Food Science of Animal Resources (한국축산식품학회지)

Korean Society for Food Science of Animal Resources (한국축산식품학회)
권호 표지
DOI QR코드

DOI QR Code

Inhibitory Effect of Genomic DNA Extracted from Pediococcus acidilactici on Porphyromonas gingivalis Lipopolysaccharide-Induced Inflammatory Responses

  • Young Hyeon, Choi (Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University) ;
  • Bong Sun, Kim (Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University) ;
  • Seok-Seong, Kang (Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University)
  • Received : 2022.07.21
  • Accepted : 2022.10.06
  • Published : 2023.01.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study aimed to assess whether genomic DNA (gDNA) extracted from Pediococcus acidilactici inhibits Porphyromonas gingivalis lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 cells. Pretreatment with gDNA of P. acidilactici K10 or P. acidilactici HW01 for 15 h effectively inhibited P. gingivalis LPS-induced mRNA expression of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein (MCP)-1. Although both gDNAs did not dose-dependently inhibit P. gingivalis LPS-induced mRNA expression of IL-6 and MCP-1, they inhibited IL-1β mRNA expression in a dose-dependent manner. Moreover, pretreatment with both gDNAs inhibited the secretion of IL-1β, IL-6, and MCP-1. When RAW 264.7 cells were stimulated with P. gingivalis LPS alone, the phosphorylation of mitogen-activated protein kinases (MAPKs) was increased. However, the phosphorylation of MAPKs was reduced in the presence of gDNAs. Furthermore, both gDNAs restored IκBα degradation induced by P. gingivalis LPS, indicating that both gDNAs suppressed the activation of nuclear factor-κB (NF-κB). In summary, P. acidilactici gDNA could inhibit P. gingivalis LPS-induced inflammatory responses through the suppression of MAPKs and NF-κB, suggesting that P. acidilactici gDNA could be effective in preventing periodontitis.

Keywords

Introduction

Probiotics, which mostly consist of lactic acid bacteria, are well known to confer beneficial effects (Chugh and Kamal-Eldin, 2020). Their physiological functions are beneficial to human health by inhibiting pathogen adhesion, enhancing the functions of the intestinal epithelial barrier, and modulating host immune responses (Lebeer et al., 2010). Moreover, the use of probiotics is a promising therapeutic approach against many inflammatory diseases (Maekawa and Hajishengallis, 2014). These effects of probiotics can be attributed to the production of bioactive molecules, such as cell wall components, bacteriocins, short-chain fatty acids, exopolysaccharides, enzymes, and amino acids (Chugh and Kamal-Eldin, 2020; Lebeer et al., 2010).

In addition to the bioactive molecules of probiotics, their genomic DNA (gDNA) has been proven to exert probiotic effects. For instance, gDNA extracted from Lactobacillus plantarum inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α secretion by suppressing the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) in THP-1 cells (Kim et al., 2012). Moreover, oligodeoxynucleotides from L. casei gDNA inhibited inflammatory responses in human intestinal epithelial cells in vitro as well as in a dextran sulfate sodium-induced murine colitis model (Hiramatsu et al., 2014). More recently, gDNA extracted from L. rhamnosus GG was found to inhibit LPS-induced interleukin (IL)-6 production in RAW 264.7 cells (Qi et al., 2020).

Periodontitis is defined as a chronic inflammatory oral disease caused by multiple microbial infections, resulting in the gradual destruction of tissues supporting teeth and the alveolar bone (Saini et al., 2009). Moreover, periodontitis is known to be associated with systemic diseases, such as cardiovascular and respiratory diseases, rheumatoid arthritis, and metabolic syndrome (Winning and Linden, 2015). Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is considered to be the main pathogen responsible for periodontitis by producing several virulence factors, such as extracellular proteases and LPS (Bostanci and Belibasakis, 2012). Among the bacteria associated with the pathogenesis of periodontitis, P. gingivalis has been extensively studied because of its unique ability to penetrate the gingivae and cause tissue destruction by inducing inflammation (How et al., 2016).

