Protective Effects of a Novel Lactobacillus brevis Strain with Probiotic Characteristics against Staphylococcus aureus Lipoteichoic Acid-Induced Intestinal Inflammatory Response

Probiotics can effectively modulate host immune responses and prevent gastrointestinal diseases. The objective of this study was to investigate the probiotic characteristics of Lactobacillus brevis KU15152 isolated from kimchi and its protective potential against intestinal inflammation induced by Staphylococcus aureus lipoteichoic acid (aLTA). L. brevis KU15152 exhibited a high survival rate in artificial gastric and bile environments. Additionally, the adhesion capability of the strain to HT-29 cells was higher than that of L. rhamnosus GG. L. brevis KU15152 did not produce harmful enzymes, such as β-glucuronidase, indicating that it could be used as a potential probiotic. The anti-inflammatory potential of L. brevis KU15152 was determined in HT-29 cells. Treatment with L. brevis KU15152 suppressed the production of interleukin-8 without inducing significant cytotoxicity. The downregulatory effects of L. brevis KU15152 were involved in the suppression of nuclear factor-kappa B activation mediated by the extracellular signal-regulated kinase and Akt signaling pathways. Collectively, these data suggest that L. brevis KU15152 can be used in developing therapeutic and prophylactic products to manage and treat aLTA-induced intestinal damage.


Determination of Cell Viability
The viability of HT-29 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with modifications [17]. Briefly, HT-29 cells (2 × 10 5 cells/well) were seeded in 24-well plates and incubated until monolayer formation. The cells were cultured with the bacterial samples for 2 h with or without 50 μg/ml aLTA for another 22 h. MTT solution was added to each well at a final concentration of 0.5 mg/ml. The cells were incubated for 30 min, the supernatant was discarded, and the resulting formazan deposits were dissolved in 1 ml dimethyl sulfoxide. The absorbance of the resultant solution was estimated at 570 nm using Thermo Scientific Multiskan GO (Thermo Scientific, USA). Cell viability was determined as a percentage relative to the absorbance of the control groups.

Measurement of IL-8 Production
The effect of Lactobacillus strains on the production of IL-8 was investigated in aLTA-induced HT-29 cells using an enzyme-linked immunosorbent assay kit (Thermo Fisher Scientific). HT-29 cells (2 × 10 5 cells/well) were plated in 24 well plates and grown until confluent monolayers were formed. The cells were pretreated with the bacterial samples. After incubation for 2 h, cells were incubated with or without 50 μg/ml aLTA for an additional 22 h. Collected cell culture supernatants were diluted appropriately with cell growth media and used to measure the amount of IL-8 according to the manufacturer's instructions.

Western Blotting
Protein expression was determined using western blotting [18]. HT-29 cells (5 × 10 5 cells/well) were plated in 6- well plates and incubated until a monolayer was formed. After 2 h of pretreatment with the bacterial samples, the cells were left untreated or stimulated with aLTA (50 μg/ml) for the indicated time. The cells were rinsed with icecold PBS and lysed with Pro-Prep lysis buffer (iNtRON Biotechnology, Korea) supplemented with protease and phosphatase inhibitor cocktails. The supernatant was collected from the lysates by centrifugation (15,000 ×g, 30 min, 4°C). Cellular proteins (20-30 μg) quantified using the DC Protein Assay Kit (Bio-Rad) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride membrane. The membranes were blocked with 5% skim milk for 1 h and incubated with appropriate primary antibodies at 4°C overnight. Primary antibodies against phosphorylated mitogen-activated protein kinase kinase (p-MEK), extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-Akt, glycogen synthase kinase (GSK) 3β, p-GSK 3β and p65 were purchased from Cell Signaling Technology, Inc. (USA). Other primary antibodies were purchased from Santa Cruz Biotechnology (USA). The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies for 2 h, at room temperature. The enhanced chemiluminescence detection kit (Bio-Rad) was used to detect the protein bands, and the density of the protein bands was quantified using the ImageJ software (National Institutes of Health, USA).

Statistical Analysis
Experimental data are presented as mean ± standard deviation (three replicates). IBM SPSS (Version 18.0, SPSS Inc., USA) was used for the statistical analysis. Statistical differences between two groups were analyzed using the unpaired one-tailed Student's t-test, and those among multiple groups, using one-way analysis of variance and Tukey's HSD test (p < 0.05).

