NOD1, and NOD2 participated in LPS-induced TNF-α production, and that the down-regulation of those receptors by pLTA pretreatment resulted in the inhibition of TNF-α production.

FIGURE 3.

The role of PRRs in pLTA-mediated tolerance. A and B, Cells were transfected with graded concentrations of pUNO-hTLR4, pUNO-hNOD1, or pUNO-hNOD2 expression vectors or the control empty vector, and incubated for 24 h. Cells pretreated with 100 μg/ml pLTA for 18 h were restimulated with 0.5 μg/ml LPS for 4 h. In all experiments, TNF-α production was examined by ELISA from culture supernatants, and data are presented as means ± SD. ∗, p < 0.05 compared with the vector only (prestimulated with 100 μg/ml pLTA) and TLR4 transfectants stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA (A). C, U937 cells were transiently cotransfected with PRR expression vectors, the pNF-κB-luc vector and the pRL-SV40 vector at the indicated concentrations. After restimulation with 0.5 μg/ml LPS for 18 h following 100 μg/ml pLTA pretreatment for 18 h, cells were lysed, and the luciferase activity was measured. Data are expressed as the mean ± SD of three independent experiments in duplicate and normalized for Renilla luciferase activity. The statistical significances (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) were compared with the vector alone (prestimulated with 100 μg/ml pLTA) and the PRR transfectants; they were also compared with the vector alone stimulated with 0.5 μg/ml LPS and the vector alone stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA.

FIGURE 3.

The role of PRRs in pLTA-mediated tolerance. A and B, Cells were transfected with graded concentrations of pUNO-hTLR4, pUNO-hNOD1, or pUNO-hNOD2 expression vectors or the control empty vector, and incubated for 24 h. Cells pretreated with 100 μg/ml pLTA for 18 h were restimulated with 0.5 μg/ml LPS for 4 h. In all experiments, TNF-α production was examined by ELISA from culture supernatants, and data are presented as means ± SD. ∗, p < 0.05 compared with the vector only (prestimulated with 100 μg/ml pLTA) and TLR4 transfectants stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA (A). C, U937 cells were transiently cotransfected with PRR expression vectors, the pNF-κB-luc vector and the pRL-SV40 vector at the indicated concentrations. After restimulation with 0.5 μg/ml LPS for 18 h following 100 μg/ml pLTA pretreatment for 18 h, cells were lysed, and the luciferase activity was measured. Data are expressed as the mean ± SD of three independent experiments in duplicate and normalized for Renilla luciferase activity. The statistical significances (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) were compared with the vector alone (prestimulated with 100 μg/ml pLTA) and the PRR transfectants; they were also compared with the vector alone stimulated with 0.5 μg/ml LPS and the vector alone stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA.

Close modal

To determine the variation of TLR-negative regulators, we examined the mRNA levels of ST-2, IRAK-M, suppressor of cytokine signaling 1, MyD88s, and single immunoglobulin domain IL-1R-related from pLTA and LPS tolerance cells. THP-1 cells did not express ST-2, a negative regulator of TLR4: pLTA and LPS tolerant cells had a slight reduction in suppressor of cytokine signaling 1, MyD88s, and single immunoglobulin domain IL-1R-related production compared with LPS treatment alone (data not shown). Therefore, these molecules were not considered to be involved in pLTA tolerance. IRAK-M, which inhibits the release of the IRAK-TNFR-associated factor 6 complex from MyD88, was induced by pLTA pretreatment as compared with LPS treatment alone (Fig. 4,A). The expression of IRAK-M might be affected by pLTA because its expression was dependent on the concentration of pLTA (Fig. 4 B).

FIGURE 4.

Specific down-regulation of IRAK-M reinstated the production of TNF-α in pLTA tolerant cells. A, THP-1 cells stimulated with 100 μg/ml pLTA or the medium for 18 h, and then the cells were restimulated with 0.5 μg/ml LPS for the indicated amounts of time (h) or (B) THP-1 cells were stimulated with the indicated concentration of pLTA for 18 h, and the total cell lysates were blotted with a specific Ab for IRAK-M. C, THP-1 cells were transfected with 0.1–8 μg of siRNAs for IRAK-M or control RNAs, and incubated for 24 h. The cell lysates were then blotted with a specific Ab for IRAK-M. D, THP-1 cells were transfected with graded concentrations of siRNAs for IRAK-M or control RNAs, and incubated for 24 h. Cells pretreated with 100 μg/ml pLTA for 18 h were restimulated with 0.5 μg/ml LPS for 4 h. The amount of TNF-α was measured by ELISA. The data represent the mean ± SD, and the data shown are representative of three independent experiments. The statistical significance (∗, p < 0.05) was compared with the control siRNA (prestimulated with 100 μg/ml pLTA) and IRAK-M siRNA transfectants; these were compared with the control siRNA stimulated with 0.5 μg/ml LPS alone, and control siRNA stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA.