The findings of in vitro and in vivo analyses have confirmed that probiotics can alleviate periodontal diseases (Nguyen et al., 2021). However, probiotics need to be viable to perform their biological functions. Moreover, the use of probiotics may cause some adverse effects, such as antibiotic resistance and systemic infections in immunocompromised patients (Doron and Snydman, 2015). Therefore, molecules with functional bioactivities, also known as postbiotics, are considered promising alternatives to probiotics. Although the health benefits of probiotics, particularly lactobacilli, are well known, pediococci are also exhibit certain probiotic effects. Bioactive molecules, such as exopolysaccharides and bacteriocins, of Pediococcus acidilactici exhibit probiotic effects, including antioxidant, lipid-lowering, antimicrobial, and anti-inflammatory effects (Moon et al., 2014; Seo and Kang, 2020; Song et al., 2021). In addition, our studies have shown the beneficial effects of postbiotics derived from P. acidilactici K10 and P. acidilactici HW01, such as bacteriocin and bacterial lysates. Bacteriocins of P. acidilactici K10 and P. acidilactici HW01 inhibit biofilm formation of foodborne pathogenic bacteria, including Enterococcus faecalis and Pseudomonas aeruginosa (Lee et al., 2020; Yoon and Kang, 2020). More recently, bacterial lysates of both P. acidilactici K10 and P. acidilactici HW01 exert antibiofim, anti-adhesive and anti-invasive activities against Listeria monocytogenes (Lee et al., 2022). Therefore, this study aimed to assess the anti-inflammatory effect of gDNA extracted from P. acidilactici K10 and P. acidilactici HW01 against P. gingivalis LPS-induced inflammatory responses.

Materials and Methods

Bacterial culture and gDNA purification

P. acidilactici K10 and P. acidilactici HW01, which were isolated from kimchi and malt (Ahn et al., 2017; Kwon et al., 2002), were maintained in Man−Rogosa−Sharpe medium (Neogen, Lansing, MI, USA) at 37℃. The gDNAs of P. acidilactici K10 and P. acidilactici HW01 (K10 gDNA and HW01 gDNA, respectively) were extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions, with some modifications. In brief, bacterial pellets were harvested by centrifugation at 13,000×g for 10 min and extensively washed with phosphate-buffered saline (PBS). The bacterial pellets were resuspended in 480 μL of 50 mM EDTA and 120 μL of lysozyme (10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37℃ for 30 min. After centrifuging at 13,000×g for 10 min, nuclei lysis solution (600 μL) was added to the pellets and incubated at 80℃ for 5 min to lyse the cell membrane. RNase solution (3 μL) was then added to the lysates, which were mixed by inverting the tubes five times and then incubated at 37℃ for 40 min. Protein precipitation solution (200 μL) was added to the mixture and incubated on ice for 5 min to purify the gDNA. After centrifuging at 13,000×g for 3 min, the supernatants were transferred to a clean microtube and gently mixed with 600 μL of isopropanol. After centrifuging at 13,000×g for 3 min, the supernatants were removed and the remaining pellets were washed with 70% ethanol. Finally, the ethanol was discarded and the pellets containing gDNA were rehydrated by adding DNA rehydration solution. The concentration and purity of gDNA were determined using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies, Hampton, NH, USA).

Cell culture

RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) containing 10% heat-inactivated fetal bovine serum (Gibco, Burlington, ON, Canada), 100 U/mL penicillin, and 100 µg/mL streptomycin (HyClone, Logan, UT, USA) at 37℃ in a 5% CO2-humidified incubator

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RAW 264.7 cells (1×105 cells/mL) were plated on a 12-well culture plate and incubated at 37℃ for 24 h. The cells were pretreated with K10 gDNA or HW01 gDNA (1 μg/mL) for 3, 9, or 15 h and stimulated with 1 µg/mL of P. gingivalis LPS for 3 h. In a separate experiment, RAW 264.7 cells (1×105 cells/mL) were plated on a 12-well culture plate and incubated at 37℃ for 24 h. The cells were pretreated with various concentrations of K10 gDNA or HW01 gDNA (0.1, 1, and 10 μg/mL) for 15 h and stimulated with 1 µg/mL of P. gingivalis LPS for 3 h. Subsequently, spent culture supernatants were discarded and total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Total RNA was reversed transcribed to complementary DNA (cDNA) using random hexamers and reverse transcriptase (Promega). cDNA was amplified by qPCR with SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan) using the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Specific genes responsible for inflammatory responses, namely those encoding IL-1β, IL-6, and monocyte chemoattractant protein (MCP)-1, were amplified under the following conditions: Denaturation at 95℃ for 10 s, amplification for 40 cycles at 95℃ for 5 s and 60℃ for 31 s. The sequences of specific primers were as follows: IL-1β, forward, 5′-CTCACAAGCAGAGCACAAGC-3′ and reverse, 5′-TCTTGGCCGAGGACTAAGGA-3′; IL-6, forward, 5′-TCCTACCCCAATTTCCAATGCT-3′ and reverse, 5′-TCTGACCACAGTGAGGAATGTC-3′; MCP-1, forward, 5′-AGCCAACTCTCACTGAAGCC-3′ and reverse, 5′-TCTCCAG CCTACTCATTGGGA-3′; and β-actin, forward, 5′-TACAGCTTCACCACCACAGC-3′ and reverse, 5′-GGAAAAGAGCCT CAGGGCAT-3′. The relative mRNA expression levels of IL-1β, IL-6, and MCP-1 were normalized to that of β-actin using the 2−ΔΔCt method.