Tolerance to Artificial Digestive Conditions and Adhesion to HT-29 Cells
Resistance to artificial gastric juice and bile salt is necessary for LAB to function as probiotics. The tolerance to artificial digestive conditions is shown in Table 1. The survival rates of LGG and L. brevis KU15152 after exposure to artificial gastric acid (pH 2.5, 0.3% pepsin) for 3 h were 98.35% and 99.36%, respectively. In addition, L. brevis KU15152 exhibited high tolerance to artificial bile salt conditions (0.3% oxgall) with a viability rate of 108.24%. The rate was higher than that of LGG (100.48%).
Adhesion to intestinal epithelial cells is an important criterion for identifying potential probiotic bacteria. The adhesion of LGG and L. brevis KU15152 to HT-29 intestinal cells was investigated ( Table 1). The adhesion rates of the two strains were 12.04% and 12.92%, respectively. The results implicated L. brevis KU15152 as a potential probiotic.

Enzyme Production
The enzyme production of the LAB strains was assessed using the API ZYM kit ( Table 2). Compared to LGG, L. brevis KU15152 did not produce α-galactosidase and N-acetyl-β-glucosaminidase. However, L. brevis KU15152 produced a remarkably higher amount of α-glucosidase, which catalytically mediates the bioconversion process. Additionally, the strain did not produce β-glucuronidase which is considered as a carcinogenic enzyme.

Effect of L. brevis KU15152 on IL-8 Production in Intestinal Cells
To investigate the effect of LTA from S. aureus (aLTA) and Lactobacillus strains with probiotic characteristics on inflammatory responses in intestinal cells, HT-29 cells were stimulated with aLTA in the presence of Lactobacillus strains. When treated with 8 log CFU/ml L. brevis KU15152, viability was 86.97 ± 1.60%. However, none of the tested groups (7 log CFU/ml) showed significant cytotoxicity in HT-29 cells (Fig. 1A). Compared to the aLTAnegative group, treatment with 50 μg/ml aLTA markedly increased the production of IL-8. However, pretreatment with Lactobacillus strains suppressed the production of IL-8 (Fig. 1B). Additionally, L. brevis KU15152 exhibited the most significant downregulation of IL-8 secretion. These findings suggest that L. brevis KU15152 inhibits IL-8 production, compared to L. brevis KU15153 and LGG without adversely affecting cell viability.

Effect of L. brevis KU15152 on NF-κB Activation in Intestinal Epithelial Cells
L. brevis KU15152 suppressed the production of IL-8 in aLTA-stimulated HT-29 cells (Fig. 1). Thus, the effect of Lactobacillus strains on the activation of NF-κB was determined. Cells incubated with 50 μg/ml aLTA showed markedly increased phosphorylation of p65 compared to cells incubated in the absence of aLTA. In contrast, the phosphorylation of p65 was downregulated by pretreatment with the Lactobacillus strains (Fig. 2). In particular, L. brevis KU15152 significantly inhibited the activation of NF-κB in HT-29 cells, indicating that the inhibitory effect of L. brevis KU15152 on aLTA-induced inflammatory response was associated with the downregulation of NF-κB activation.

Effect of L. brevis KU15152 on MEK/ERK Signaling Pathways in HT-29 Cells
To determine the signaling mechanisms related to the effect of L. brevis KU15152 on the inflammatory response in intestinal epithelial cells, the expression levels of the MEK/ERK signaling-related proteins were examined (Fig. 3). Stimulation with aLTA significantly upregulated the expression of phosphorylated MEK and ERK 1/2 in HT-29 cells. However, MEK and ERK 1/2 phosphorylation was alleviated by treatment with L. brevis KU15152 for 20 min.

Effect of L. brevis KU15152 on Akt/GSK 3β Signaling Pathways in HT-29 Cells
IL-8 is reportedly associated with the activation of Akt, which leads to phosphorylation of GSK 3β. As shown in Fig. 4, aLTA stimulation promoted the phosphorylation of Akt. Treatment with L. brevis KU15152 reduced the phosphorylation of Akt induced by aLTA. The aLTA-induced activation of GSK 3β was mitigated by L. brevis KU15152. These findings indicate that L. brevis KU15152 can suppress inflammatory responses in intestinal epithelial cells by regulating the Akt/GSK 3β signaling pathways.