FIGURE 4.

Specific down-regulation of IRAK-M reinstated the production of TNF-α in pLTA tolerant cells. A, THP-1 cells stimulated with 100 μg/ml pLTA or the medium for 18 h, and then the cells were restimulated with 0.5 μg/ml LPS for the indicated amounts of time (h) or (B) THP-1 cells were stimulated with the indicated concentration of pLTA for 18 h, and the total cell lysates were blotted with a specific Ab for IRAK-M. C, THP-1 cells were transfected with 0.1–8 μg of siRNAs for IRAK-M or control RNAs, and incubated for 24 h. The cell lysates were then blotted with a specific Ab for IRAK-M. D, THP-1 cells were transfected with graded concentrations of siRNAs for IRAK-M or control RNAs, and incubated for 24 h. Cells pretreated with 100 μg/ml pLTA for 18 h were restimulated with 0.5 μg/ml LPS for 4 h. The amount of TNF-α was measured by ELISA. The data represent the mean ± SD, and the data shown are representative of three independent experiments. The statistical significance (∗, p < 0.05) was compared with the control siRNA (prestimulated with 100 μg/ml pLTA) and IRAK-M siRNA transfectants; these were compared with the control siRNA stimulated with 0.5 μg/ml LPS alone, and control siRNA stimulated with 0.5 μg/ml LPS following 100 μg/ml pLTA.

Close modal

To examine whether the down-regulation of IRAK-M expression would alter pLTA-mediated tolerance, the IRAK-M expression was degraded in THP-1 cells transfected with a specific small interference RNA (siRNA) for IRAK-M (Fig. 4,C). Although TNF-α production was inhibited in control siRNA-transfected cells, which were pretreated with 100 μg/ml pLTA followed by 0.5 μg/ml LPS restimulation, it was reinstated in THP-1 cells transfected with IRAK-M siRNAs in a dose-dependent manner (Fig. 4 D). This result indicates that IRAK-M, which was up-regulated by pLTA pretreatment, played an important role as a negative regulator for TLR4 signaling and inhibited LPS-induced TNF-α production.

LPS-mediated phosphorylation of p38, ERK1/2, and JNK1/2 in medium-pretreated THP-1 cells was evident after 15 min, and optimal responses were reached by 30 min of LPS stimulation. MAPK phosphorylation, however, was significantly inhibited in pLTA-pretreated THP-1 cells following restimulation with 0.5 μg/ml LPS (Fig. 5,A). Although LPS caused a degradation of IκBα and IκBβ after 60 min in the untolerized cells, pLTA-pretreated cells exhibited a markedly suppressed degradation of both IκBα and IκBβ in response to LPS restimulation (Fig. 5,A). As shown in Fig. 5,B, pLTA pretreatment inhibited the capacity of U937 cells to induce NF-κB-dependent transcription of the luciferase reporter gene in response to subsequent stimulation with LPS. THP-1 cells stimulated with LPS induced a high level of LITAF production as compared with untreated or pLTA-treated cells, whereas pLTA pretreatment reduced LPS-induced LITAF production (Fig. 5,C). As expected, the protein level of LITAF was also induced by LPS stimulation. pLTA pretreatment, however, inhibited LPS-induced LITAF protein production (Fig. 5 D). Taken together, these results suggest that the activation of MAPKs, NF-κB and especially LITAF, were involved in LPS-induced TNF-α production, and that pLTA tolerance inhibited TNF-α production via the down-regulation of signal transduction associated with the MAPKs, NF-κB and LITAF.