Enzyme-linked immunosorbent assay (ELISA)

RAW 264.7 cells (1×105 cells/mL) were plated on a 96-well culture plate and incubated at 37℃ for 24 h. The cells were pretreated with 1 µg/mL of K10 gDNA or HW01 gDNA at 37℃ for 15 h and stimulated with P. gingivalis LPS (1 µg/mL) at 37℃ for further 24 h. Subsequently, spent culture supernatants were collected and the secretion levels of IL-1β, IL-6, and MCP-1 were measured using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Western blot analysis

RAW 264.7 cells (1×105 cells/mL) were plated on a 6-well culture plate and incubated at 37℃ for 24 h. The cells were pretreated with 1 µg/mL of K10 gDNA or HW01 gDNA at 37℃ for 15 h and stimulated with P. gingivalis LPS (1 µg/mL) at 37℃ for 30 min. The cells were then washed with PBS and lysed with a lysis buffer (1 M HEPES, 1 M NaCl, 1% IGEPAL®-CA 630, 0.75% sodium deoxycholate, and 10% glycerol) containing protease and phosphatase inhibitors. Equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibodies specific to extracellular signal-regulated kinase (ERK), phosphorylated ERK, p38 kinase, phosphorylated p38 kinase, c-Jun-N-terminal kinase (JNK), phosphorylated JNK, IκBα (Cell Signaling Technology, Danvers, MA, USA), or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4℃ for 15 h. The membranes were washed with TBST and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) at room temperature for 1 h. Immunoreactive proteins were detected using an enhanced chemiluminescence reagent (Dyne Bio, Seongnam, Korea) and visualized using a C-DiGit blot scanner (Li-Cor Bioscience, Lincoln, NE, USA).

Statistical analysis

All experiments were performed thrice. The results are expressed as the means±SDs. Statistical differences between the appropriate control and gDNA-treated groups were assessed by performing unpaired two-tailed t-tests using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).

Results

K10 gDNA and HW01 gDNA inhibit Porphyromonas gingivalis lipopolysaccharide (LPS)-induced mRNA expression of IL-1β, IL-6, and monocyte chemoattractant protein (MCP)-1

When the cells were pretreated with K10 gDNA and HW01 gDNA for 3 h, the gDNAs did not inhibit P. gingivalis-induced mRNA expression of IL-1β (Fig. 1A), IL-6 (Fig. 1B), and MCP-1 (Fig. 1C). However, when the cells were pretreated with K10 gDNA and HW01 gDNA for 9 h, the gDNAs partially suppressed inflammatory responses. As shown in Fig. 1D, P. gingivalis LPS-induced IL-1β mRNA expression was significantly downregulated in the presence of K10 gDNA and HW01 gDNA (p<0.05), whereas IL-6 and MCP-1 mRNA expression was not inhibited by these gDNAs (Figs. 1E and 1F, respectively). When RAW 264.7 cells were pretreated with gDNAs for 15 h, the gDNAs significantly inhibited the mRNA expression of IL-1β (Fig. 1G), IL-6 (Fig. 1H), and MCP-1 (Fig. 1I; p<0.05). As 15 h-pretreatment with gDNAs effectively inhibited P. gingivalis LPS-induced inflammatory responses, all subsequent experiments were performed using cells pretreated with gDNAs for 15 h.

CSSPBQ_2023_v43n1_101_f0001.png 이미지

Fig. 1. Inhibition of mRNA expression of inflammatory mediators by Pediococcus acidilactici gDNAs in RAW 264.7 cells stimulated with Porphyromonas gingivalis LPS. RAW 264.7 cells were pretreated with K10 gDNA or HW01 gDNA (1 µg/mL) for 3, 6, or 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 3 h. The mRNA expression levels of IL-1β, IL-6, and MCP-1 were determined using RT-qPCR. The results are expressed as the means±SDs from three independent experiments. * Indicates a statistically significant difference (p<0.05) compared with the control. LPS: P. gingivalis LPS; K10: K10 gDNA; HW01: HW01 gDNA. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; LPS, lipopolysaccharide; gDNA, genomic DNA.