Discussion
Probiotics are used to maintain and improve human health. To function as probiotics, LAB strains should survive the transit through the gastrointestinal tract and adhere to the intestine for a certain period [19]. In the present study, we demonstrated that L. brevis KU15152 had a high survival rate of 99.36% after incubation at pH 2.5 for 3 h. The tolerance to the gastrointestinal conditions of low pH (pH 2.5-3.5) is essential for probiotic strains, because they must go through the stomach [20]. The pH of gastric acid is usually 2.5, which can disrupt the bacterial cell envelope. Bile has a bacteriostatic role within the gut, and its effects are regulated by bile salts. However, bile salts can damage the structure of the bacterial cell membrane and kill bacteria [21]. The present study findings demonstrated the capability of L. brevis KU15152 to survive under condition mimicking bile salt with essentially no decline in viability after incubation in MRS broth with 0.3% oxgall for 24 h. These results are comparable with those of a previous study on the probiotic characteristics of L. brevis strains [15]. Furthermore, bile salt hydrolase produced by Lactobacillus bacteria increases the number of viable bacteria after incubation with bile salts [22]. As previously reported, probiotic organisms compete with pathogens for binding sites in the intestine, and for probiotics, intestinal adhesion is an important criterion [23]. L. brevis KU15152 and LGG exhibited adhesion rates of 12.92% and 12.04%, respectively. Compared with the adhesion abilities of various probiotic strains in a previous study [24], L. brevis KU15152 avidly adhered to intestinal epithelial cells. The strain produced various enzymes but did not produce hazardous enzymes including β-glucuronidase. These results indicate the likelihood that L. brevis KU15152 can survive in the human gastrointestinal tract and function as a probiotic.
LTA from S. aureus stimulates host cells to produce inflammatory cytokines such as IL-8 and tumor necrosis factor-α by the activation of NF-κB [25,26]. LTAs from pathogenic bacteria, including S. aureus, S. epidermidis, and Streptococcus pneumonia could activate macrophages. In contrast, LTAs from probiotics and non-pathogenic bacteria, such as L. plantarum, have weak immunostimulatory effects [27]. In the present study, 50 μg/ml aLTA stimulated the secretion of IL-8 from HT-29 cells following NF-κB activation, and L. brevis KU15152 had an inhibitory effect on this activation. IL-8 is a chemokine that attracts leukocytes, such as basophils and neutrophils, to lesions and is associated with angiogenesis, metastasis, and inflammation [28]. After proinflammatory stimulation, a signaling cascade begins, leading to the translocation of NF-κB into the nucleus. In a previous study, decreased IL-8 production after glutamine pretreatment in human intestinal epithelial cells was accompanied by a reduction in the ubiquitination of IκB-α, cellular inhibitor of NF-κB [29].
In epithelial cells, Chlamydia trachomatis infection upregulates IL-8 secretion through the ERK signaling pathway [30,31]. MEK1 and MEK2 are associated with the regulation of cellular processes [32]. MEK plays a role in the initiation of the MEK/ERK pathway. In addition, ERK 1/2 reacts with various substrates in the cytoplasm and nucleus and activated ERK catalyzes the activation of transcription factors, which results in the expression of several genes related to cell differentiation, proliferation, and apoptosis [33]. In the present study, the expression levels of phosphorylated MEK and ERK 1/2 increased in aLTA-stimulated HT-29 cells compared to those in the aLTA-negative group. L. brevis KU15152 pretreatment downregulated such expression.
The binding of IL-8 to its receptors activates the Akt signaling pathway [34]. Akt can activate B-cell lymphomarelated X protein and decrease mitochondrial membrane translocation and apoptosis [35]. Akt phosphorylates GSK 3β, which is important in cell death. For example, increased phosphorylated GSK 3β can cause apoptosis in neuronal PC12 cells and fibroblasts [36]. Furthermore, ginsenoside Rg1 modulates innate immune responses by regulating the PI3K/Akt pathway [37]. In the present study, pretreatment with L. brevis KU15152 reduced the extent of phosphorylation of Akt and GSK 3β induced by aLTA in intestinal epithelial cells.
In conclusion, this study demonstrates the anti-inflammatory potential and probiotic characteristics of L. brevis KU15152 using the representative intestinal epithelial cells, HT-29. L. brevis KU15152 tolerated artificial gastric acid and bile salts, and avidly adhered to HT-29 cells. L. brevis KU15152 alleviated aLTA-induced IL-8 production by suppressing NF-κB activation. The inhibitory effect of L. brevis KU15152 on intestinal inflammatory response was mediated by the regulation of MEK/ERK and Akt/GSK 3β signaling pathways. Although further studies are required, these findings provide underlying molecular evidence for the protective effects of probiotics on the intestine. The collective findings indicate the potential of L. brevis KU15152 as a functional probiotic product.