FIGURE 5.

pLTA tolerance down-regulated the activated signaling pathways. A, THP-1 cells were pretreated with either the medium or 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for the indicated amounts of time (min). The cell lysates were blotted with phosphospecific Abs for ERK1/2, p38, JNK1/2, IκB-α, and IκBβ. To verify the amount of loaded protein, they were also probed with anti-ERK1/2. B, U937 cells were transiently cotransfected with the pNF-κB-luc and pRL-SV40 control vectors. They were preincubated with the medium or 100 μg/ml pLTA for 18 h, and then restimulated with LPS at the indicated concentrations for 20 h. Cells were lysed and firefly and Renilla luciferase activities were measured. Data are expressed as the mean ± SD of three experiments performed in triplicate and normalized for Renilla luciferase activity. C, THP-1 cells were pretreated with 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. The total RNA was extracted, and cDNA synthesis was performed. The level of LITAF was examined by quantitative real-time PCR methods using specific primers for human LITAF. Data are expressed as the mean ± SD of three independent experiments. ∗, p < 0.05 vs untreated cells, indicated as C. D, THP-1 cells were pretreated with 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. The cell lysates were blotted with a specific Ab for human LITAF (Upstate Biotechnology).

FIGURE 5.

pLTA tolerance down-regulated the activated signaling pathways. A, THP-1 cells were pretreated with either the medium or 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for the indicated amounts of time (min). The cell lysates were blotted with phosphospecific Abs for ERK1/2, p38, JNK1/2, IκB-α, and IκBβ. To verify the amount of loaded protein, they were also probed with anti-ERK1/2. B, U937 cells were transiently cotransfected with the pNF-κB-luc and pRL-SV40 control vectors. They were preincubated with the medium or 100 μg/ml pLTA for 18 h, and then restimulated with LPS at the indicated concentrations for 20 h. Cells were lysed and firefly and Renilla luciferase activities were measured. Data are expressed as the mean ± SD of three experiments performed in triplicate and normalized for Renilla luciferase activity. C, THP-1 cells were pretreated with 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. The total RNA was extracted, and cDNA synthesis was performed. The level of LITAF was examined by quantitative real-time PCR methods using specific primers for human LITAF. Data are expressed as the mean ± SD of three independent experiments. ∗, p < 0.05 vs untreated cells, indicated as C. D, THP-1 cells were pretreated with 100 μg/ml pLTA for 18 h, and then restimulated with 0.5 μg/ml LPS for 4 h. The cell lysates were blotted with a specific Ab for human LITAF (Upstate Biotechnology).

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To examine the inhibitory effects of pLTA on endotoxin shock induced by LPS in BALB/c mice, we examined the survival rate of endotoxin shock mice. In the absence of pLTA or LPS, ∼85% of the mice died within 2 days of an i.p. injection of the aqueous solution containing 40–45 mg/kg LPS (Fig. 6,A). When 15 mg/kg LPS was injected i.p. 24 h before the i.p. injection of 45 mg/kg LPS, the rate of survival was improved by 58%. Interestingly, the i.p. preinjection of 300 mg/kg pLTA, followed 24 h later by i.p. injection of 45 mg/kg LPS, significantly increased the survival rate of endotoxin shock mice (Fig. 6,B). When mice were preinjected i.p. with various concentrations of pLTA 24 h before i.p. injection of 45 mg/kg LPS, the survival rate increased in a dose-dependent manner; mice injected with over 100 mg/kg pLTA did not show mortality (Fig. 6,C). In this study, mice preinjected i.p. with over 100 mg/kg pLTA recovered their health within 2 days, whereas mice preinjected with below 50 mg/kg pLTA recovered painfully after 4 days. Mice preinjected with 300 mg/kg pLTA recovered their health most rapidly among the experimental groups. LPS-tolerized mice, however, showed severe diarrhea and hardly recovered their health after LPS restimulation, as compared with the pLTA-tolerized mice. In addition, the plasma TNF-α levels 24 h after i.p. injection with 45 mg/kg LPS, following a preinjection of 300 mg/kg pLTA or 15 mg/kg LPS 24 h earlier, significantly decreased compared with the preinjection of PBS (Fig. 6,D). In the PBS-preinjected mice, the serum levels of ALT and triacylglycerol were markedly augmented, but pLTA strikingly inhibited the increase in these levels. However, LPS preinjection did not alter the triacylglycerol level, while reducing the serum ALT level slightly, as compared with PBS preinjection (Fig. 6, E and F). Serum AST and serum total cholesterol levels in the LPS and pLTA preinjection groups did not show significant difference from the PBS preinjection group, although these levels in the LPS and pLTA preinjection groups were higher than those in the control group (data not shown). These results suggest that pLTA and LPS significantly suppress lethality in endotoxin shock mice induced by LPS, probably resulting from the reduction of hepatic inflammation.