K10 genomic DNA and HW01 gDNA dose-dependently inhibit Porphyromonas gingivalis lipopolysaccharide (LPS)-induced mRNA expression of IL-1β but not that of IL-6 and monocyte chemoattractant protein (MCP)-1

To assess the inhibitory effect of different concentrations of gDNAs on P. gingivalis LPS-induced inflammatory responses in RAW 264.7 cells, the cells were pretreated with gDNAs (0.1, 1, and 10 µg/mL) for 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 3 h. As expected, P. gingivalis LPS alone induced IL-1β mRNA expression. However, both K10 gDNA and HW01 gDNA dose-dependently inhibited P. gingivalis LPS-induced IL-1β mRNA expression (Figs. 2A and 2D, respectively). Pretreatment with 0.1 and 1 µg/mL of K10 gDNA and HW01 gDNA significantly inhibited P. gingivalis LPS-induced IL-6 mRNA expression (p<0.05; Figs. 2B and 2E, respectively). However, 10 µg/mL of both gDNAs failed to inhibit P. gingivalis LPS-induced IL-6 mRNA expression (Figs. 2B and 2E). Similarly, 0.1 and 1 µg/mL of K10 gDNA significantly inhibited P. gingivalis LPS-induced MCP-1 mRNA expression, whereas 10 µg/mL did not (Fig. 2C). Moreover, 1 µg/mL of HW01 gDNA inhibited P. gingivalis LPS-induced MCP-1 expression, whereas 0.1 and 10 µg/mL did not (Fig. 2F). Therefore, we decided that the optimal concentration of gDNAs to inhibit P. gingivalis LPS-induced inflammatory responses was 1 µg/mL and used this concentration for further analysis.

CSSPBQ_2023_v43n1_101_f0002.png 이미지

Fig. 2. Inhibition of mRNA expression of inflammatory mediators by various concentrations of Pediococcus acidilactici gDNAs in RAW 264.7 cells stimulated with Porphyromonas gingivalis LPS. RAW 264.7 cells were pretreated with K10 gDNA or HW01 gDNA (0.1, 1, and 10 µg/mL) for 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 3 h. The mRNA expression levels of IL-1β, IL-6, and MCP-1 were determined using RT-qPCR. The results are expressed as the means±SDs from three independent experiments. * Indicates a statistically significant difference (p<0.05) compared with the control. LPS: P. gingivalis LPS; K10: K10 gDNA; HW01: HW01 gDNA. RT-qPCR reverse transcription-quantitative polymerase chain reaction; LPS, lipopolysaccharide; gDNA, genomic DNA.

K10 gDNA and HW01 genomic DNA inhibit Porphyromonas gingivalis lipopolysaccharide (LPS)-induced secretion of IL-1β, IL-6, and monocyte chemoattractant protein (MCP)-1

To further confirm the inhibition of inflammatory responses at the protein level, RAW 264.7 cells were pretreated with gDNAs (1 µg/mL) for 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 24 h. Consistent with the results of mRNA expression, both gDNAs significantly inhibited IL-1β (Fig. 3A), IL-6 (Fig. 3B), and MCP-1 (Fig. 3C) secretion induced by P. gingivalis LPS (p<0.05). These results indicate that K10 gDNA and HW01 gDNA effectively inhibit P. gingivalis LPS-induced inflammatory responses.

CSSPBQ_2023_v43n1_101_f0003.png 이미지

Fig. 3. Inhibition of protein expression of inflammatory mediators by Pediococcus acidilactici gDNAs in RAW 264.7 cells stimulated with Porphyromonas gingivalis LPS. RAW 264.7 cells were pretreated with K10 gDNA or HW01 gDNA (1 µg/mL) for 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 24 h. The secretion levels of IL-1β, IL-6, and MCP-1 were determined using ELISA. The results are expressed as the means±SDs from three independent experiments. * Indicates a statistically significant difference (p<0.05) compared with the control. LPS: P. gingivalis LPS; K10: K10 gDNA; HW01: HW01 gDNA. ELISA, enzyme-linked immunosorbent assay; gDNA, genomic DNA; LPS, lipopolysaccharide.

