Probiotic genomic DNA reduces the production of pro-inflammatory cytokine tumor necrosis factor-alpha

Authors


Correspondence: Dae Kyun Chung, School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 449-701, South Korea. Tel.: +82 31 2012465; fax: +82 31 2028333; e-mail: dkchung@khu.ac.kr

Abstract

The effect of Lactobacillus plantarum genomic DNA on lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, nuclear factor-kappa B activation, and the expressions of tumor necrosis factor-alpha, interleukin-1 receptor-associated kinase M, and the pattern recognition receptor were examined. Pretreatment of p-gDNA inhibited the phosphorylation of MAPKs and nuclear factor-kappa B, and also inhibited LPS-induced TNF-α production in response to subsequent LPS stimulation. L. plantarum genomic DNA-mediated inhibition of signaling pathway and tumor necrosis factor-alpha was accompanied by the suppression of toll-like receptor (TLR) 2, TLR4, and TLR9 and the induction of interleukin-1 receptor-associated kinase M, a negative regulator of TLR. This study can extend our understanding of the biological function of probiotic genomic DNA as an anti-inflammatory agent.

Introduction

Probiotics are microbial organisms that are beneficial to host health (Bengmark, 2000; Isolauri, 2001). Lactobacillus plantarum produces lipoteichoic acid (LTA), which reportedly reduces lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-α) production (Kim et al., 2008). Other bacterial components and products, including bacterial DNA, can also stimulate innate cellular immunity. Recent studies have identified toll-like receptor (TLR) 9 as the mammalian receptor for bacterial DNA (Hemmi et al., 2000). The functional consequences and signal transduction mechanisms that occur in response to bacterial DNA ligation of TLR9 on cells of the innate immune system are beginning to be elucidated (Takeshita et al., 2001). Although the benefit of Lactobacillus to the human body is well known, the effect of Lactobacillus DNA has not been established.

The number of reported cases of sepsis and septic shock caused by Gram-negative and Gram-positive bacteria, viruses, fungi, and parasites is increasing every year (Glauser et al., 1991). According to some reports, sepsis is due to Gram-negative bacteria in 30–80% of cases and Gram-positive bacteria in 6–24% of cases. Death rates in patients with septic shock vary from 20% to 80% (Geerdes et al., 1992; Bates et al., 1995). TNF-α production initiated by bacterial components such as LPS, lipoteichoic acid (LTA), and peptidoglycan (PGN) can lead to the development of systemic inflammatory response syndrome. If the molecular pathways leading to an inflammatory response can be determined, treatment targets can be identified to reduce harmful immune function during clinical sepsis. Recent reports have explained a general pathway involving the interaction between LPS and TLR (Ulevitch & Tobias, 1995; Lakhani & Bogue, 2003). DNA binding to the endosomally localized TLR9 leads to activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which stimulate not only potent pro-inflammatory activities but also the interferon regulatory factor pathway that induces anti-inflammatory activities (Kumagai et al., 2008). The extent of the immune response to different bacterial DNA also varies significantly among species, and recognition of bacterial DNA may further differ depending on cell type (Dalpke et al., 2006).

In this study, we identified the role of probiotic genomic DNA in the reduction of endotoxin-mediated excessive inflammation, and examined the variation of signaling pathway and receptor expression involved in this tolerance.

Materials and methods

Cell lines

THP-1, human monocyte-like cells, were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U mL−1 penicillin, and 100 g mL−1 streptomycin. THP-1 cells were seeded onto 96- or 12-well plates. After incubation for 6 h, the THP-1 cells were stimulated with gDNA and/or LPS (Escherichia coli 055:B5; Sigma-Aldrich, St. Louis, MO).