FIGURE 6.

pLTA improved the survival rate of endotoxin shock mice induced by LPS. A, BALB/c mice (6 wk old, 8 mice) were injected i.p. with the indicated concentrations of LPS. B, Mice (16 mice) were injected i.p. with 15 mg/kg LPS or 300 mg/kg pLTA. After 24 h, they were reinjected i.p. with 45 mg/kg LPS. C, Mice (eight mice) were preinjected i.p. with the indicated concentrations of pLTA or PBS and, after 24 h, reinjected i.p. with 45 mg/kg LPS. ∗, p < 0.05 compared with PBS preinjection. D, The effect of pLTA on plasma TNF-α level in endotoxin shock mice. The plasma was collected 24 h after i.p. injection of 45 mg/kg LPS following pre-i.p. injection of 15 mg/kg LPS or 300 mg/kg pLTA for 24 h. The TNF-α level from plasma was determined by ELISA. Each value represents the mean ± SD of two independent experiments in duplicate. ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with PBS preinjection. E and F, The effect of pLTA on serum ALT (E) and triacylglycerol (F) levels in endotoxin shock mice 24 h after i.p. injection of 45 mg/kg LPS. Each value represents the mean ± SD of three experiments. ∗, p < 0.05, compared with PBS.

FIGURE 6.

pLTA improved the survival rate of endotoxin shock mice induced by LPS. A, BALB/c mice (6 wk old, 8 mice) were injected i.p. with the indicated concentrations of LPS. B, Mice (16 mice) were injected i.p. with 15 mg/kg LPS or 300 mg/kg pLTA. After 24 h, they were reinjected i.p. with 45 mg/kg LPS. C, Mice (eight mice) were preinjected i.p. with the indicated concentrations of pLTA or PBS and, after 24 h, reinjected i.p. with 45 mg/kg LPS. ∗, p < 0.05 compared with PBS preinjection. D, The effect of pLTA on plasma TNF-α level in endotoxin shock mice. The plasma was collected 24 h after i.p. injection of 45 mg/kg LPS following pre-i.p. injection of 15 mg/kg LPS or 300 mg/kg pLTA for 24 h. The TNF-α level from plasma was determined by ELISA. Each value represents the mean ± SD of two independent experiments in duplicate. ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with PBS preinjection. E and F, The effect of pLTA on serum ALT (E) and triacylglycerol (F) levels in endotoxin shock mice 24 h after i.p. injection of 45 mg/kg LPS. Each value represents the mean ± SD of three experiments. ∗, p < 0.05, compared with PBS.

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In the previous report, the quality and level of d-alanine substitution in LTA were shown to be key factors for cytokine induction (5, 20). Therefore, we examined whether pLTA-induced tolerance is dependent on the d-alanine contents of LTA. Although intact pLTA and dealanylated pLTA lacking d-alanine induced TNF-α production, deacylated pLTA, lacking a fatty acid chain, did not induce TNF-α production (Fig. 7,A). Similarly, the inhibition of LPS-induced TNF-α production occurred by both intact pLTA and dealanylated pLTA, but not by deacylated pLTA (Fig. 7,B). The d-alanine contents of both modified pLTA were confirmed through indirect ELISA methods using the anti-d-alanine Ab, and Fig. 7,C shows the reduction of d-alanine contents from both modified pLTAs as compared with the intact pLTA. In addition, the phosphorylation of MAPKs such as ERK1/2, JNK1/2, and p38 was inhibited in both intact pLTA- and dealanylated pLTA-pretreated THP-1 cells following 30 min of restimulation with 0.5 μg/ml LPS. In contrast, PBS- and deacylated pLTA pretreatment did not inhibit LPS-induced phosphorylation of ERK1/2, JNK1/2, or p38 (Fig. 7 D). These results suggest that the fatty acid chain of pLTA plays an important role in the inhibition of LPS-induced TNF-α production, as well as in TNF-α production. The d-alanine content of pLTA, however, may not affect the TNF-α production or the inhibition of LPS-induced TNF-α production.

FIGURE 7.