K10 genomic DNA and HW01 gDNA alleviate mitogen-activated protein kinases (MAPK) phosphorylation and nuclear factor-κB (NF-κB) activation

MAPK pathways, such as ERK 1/2, p38 kinase, JNK, and NF-κB pathways, are known to be common inflammatory signaling pathways that cause the release of proinflammatory cytokines, such as IL-1β, and consequently lead to inflammation (Jayakumar et al., 2021). Therefore, the activation of MAPK and NF-κB was assessed in RAW 264.7 cells pretreated with or without gDNAs. As shown in Fig. 4, the phosphorylation of ERK 1/2, p38 kinase, and JNK was higher in RAW 264.7 cells stimulated with P. gingivalis LPS than in those not treated with P. gingivalis LPS. However, pretreatment with K10 gDNA and HW01 gDNA abrogated the phosphorylation of ERK 1/2, p38 kinase, and JNK, indicating that ERK 1/2, p38 kinase, and JNK signaling pathways were blocked on pretreatment with both these gDNAs and consequently inhibited P. gingivalis LPS-induced inflammatory responses. Moreover, inhibitors of NF-κB, such as IκBα, are associated with the downregulation of NF-κB (Wang et al., 2020). P. gingivalis LPS alone inhibited IκBα degradation, indicating that the inflammatory responses were augmented by NF-κB activation. However, pretreatment of RAW 264.7 cells with K10 gDNA or HW01 gDNA (Fig. 4) restored IκBα degradation, suggesting that both these gDNAs attenuated NF-κB activation, which was associated with P. gingivalis LPS-induced inflammatory responses.

CSSPBQ_2023_v43n1_101_f0004.png 이미지

Fig. 4. Inhibition of MAPK phosphorylation and NF-κB activation by Pediococcus acidilactici gDNAs in RAW 264.7 cells stimulated with Porphyromonas gingivalis LPS. RAW 264.7 cells were pretreated with K10 gDNA or HW01 gDNA (1 µg/mL) for 15 h and then stimulated with P. gingivalis LPS (1 µg/mL) for 30 min. The phosphorylation of ERK 1/2, p38, and JNK and the degradation of IκBα were assessed using western blot analysis. MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; gDNA, genomic DNA; LPS, lipopolysaccharide; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase.

Discussion

Periodontitis is the most prevalent oral disease that is characterized by gingival inflammation and the loss of connective tissue attachment, resulting in alveolar bone resorption and tooth loss (Xu et al., 2020). Although the role of probiotics in modulating periodontal diseases is not fully understood, several studies have reported the protective effects of probiotics, particularly Lactobacillus spp., on periodontal diseases based on in vitro and in vivo analyses (Gatej et al., 2018; Nguyen et al., 2021). P. acidilactici is frequently found in fermented vegetables and meat-based products and is considered a potential probiotic with antimicrobial, antioxidant, and immunomodulatory effects (Feng et al., 2016; Papagianni and Anastasiadou, 2009; Song et al., 2021). Furthermore, the present study demonstrated that K10 gDNA and HW01 gDNA markedly inhibited P. gingivalis LPS-induced inflammatory responses by suppressing MAPK and NF-κB signaling pathways in RAW 264.7 cells, suggesting that P. acidilactici could also be a potential probiotic that could be beneficial to oral health.

Several lactobacilli have been reported to inhibit inflammatory responses induced by periodontal pathogenic bacteria, such as P. gingivalis. Co-culture with P. gingivalis and L. acidophilus significantly decreased the mRNA and protein expression levels of IL-1β, IL-6, and IL-8 in gingival epithelial cells in a dose-dependent manner (Zhao et al., 2012). More recently, mono-infection of gingival epithelial cells with P. gingivalis was found to increase the expression of IL-1β and TNF-α. However, treatment with L. rhamnosus Lr-32 or L. acidophilus LA-5 markedly decreased the expression of IL-1β and TNF-α (Albuquerque‐Souza et al., 2019). In fact, P. acidilactici has displayed anti-inflammatory activities against various pathogenic bacteria. We previously found that bacteriocins isolated from P. acidilactici K10 and HW01 suppressed IL-8 production in the human intestinal epithelial cell line Caco-2 in response to Enterococcus faecalis infection (Yoon and Kang, 2020). Cell-free supernatants of P. acidilactici TMAB26 effectively inhibited Klebsiella pneumoniae LPS-induced IL-6 and TNF-α mRNA expression in HT-29 cells and peripheral blood mononuclear cells (Barigela and Bhukya, 2021). However, the antagonistic effect of P. acidilactici or its component(s) on P. gingivalis-induced inflammatory responses has not been elucidated. In addition, gDNA derived from Lactobacillus spp. has been found to have anti-inflammatory properties. For example, high-frequency oligodeoxynucleotides of L. casei gDNA inhibited IL-8 production in Caco-2 cells in vitro and alleviated dextran sulfate sodium-induced murine colitis (Hiramatsu et al., 2014). Consistent with the present findings, pretreatment with L. rhamnosus GG gDNA was found to alleviate mRNA expression levels of inflammatory cytokines in RAW 264.7 cells upon LPS challenge (Qi et al., 2020). However, whether gDNAs of probiotic strains exhibit anti-inflammatory effects against oral pathogenic bacteria remains unclear. We found that P. acidilactici gDNA can inhibit P. gingivalis LPS-induced inflammatory responses.