Bacterial strains and purification of gDNA

gDNA was isolated from L. plantarum (KCTC 10887BP) and Staphylococcus aureus (KCTC 1621) for stimulation of THP-1 cells. Lactobacillus plantarum was cultured with de Man, Rogosa and Sharpe (MRS) broth and S. aureus with brain heart infusion (BHI) broth at 37 °C for 18 h. Bacteria were harvested by centrifugation at 13 000 g for 10 min and washed with phosphate-buffered saline (WelGENE, Daegu, Korea). The pellet was resuspended in TE buffer (100 mM Tris–Cl, 10 mM EDTA) and then incubated at 37 °C for 4 h with addition of 200 μL lysozyme (20 mg mL−1; Sigma) and 3 μL RNase (Qiagen, Valenica, CA). Next, 3 μL proteinase K (20 mg mL−1; Sigma) and 10% SDS were added, followed by further incubation at 37 °C for an additional hour. gDNA was isolated by repeated extraction with phenol-chloroform to exclude protein contamination and precipitated with isopropanol. After washing with 70% ethanol, gDNA was separated again using a centrifugal separator and all ethanol was removed. The DNA preparations were resuspended with nuclease-free water for use in our experiments, and protein/LPS contamination was examined by silver staining and the Limulus amebocyte lysate QCL-1000® kit (Lonza, Allendale, NJ).

ELISA

After cells were stimulated with gDNA and/or LPS, cell supernatants were collected and assayed for cytokine production by standard sandwich ELISA. TNF-α production was determined using monoclonal anti-human mouse IgG1, clone 28401, and biotinylated anti-mouse TNF-α specific polyclonal Ab (goat IgG) for human TNF-α detection (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. The optical density of the samples was determined using a microplate reader (Eppendorf BioPhotometer, Hauppauge, NY) set to 450 nm with a wavelength correction of 540 nm.

Western blot analysis

Cellular extracts were prepared as described with minor modifications (Medvedev et al., 2000). Ten micrograms of total protein were resuspended in a Proprep buffer (iNtRON Biotechnology, Seongnam, Korea), boiled for 5 min, resolved by 12% SDS-PAGE in a Tris/glysine/SDS buffer (25 mM Tris, 250 mM glysine, 0.1% SDS), and blotted onto nitrocellulose membranes (100 V, 2 h, 4 °C). After blocking for 1 h in TBS-T (20 mM Tris–HCL, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBS-T and probed overnight with anti-phospho-MAPK Ab (Cell signaling, Danvers, MA), in TBS-T containing 5% BSA. After being washed three times with Tris-buffered saline-Tween (TBS-T), the membranes were incubated with secondary horseradish-peroxidase (HRP)-conjugated donkey anti-rabbit Ig for 2 h and washed five times in TBS-T; target proteins were detected using ECL reagents (GE Healthcare Biosciences) according to the manufacturer's description.

Reverse transcription (RT)-PCR analysis

THP-1 cells were seeded at a density of 2 × 106 cells mL−1 in six-well tissue culture plates and stimulated with gDNA and/or LPS. Untreated cells were used as controls. Total cellular RNA was extracted using RNA isolation Solvent RNA-Bee (iNtRON Biotechnology), according to the manufacturer's protocol. The amount and quality of RNA were determined by spectrophotometry. cDNAs from total RNA were prepared with the ImProm-II™ Reverse Transcription System (Promega, Madison, WI) according to the manufacturer's instructions. RT-PCR was performed using specific primers for the selected genes, and mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR products were analyzed on 1% agarose gels visualized with ethidium bromide.

Statistical analysis

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

Results

gDNA inhibited LPS-induced TNF-α production

We examined the hypothesis that Lactobacillus gDNA (p-gDNA) would inhibit TNF-α production based on our previous observation that Lactobacillus LTA reduces LPS-induced TNF-α production. THP-1 cells pretreated with 1 and 10 μg mL−1 of p-gDNA or S. aureus genomic DNA (a-gDNA) followed by re-stimulation with 0.5 μg mL−1 of LPS displayed significantly less LPS-induced TNF-α production (Fig. 1a). The inhibitory efficiency of gDNAs increased gradually with the gDNA pretreatment time (Fig. 1b).