Effect of modified pLTA on LPS-induced TNF-α production. A, THP-1 cells were treated with intact pLTA, dealanylated pLTA, or deacylated pLTA at the indicated concentrations for 4 h. The cells treated with neither intact pLTA nor modified pLTA are indicated as C. B, THP-1 cells were pretreated with the indicated concentration of intact pLTA, dealanylated pLTA, or deacylated pLTA for 18 h before restimulation with 0.5 μg/ml LPS for 4 h. The TNF-α level in the culture supernatants was determined by ELISA. C, The d-alanine contents of intact pLTA and modified pLTA were determined by indirect ELISA methods with the anti-d-alanine Ab. In all of the experiments, the data are represented as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs C, PBS or intact. A typical result is represented from three independent experiments in triplicate. D, THP-1 cells were pretreated with 100 μg/ml intact pLTA, dealanylated pLTA, deacylated pLTA, or PBS for 18 h before restimulation with 0.5 μg/ml LPS, and harvested after 30 min to detect MAPKs. The cell lysates were blotted with phosphor-specific Abs for ERK, JNK, and p38. To verify the amount of loaded protein, they were also probed with anti-ERK1/2. In the figure, dealanylated pLTA is represented as pH 8.5; deacylated pLTA is represented as 0.1 M NaOH; and unmodified pLTA is represented as intact.

FIGURE 7.

Effect of modified pLTA on LPS-induced TNF-α production. A, THP-1 cells were treated with intact pLTA, dealanylated pLTA, or deacylated pLTA at the indicated concentrations for 4 h. The cells treated with neither intact pLTA nor modified pLTA are indicated as C. B, THP-1 cells were pretreated with the indicated concentration of intact pLTA, dealanylated pLTA, or deacylated pLTA for 18 h before restimulation with 0.5 μg/ml LPS for 4 h. The TNF-α level in the culture supernatants was determined by ELISA. C, The d-alanine contents of intact pLTA and modified pLTA were determined by indirect ELISA methods with the anti-d-alanine Ab. In all of the experiments, the data are represented as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs C, PBS or intact. A typical result is represented from three independent experiments in triplicate. D, THP-1 cells were pretreated with 100 μg/ml intact pLTA, dealanylated pLTA, deacylated pLTA, or PBS for 18 h before restimulation with 0.5 μg/ml LPS, and harvested after 30 min to detect MAPKs. The cell lysates were blotted with phosphor-specific Abs for ERK, JNK, and p38. To verify the amount of loaded protein, they were also probed with anti-ERK1/2. In the figure, dealanylated pLTA is represented as pH 8.5; deacylated pLTA is represented as 0.1 M NaOH; and unmodified pLTA is represented as intact.

Close modal

Inflammation is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis after injury. Excessive inflammation can, however, cause inflammatory diseases such as septic shock, Legionnaire’s disease, systemic lupus erythematosus, Crohn’s disease, atherosclerosis, and so on (21). To treat these inflammatory diseases, researchers have tried to induce tolerance against endotoxin, which induces excessive inflammation, and several reports have shown that bacterial cell wall components induced homologous tolerance (15, 16, 22, 23). However, most bacterial cell walls used in tolerance-induction tests were isolated from pathogenic bacteria, making them harmful for use in clinical applications. The reason for this is that it is difficult to decide the proper concentration to induce the tolerance, and the composition of these bacterial cell walls can cause synergistical inflammation or septic shock (17, 18). In our present experiments, the treatment of LPS or aLTA in THP-1 cells induced excessive proinflammatory cytokine production, and the mouse survival rate was reduced by 50% in the LPS-induced tolerance as compared with endotoxin shock mice, indicating the risk of pathogenic bacterial cell walls as a clinical material. Therefore, we selected an LTA isolated from L. plantarum as a material that would induce tolerance without the trouble of side effects. Unlike LPS or aLTA, pLTA did not induce the high level of proinflammatory cytokines and showed low cytotoxicity (data not shown). In contrast, pLTA efficiently inhibited LPS-induced TNF-α production, and results in a high survival rate among mice with endotoxin shock.