During the development of periodontitis, periodontal pathogens trigger innate immune responses by releasing various cytokines and chemokines, which play a pivotal role in the progression of inflammation (Ramadan et al., 2020). Excessive IL-6 production may be associated with periodontal tissue destruction by inducing matrix metalloproteinase (MMP)-1 secretion during the progression of periodontitis (Sundararaj et al., 2009). In addition, IL-1β is involved in the production of MMP-1, leading to tissue damage (Cheng et al., 2020). MCP-1 is produced by macrophages and fibroblasts and acts as a chemoattractant for monocytes and neutrophils (Strecker et al., 2011). Excessive neutrophil activation can result in the production of toxic compounds and reactive oxygen species that can also affect aggressive periodontitis (Ramadan et al., 2020). We found that both K10 gDNA and HW01 gDNA significantly suppressed these cytokines/chemokines by regulating MAPK and NF-κB signaling pathways, suggesting that they could relieve inflammation and consequently prevent periodontitis. Although the precise mechanisms by which P. acidilactici gDNAs inhibit the production of cytokines/chemokines responsible for periodontitis are not fully understood, it can be speculated that cell signaling mediated by toll-like receptor 9 (TLR9) negatively regulates inflammatory responses. TLR9 is a critical receptor for recognizing bacterial DNA (Chockalingam et al., 2012). Li et al. (2017) reported that TLR9 deficiency enhanced the activation of NF-κB, resulting in increased IL-1β expression in intestinal epithelial cells. Moreover, the use of CpG oligodeoxynucleotides as a TLR9 ligand inhibited MCP-1 expression in adipocytes (Thomalla et al., 2019). Although TLR signaling pathways mainly induce inflammatory responses, TLR9 signaling promotes the accumulation of IκBα in the cytoplasm, thereby suppressing NF-κB activation (Li et al., 2017). Therefore, TLR9 activation by P. acidilactici gDNAs may negatively regulate intracellular signaling pathways, such as MAPK and NF-κB pathways, thereby attenuating inflammatory responses.

Conclusion

In conclusion, the present study demonstrated the inhibitory effect of P. acidilactici gDNA on P. gingivalis LPS-induced inflammatory responses. Several probiotic lactobacilli have shown anti-inflammatory activities against P. gingivalis. Our results provide important insights into the inhibition of periodontal inflammation using P. acidilactici gDNA. Thus, P. acidilactici gDNA could be a potential therapeutic option against periodontitis.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2020R1A2C1010010). This work was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Technology Commercialization Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (821028-03-2-HD040).

Author Contributions

Conceptualization: Choi YH, Kim BS, Kang SS. Data curation: Choi YH, Kim BS. Formal analysis: Choi YH, Kim BS. Methodology: Choi YH, Kim BS. Software: Choi YH, Kim BS. Validation: Choi YH, Kim BS. Investigation: Choi YH, Kim BS. Writing - original draft: Choi YH. Writing - review & editing: Choi YH, Kim BS, Kang SS.

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.