Figure 1.

gDNA inhibited LPS-induced TNF-α production. (a) THP-1 cells were pretreated for 24 h with p-gDNA or a-gDNA at the concentrations listed, and re-stimulated with 0.5 μg mL−1LPS for 6 h. (b) Cells were pretreated with 10 μg mL−1 p-gDNA or a-gDNA for the indicated times, and re-stimulated with 0.5 μg mL−1LPS for 6 h. In all experiments, the TNF-α level in the culture supernatants was determined using ELISA.

p-gDNA did not increase TNF-α production

THP-1 cells treated with various concentrations of a-gDNA for 6 h showed a dose-dependent increase of TNF-α production, whereas p-gDNA barely produced TNF-α compared to a-gDNA-treated cells (Fig. 2a). TNF-α production from THP-1 cells treated with 10 μg mL−1 of a-gDNA peaked at 6 h after stimulation and slowly decreased (Fig. 2b). As THP-1 cells are very sensitive to endotoxin, we tried to exclude endotoxin contamination from prepared gDNA. All gDNA preparations were confirmed for the presence of endotoxin using a Limulus amebocyte lysate assay kit. Although endotoxin concentration remained below stimulatory levels (0.05 ng mL−1) throughout the study, we treated the prepared gDNA with polymyxin B before incubation with THP-1 cells to test whether the experiments were affected by contamination. As shown in Fig. 2c, endotoxin-induced TNF-α decreased after pretreatment with 50 μg mL−1polymyxin B, but p-gDNA- or a-gDNA-mediated TNF-α production was not affected by polymyxin B, demonstrating that the media and gDNAs were not contaminated with endotoxin. To confirm whether gDNA can induce TNF-α production from THP-1 cells, prepared gDNA was treated with DNase. Control aDNA induced TNF-α but DNase-treated aDNA did not. p-gDNA modestly induced TNF-α production in both the DNase treated and untreated tests (Fig. 2d). In another experiment, DNase treatment of gDNA significantly inhibited DNA-mediated tolerance, further confirming that gDNA is responsible for the induction of TNF-α and the inhibition of LPS-induced TNF-α production (Fig. 2e).

Figure 2.

p-gDNA did not increase TNF-α production. (a) THP-1 cells were treated with p-gDNA or a-gDNA at the indicated concentrations for 6 h. (b) Cells were treated with 10 μg mL−1 p-gDNA or a-gDNA for the indicated times. (c) Cells were pretreated for 1 h with 50 μg mL−1polymyxin B and re-stimulated with 0.5 μg mL−1 p-gDNA, a-gDNA or LPS for 6 h. DNA preparations were treated with DNase (Sigma-Aldrich). DNase activity was terminated by incubating the preparation at 70 °C for 10 min. (d) Cells were treated with 10 μg mL−1DNase-treated gDNA or intact gDNA for 6 h. (e) Cells were pretreated for 24 h with DNase-treated gDNA or intact gDNA and re-stimulated with 0.5 μg mL−1LPS for 6 h.

gDNA regulated NF-κB and MAPK activation

To identify which signaling pathway may be involved in gDNA-mediated TNF-α production, the signaling inhibitors were treated for 30 min before ligand stimulation. p-gDNA caused low basic TNF-α expression levels that were not affected by inhibitors. In contrast, TNF-α expression was reduced by gDNA from cells treated with NF-κB and MAPK inhibitors. This result indicates that NF-κB and MAPK are involved in the gDNA-mediated signaling pathway (Fig. 3a). LPS-mediated phosphorylation of NF-κB, p38, ERK 1/2, and JNK 1/2 in THP-1 cells was increased after 15 min treatment, and optimal responses were reached after 30 min of LPS stimulation. NF-κB and MAPK phosphorylation, however, were significantly inhibited in p-gDNA- or a-gDNA pretreated THP-1 cells followed by re-stimulation with 0.5 μg mL−1 LPS (Fig. 3b and c). We also evaluated differences between p-gDNA and a-gDNA in signaling transduction. The phosphorylation of NF-κB, p38, ERK 1/2 and JNK 1/2 was increased by a-gDNA, whereas p-gDNA treatment barely induced phosphorylation of those molecules (Fig. 3d). These results suggest that the activation of MAPK and NF-κB is involved in LPS-induced TNF-α production, and that gDNA inhibits TNF-α production through the downregulation of signaling transduction associated with the NF-κB and MAPK pathways.