A previous study has shown that the impaired expression and functions of common signaling, which intermediates LPS and IL-1 signaling, induce LPS-mediated tolerance (14). In particular, the regulation of TLR4 expression, which plays an important role in the induction of septic shock, may depend on the material that induces tolerance. The expression of the TLR4-MD2 complex was reduced in LPS-tolerized mouse peritoneal macrophages (24), whereas macrophage-activating lipoprotein 2-induced tolerance did not reduce the expression of the TLR4-MD2 complex (25). Similar to LPS-induced tolerance, pLTA-induced tolerance reduced the expression of the TLR4-MD2 complex. In addition, the expression of NOD1 and NOD2, which induces the activation of NF-κB through LPS stimulation, and resulted in the up-regulation of inflammatory cytokines (26, 27), was also reduced through pLTA tolerance. The excessive expression of NOD1 and NOD2, as well as TLR4, causes inflammatory diseases. Therefore, the reduction of TLR4, NOD1, and NOD2 may inhibit excessive inflammation through the limited interaction between those receptors and LPS. Interestingly, the expression of CD14, which plays an important role in the recognition of LPS via TLR4, increased in the pLTA-tolerized cells. This phenomenon may be explained in that CD14 also participates in the interaction between TLR2 and LTA (28). A previous study has shown that LTA competes with LPS for binding to the CD14 of human monocytes, and LPS-CD14 binding is inhibited completely by Streptococcus sanguis LTA at a 100-fold higher concentration than LPS (23). These results suggest that the increased CD14 in pLTA tolerance may interact with pLTA, and result in the decreased interaction between CD14 and LPS.

The activation of MAPKs such as ERK1/2, JNK1/2, and p38 is important in mediating many macrophage functions, including the activation of various transcription factors and the production of pro- and anti-inflammatory cytokines (29, 30). Recent studies have reported that endotoxin- or bacterial lipoprotein-induced tolerance down-regulated the phosphorylation of ERK, JNK, and p38 in mouse macrophages and human THP-1 cells, respectively (14, 31). In addition, the LPS-induced activation of the MAPK pathways plays an important role in mediating NF-κB activation. NF-κB activation is induced by the dissociation of IκBα and IκBβ, which are phosphorylated by IκB kinases. Therefore, the amount of degradation of IκBα and IκBβ is commonly used as an indicator to confirm the activation of NF-κB. In the present study, pretreatment of pLTA on THP-1 cells inhibited the phosphorylation of MAPKs and the degradation of IκBα and IκBβ. These results suggest that pLTA tolerance and LPS tolerance may share similar intracellular signal transduction pathways. As a result of the inhibition of intracellular signaling pathways, the expression of LITAF was inhibited in the pLTA-tolerized cells. LITAF is known as the LPS-induced TNF-α factor, which mediates TNF-α transcription (32, 33). It has been reported that the inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-α transcripts. These results suggest that LITAF, as the final factor in the signaling cascade, plays an important role in LPS-induced TNF-α production, and the inhibition of LITAF expression through pLTA tolerance mediates the inhibition of LPS-induced TNF-α production.

Unlike other bacterial cell walls, such as aLTA, LPS, and lipoproteins, pLTA was prepared from lactic acid bacteria, which contributes to the health of the host. pLTA had low cytotoxicity and did not induce excessive inflammation. In particular, pLTA significantly reduced the excessive TNF-α production caused by endotoxin and increased the survival rate of endotoxin shock mice. These inhibitory effects were induced by the complex mechanism of 1) the inhibition of intracellular signaling pathways such as MAPKs and NF-κB; 2) the reduction of sepsis-related PRRs such as TLR4, NOD1, and NOD2; and 3) the induction of IRAK-M. The alteration of these mechanisms reduced LITAF expression and resulted in the inhibition of excessive LPS-induced TNF-α production; thus, there was a high survival rate among endotoxin shock mice. Therefore, pLTA tolerance may be more effective in the prevention and treatment of endotoxin shock, because pLTA is safer and more inducible in the induction of tolerance compared with other bacterial cell wall components.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG05-0307-2-0), Republic of Korea.

3

Abbreviations used in this paper: LTA, lipoteichoic acid; PGN, peptidoglycan; pLTA, L. plantarum lipoteichoic acid; PRR, pattern recognition receptor; BMM, bone marrow-derived macrophage; LITAF, LPS-induced TNF-α factor; AST, aspartate aminotransferase; ALT, alanine aminotransferase; aLTA, S. aureus LTA; CX, crude extract; CW, cell wall; TA, teichoic acid; sup, supernatant; aPGN, S. aureus PGN; IRAK-M, IL-1R-associated kinase M; siRNA, small interference RNA.

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