References

  1. Ahn H, Kim J, Kim WJ. 2017. Isolation and characterization of bacteriocin-producing Pediococcus acidilactici HW01 from malt and its potential to control beer spoilage lactic acid bacteria. Food Control 80:59-66. https://doi.org/10.1016/j.foodcont.2017.04.022
  2. Albuquerque-Souza E, Balzarini D, Ando-Suguimoto ES, Ishikawa KH, Simionato MRL, Holzhausen M, Mayer MPA. 2019. Probiotics alter the immune response of gingival epithelial cells challenged by Porphyromonas gingivalis. J Periodontal Res 54:115-127. https://doi.org/10.1111/jre.12608
  3. Barigela A, Bhukya B. 2021. Probiotic Pediococcus acidilactici strain from tomato pickle displays anti-cancer activity and alleviates gut inflammation in-vitro. 3 Biotech 11:23. https://doi.org/10.1007/s13205-020-02570-1
  4. Bostanci N, Belibasakis GN. 2012. Porphyromonas gingivalis: An invasive and evasive opportunistic oral pathogen. FEMS Microbiol Lett 333:1-9. https://doi.org/10.1111/j.1574-6968.2012.02579.x
  5. Cheng R, Wu Z, Li M, Shao M, Hu T. 2020. Interleukin-1β is a potential therapeutic target for periodontitis: A narrative review. Int J Oral Sci 12:2. https://doi.org/10.1038/s41368-019-0068-8
  6. Chockalingam A, Rose WA II, Hasan M, Ju CH, Leifer CA. 2012. Cutting edge: A TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production. J Immunol 188:527-530. https://doi.org/10.4049/jimmunol.1102713
  7. Chugh B, Kamal-Eldin A. 2020. Bioactive compounds produced by probiotics in food products. Curr Opin Food Sci 32:76-82. https://doi.org/10.1016/j.cofs.2020.02.003
  8. Doron S, Snydman DR. 2015. Risk and safety of probiotics. Clin Infect Dis 60:S129-S134. https://doi.org/10.1093/cid/civ085
  9. Feng J, Wang L, Zhou L, Yang X, Zhao X. 2016. Using in vitro immunomodulatory properties of lactic acid bacteria for selection of probiotics against Salmonella infection in broiler chicks. PLOS ONE 11:e0147630. https://doi.org/10.1371/journal.pone.0147630
  10. Gatej SM, Marino V, Bright R, Fitzsimmons TR, Gully N, Zilm P, Gibson RJ, Edwards S, Bartold PM. 2018. Probiotic Lactobacillus rhamnosus GG prevents alveolar bone loss in a mouse model of experimental periodontitis. J Clin Periodontol 45:204-212. https://doi.org/10.1111/jcpe.12838
  11. Hiramatsu Y, Satho T, Hyakutake M, Irie K, Mishima K, Miake F, Kashige N. 2014. The anti-inflammatory effects of a highfrequency oligodeoxynucleotide from the genomic DNA of Lactobacillus casei. Int Immunopharmacol 23:139-147. https://doi.org/10.1016/j.intimp.2014.08.013
  12. How KY, Song KP, Chan KG. 2016. Porphyromonas gingivalis: An overview of periodontopathic pathogen below the gum line. Front Microbiol 7:53. https://doi.org/10.3389/fmicb.2016.00053
  13. Jayakumar T, Lin KC, Chang CC, Hsia CW, Manubolu M, Huang WC, Sheu JR, Hsia CH. 2021. Targeting MAPK/NF-κB pathways in anti-inflammatory potential of rutaecarpine: Impact on Src/FAK-mediated macrophage migration. Int J Mol Sci 23:92. https://doi.org/10.3390/ijms23010092
  14. Kim CH, Kim HG, Kim JY, Kim NR, Jung BJ, Jeong JH, Chung DK. 2012. Probiotic genomic DNA reduces the production of pro-inflammatory cytokine tumor necrosis factor-alpha. FEMS Microbiol Lett 328:13-19. https://doi.org/10.1111/j.1574-6968.2011.02470.x
  15. Kwon DY, Koo MS, Ryoo CR, Kang CH, Min KH, Kim WJ. 2002. Bacteriocin produced by Pediococcus sp. in kimchi and its characteristics. J Microbiol Biotechnol 12:96-105.
  16. Lebeer S, Vanderleyden J, De Keersmaecker SCJ. 2010. Host interactions of probiotic bacterial surface molecules: Comparison with commensals and pathogens. Nat Rev Microbiol 8:171-184. https://doi.org/10.1038/nrmicro2297
  17. Lee DH, Kim BS, Kang SS. 2020. Bacteriocin of Pediococcus acidilactici HW01 inhibits biofilm formation and virulence factor production by Pseudomonas aeruginosa. Probiotics Antimicrob Proteins 12:73-81. https://doi.org/10.1007/s12602-019-09623-9
  18. Lee HB, Kim KH, Kang GA, Lee KG, Kang SS. 2022. Antibiofilm, antiadhesive and anti-invasive activities of bacterial lysates extracted from Pediococcus acidilactici against Listeria monocytogenes. Foods 11:2948. https://doi.org/10.3390/foods11192948