Figure 3.

gDNA downregulated activated signaling pathways and a-gDNA induced strong signal transduction. (a) THP-1 cells were pretreated with 10 μM NF-κB inhibitor (Merckbiosciences, 4-hydroxyphenethylaminoquinazoline), ERK inhibitor (Merckbiosciences, PD 98059), JNK inhibitor (Merckbiosciences, SP600125) or p38 kinase inhibitor (Merckbiosciences, SB 203580) for 30 min followed by re-stimulation with 10 μg mL−1gDNA for 6 h. (b,c) Cells were pretreated for 24 h with PBS, p-gDNA or a-gDNA, and re-stimulated with 0.5 μg mL−1LPS for the indicated times. The cell lysates were blotted with phospho-specific antibodies for NF-κB, ERK1/2, JNK1/2 and p38. To verify the amount of loaded protein, they were also probed with β-actin. (d) Cells were treated with 10 μg mL−1 p-gDNA or a-gDNA for the indicated times.

gDNA downregulated the production of sepsis-related receptors

LPS induces septic shock through pattern recognition receptors (PRRs), especially TLR4 (Lakhani & Bogue, 2003). Therefore, we examined the role of gDNA pretreatment on the expression of PRRs. The mRNA level of TLR2, TLR4 and TLR9 was downregulated in THP-1 cells pretreated with gDNA followed by re-stimulation with 0.5 μg mL−1 LPS for 4 h. LPS increased TLR expression after 15 min, whereas TLR expression was reduced in THP-1 cells pretreated with p-gDNA or a-gDNA compared to LPS alone (Fig. 4a and b). Extracellular treatment of THP-1 cells with gDNA induced TLR2, TLR4 and TLR9 expression, although there were differences between strains. Expression levels of TLR2 and TLR9 after a-gDNA treatment were higher than after p-gDNA treatment. A low level of TLR4 expression was shown in both p-gDNA- and a-gDNA-treated cells; however, it was slightly increased by p-gDNA in a time-dependent manner, and a-gDNA showed a tendency to decrease after reaching a peak at 15 min (Fig. 4c).

Figure 4.

gDNA downregulated the LPS-induced sepsis-related receptors. (a,b) THP-1 cells were pretreated for 24 h with PBS, p-gDNA or a-gDNA, re-stimulated with 0.5 μg mL−1LPS for the indicated times, and then cDNAs were synthesized with extracted total RNA. The amount of TLR expression was examined with reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for TLRs. (c) Cells were stimulated with 10 μg mL−1 p-gDNA or a-gDNA for the indicated times. The primers used in PCR were: TLR9 forward, 5′ CAC TCG ATG AGA CCA CGC TC 3′ and TLR9 reverse, 5′ AGT CGT GGT AGC TCC GTG AA 3′; TLR4 forward, 5′ GAT AGC GAG CCA CGC ATT CA 3′ and TLR4 reverse, 5′ TAT TAG GAA CCA CCT CCA CGC A 3′; TLR2 forward, 5′ ACT CCA TTC CCT CAG GGC TC 3′ and TLR2 reverse, 5′ TGG AAT ATG CAG CCT CCG GA 3′; GAPDH forward, 5′ AAG GTC GGA GTC AAC GGA TTT 3′ and GAPDH reverse, 5′ GCA GTG AGG GTC TCT CTC CT 3′.

p-gDNA upregulated the production of IRAK-M, a negative regulator of TLR signaling

Although both p-gDNA and a-gDNA reduced LPS-induced TNF-α production, they displayed different trends in TNF-α induction. To further evaluate the differences between p-gDNA and a-gDNA, we examined the variation of TLR-negative regulators and examined the mRNA levels of IRAK-M, IRAK4, IRAK1 and IRAK2 in THP-1 cells. IRAK-M blocks the pathway in which IRAK4 is processed to IRAK1, and IRAK1 promotes IRAK2. The expression of IRAK-M increased along with treatment time in p-gDNA-treated cells, whereas it peaked at 30 min after treatment with a-gDNA and then slightly declined (Fig. 5a). IRAK-M blocked IRAK4 activation and subsequent IRAK1 phosphorylation (Miggin & O'Neill, 2006). When THP-1 cells were treated with p-gDNA, IRAK-4 was increased in a time-dependent manner, whereas IRAK1 and IRAK2 increased slightly and then disappeared after about 120 min. For a-gDNA, the IRAK4 reached its highest level at 30 min and then stabilized, whereas the expression of IRAK1 and IRAK2 dramatically increased after 120 min (Fig. 5b). These results indicate that IRAK-M, which was upregulated by gDNA, plays an important role as a negative regulator for TLR signaling, and it may be involved in gDNA-mediated tolerance.