  19. Li Y, Liu M, Zuo Z, Liu J, Yu X, Guan Y, Zhan R, Han Q, Zhang J, Zhou R, Sun R, Tian Z, Zhang C. 2017. TLR9 regulates the NF-κB-NLRP3-IL-1β pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation. J Immunol 199:761-773. https://doi.org/10.4049/jimmunol.1601416
  20. Maekawa T, Hajishengallis G. 2014. Topical treatment with probiotic Lactobacillus brevis CD2 inhibits experimental periodontal inflammation and bone loss. J Periodontal Res 49:785-791. https://doi.org/10.1111/jre.12164
  21. Moon YJ, Baik SH, Cha YS. 2014. Lipid-lowering effects of Pediococcus acidilactici M76 isolated from Korean traditional makgeolli in high fat diet-induced obese mice. Nutrients 6:1016-1028. https://doi.org/10.3390/nu6031016
  22. Nguyen T, Brody H, Radaic A, Kapila Y. 2021. Probiotics for periodontal health: Current molecular findings. Periodontol 2000 87:254-267. https://doi.org/10.1111/prd.12382
  23. Papagianni M, Anastasiadou S. 2009. Pediocins: The bacteriocins of pediococci. Sources, production, properties and applications. Microb Cell Fact 8:3. https://doi.org/10.1186/1475-2859-8-3
  24. Qi SR, Cui YJ, Liu JX, Luo X, Wang HF. 2020. Lactobacillus rhamnosus GG components, SLP, gDNA and CpG, exert protective effects on mouse macrophages upon lipopolysaccharide challenge. Lett Appl Microbiol 70:118-127. https://doi.org/10.1111/lam.13255
  25. Ramadan DE, Hariyani N, Indrawati R, Ridwan RD, Diyatri I. 2020. Cytokines and chemokines in periodontitis. Eur J Dent 14:483-495. https://doi.org/10.1055/s-0040-1712718
  26. Saini R, Marawar PP, Shete S, Saini S. 2009. Periodontitis, a true infection. J Glob Infect Dis 1:149-150. https://doi.org/10.4103/0974-777X.56251
  27. Seo HJ, Kang SS. 2020. Inhibitory effect of bacteriocin produced by Pediococcus acidilactici on the biofilm formation of Salmonella typhimurium. Food Control 117:107361. https://doi.org/10.1016/j.foodcont.2020.107361
  28. Song YR, Lee CM, Lee SH, Baik SH. 2021. Evaluation of probiotic properties of Pediococcus acidilactici M76 producing functional exopolysaccharides and its lactic acid fermentation of black raspberry extract. Microorganisms 9:1364. https://doi.org/10.3390/microorganisms9071364
  29. Strecker JK, Minnerup J, Gess B, Bernd Ringelstein E, Schabitz WR, Schilling M. 2011. Monocyte chemoattractant protein1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice. PLOS ONE 6:e25863. https://doi.org/10.1371/journal.pone.0025863
  30. Sundararaj KP, Samuvel DJ, Li Y, Sanders JJ, Lopes-Virella MF, Huang Y. 2009. Interleukin-6 released from fibroblasts is essential for up-regulation of matrix metalloproteinase-1 expression by U937 macrophages in coculture: Cross-talking between fibroblasts and u937 macrophages exposed to high glucose. J Biol Chem 284:13714-13724. https://doi.org/10.1074/jbc.M806573200
  31. Thomalla M, Schmid A, Neumann E, Pfefferle PI, Muller-Ladner U, Schaffler A, Karrasch T. 2019. Evidence of an antiinflammatory toll-like receptor 9 (TLR 9) pathway in adipocytes. J Endocrinol 240:325-343. https://doi.org/10.1530/JOE-18-0326
  32. Wang X, Peng H, Huang Y, Kong W, Cui Q, Du J, Jin H. 2020. Post-translational modifications of IκBα: The state of the art. Front Cell Dev Biol 8:574706. https://doi.org/10.3389/fcell.2020.574706
  33. Winning L, Linden GJ. 2015. Periodontitis and systemic disease. BDJ Team 2:15163. https://doi.org/10.1038/bdjteam.2015.163
  34. Xu W, Zhou W, Wang H, Liang S. 2020. Roles of Porphyromonas gingivalis and its virulence factors in periodontitis. Adv Protein Chem Struct Biol 120:45-84. https://doi.org/10.1016/bs.apcsb.2019.12.001
  35. Yoon JW, Kang SS. 2020. In vitro antibiofilm and anti-inflammatory properties of bacteriocins produced by Pediococcus acidilactici against Enterococcus faecalis. Foodborne Pathog Dis 17:764-771. https://doi.org/10.1089/fpd.2020.2804
  36. Zhao J, Feng X, Zhang X, Le K. 2012. Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on secretion of IL1B, IL6, and IL8 by gingival epithelial cells. Inflammation 35:1330-1337. https://doi.org/10.1007/s10753-012-9446-5