Figure 5.

p-gDNA upregulated the production of IRAK-M, a negative regulator of TLR. (a,b) THP-1 cells were stimulated with 10 μg mL−1 p-gDNA or a-gDNA for the indicated times. The primers used in PCR were: IRAK-M forward, 5′ GAG TAC ATC AGA CAG GGG AA 3′ and IRAK-M reverse, 5′ AAT TCT CTA AGG AGA TCC CG 3′; IRAK4 forward, 5′ CCG GGC AGG AAT AGA AGA TGA ACA 3′ and IRAK4 reverse, 5′ GTG GTG CCC CAG TCA AAC AG 3′; IRAK2 forward, 5′ TCC ACA GCA ACG TCA AGA GC 3′ and IRAK2 reverse, 5′ GTC CAC TCG CTT TGT CAG CT 3′; IRAK1 forward, 5′ GGA GTG GCT TTG AGA AGC AC 3′ and IRAK1 reverse, 5′ TAG GAG TTC TCC TGC GGG GA 3′.

Discussion

Inflammation is a frontline defense mechanism against infection and injury, restoring the body to homeostasis. Excessive expression of inflammatory cytokines, however, causes inflammatory diseases such as septic shock (Cook et al., 2004). To treat inflammatory diseases, researchers have tried to induce tolerance against endotoxins that induce excessive inflammation. Several reports have shown that bacterial cell wall components induce homologous tolerance (Sugawara et al., 1999; Lehner et al., 2001; Yang et al., 2001; Jacinto et al., 2002). Treatment of THP-1 cells with LPS or a-gDNA significantly increases the production of pro-inflammatory cytokine, TNF-α, illustrating the risk of pathogenic bacterial gDNA.

We purified gDNA from L. plantarum, predicting that it would induce tolerance. Whereas p-gDNA did not stimulate much pro-inflammatory cytokine expression and showed low levels of cytotoxicity, p-gDNA efficiently inhibited LPS-induced TNF-α production from THP-1 cells. Further, p-gDNA reduced the expression of TLR2, TLR4 and TLR9, which induced the activation of NF-κB through the LPS signaling pathway, leading to the upregulation of inflammatory cytokines (Verstrepen et al., 2008). The activation of MAPKs such as ERK1/2, JNK1/2 and p38 is necessary to mediate many macrophage functions, including the activation of various transcription factors and the production of pro- and anti-inflammatory cytokines (Payne et al., 1991; DeFranco et al., 1998). In addition, the LPS-induced activation of the MAPK pathways plays an important role in the NF-κB activation. In the present study, pretreatment of THP-1 cells with p-gDNA inhibited the phosphorylation of MAPKs and NF-κB. The expression of IRAK-M by gDNA may also block the signaling transfer from IRAK4 to IRAK1, which reduces the downstream expression of TNF-α.

Unlike a-gDNA and LPS, p-gDNA was prepared from a probiotic organism. p-gDNA did not induce the overexpression of inflammatory cytokines. The inhibitory effects of p-gDNA resulted from the complex mechanisms of (1) the inhibition of intercellular signaling pathways such as the MAPK and NF-κB pathways; (2) the reduction of sepsis-related PRRs such as TLR2, TLR4, and TLR9; and (3) the induction of IRAK-M. Accordingly, p-gDNA tolerance may offer an effective approach for the prevention and treatment of endotoxin-induced shock.

Acknowledgements

This research was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean government (Ministry of Education, Science and Technology) (KRF-2008-313-F00132).

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