Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α

Abstract

AIM: To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-α (TNF-α) on intestinal epithelial cells.

METHODS: Caco-2 cells were incubated with TNF-α alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 (IL-8) secretion by intestinal epithelial cells was measured using an ELISA. Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase (ERK), anti-phospho-ERK and anti-IκB-α.

RESULTS: A TNF-α-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF-α-induced IL-8 secretion was reduced by L. plantarum. L. plantarum inhibited the activation of ERK and the degradation of IκB-α in TNF-α-treated Caco-2 cells.

CONCLUSION: Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-α is inhibited by L. plantarum. Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.

Key Words: Lactobacillus plantarum, Tumor necrosis factor-α, Epithelial barrier, Interleukin-8, ERK, IκB-α

INTRODUCTION

Probiotics are defined as living microorganisms that exert beneficial effects on human health[1]. They are effective in shortening the duration of infectious diarrhea in children, and preventing antibiotics-associated diarrhea[2,3]. Probiotics have been shown to prevent a relapse of postoperative pouchitis in ulcerative colitis[4].

Tumor necrosis factor-α (TNF-α) is a proinflamma-tory cytokine and plays a central role in intestinal inflammation in Crohn disease. TNF-α levels in serum, stool and intestinal tissues are elevated in patients with Crohn’s disease[5,6]. In Crohn’s disease, the elevation in epithelial permeability of the ileal mucosa may be mediated by TNF-α[7]. Treatment with anti-TNF-α antibody is effective in cases of intractable Crohn’s disease[8].

As disturbance of the intestinal microflora plays an important role in the pathogenesis of murine experimental colitis and human inflammatory bowel disease[3], probiotics have been used to modify the bacterial flora of the gut. Lactobacillus plantarum is isolated from Kimchi, a traditional Korean food made from fermented vegetables[9]. L. plantarum attenuates intestinal inflammation in the interleukin (IL) 10 gene-deficient mouse model, which spontaneously develops enterocolitis[10].

The mechanisms of action of probiotics include improvement of epithelial barrier function and immunoregulatory effects[11]. Each probiotic species may have an individual mechanism of action. The combination probiotic, VSL3 contains L. plantarum and enhances human intestinal epithelial barrier function[12]. Intestinal epithelial cells release potent neutrophil attractant chemokines such as IL-8 when stimulated by TNF-α. Secretion of IL-8 by epithelial cells has been suggested to be important in the pathogenesis of inflammatory bowel diseases, because IL-8 induces migration of inflammatory cells into the mucosa. Some lactobacilli inhibit the induction of IL-8 production by TNF-α in human intestinal epithelial cells[13-15]. TNF-α-stimulated IL-8 secretion by intestinal epithelial cells is mediated by extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB)[16].

The aim of this study was to determine whether L. plantarum reverses the deleterious effects of TNF-α on intestinal epithelial cells. We performed an in vitro study in which Caco-2 cells were treated with TNF-α alone or with TNF-α plus L. plantarum. We investigated the effect of L. plantarum on TNF-α-induced alteration of epithelial barrier function, IL-8 production, and ERK/NF-κB pathway dynamics.

MATERIALS AND METHODS

Cell lines

Caco-2 cells, an established cell line model for mature differentiated enterocytes, were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured in 25 mmol/L glucose-Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 1% nonessential amino acids, and 4 mmol/L glutamine. Cultures were maintained at 37°C in an incubator containing an atmosphere of 5% CO₂. Cells were used within 14 d of seeding or within five days of confluency. The Caco-2 cell culture medium was replaced with antibiotic-free culture medium 24 h before experiments.

Probiotics

L. plantarum (ATCC 8014) was incubated in Lactobacillus MRS broth at 37°C for 24 h, then diluted in MRS broth to a density of 0.5 absorbance units at a wavelength of 600 nm. Then, 1 × 10⁷ colony-forming units of L. plantarum per mL were added at a multiplicity of 10:1 to the Caco-2 cells. Untreated cells were used as controls in all experiments.

Electrical resistance measurements

Caco-2 cells were grown as polarized monolayers on 6.5 mm transwell plates (0.4 μm pores; Corning Incorporated, Acton, MA, USA). Caco-2 monolayers with epithelial resistance greater than 500 Ωcm² were used, and L. plantarum was added apically to the polarized monolayers. TNF-α (10 ng/mL) was simultaneously added to the basolateral side of the cell monolayers. Electrical resistance across the monolayers was measured at various times using an epithelial volt-ohm meter (World Precision Instruments, Sarasota, FL, USA). Measurements were expressed in Ωcm² after subtracting mean values for resistance obtained from cell-free inserts.

ELISA for IL-8 measurement

TNF-α (10 ng/mL) and L. plantarum were added simultaneously to Caco-2 cells and incubated for 5 h. Culture medium was collected and centrifuged for 10 min to pellet residual bacteria. The supernatant was collected for determination of IL-8 concentration using an ELISA (Pierce, Rockford, IL, USA). Cytokine concentrations were determined using 96-well plates as described by the manufacturer.

Western blotting

TNF-α (10 ng/mL) and L. plantarum were added simultaneously to Caco-2 cells. The treated and untreated cells were washed with PBS and scraped into cell lysis buffer (20 mmol/L HEPES, 0.1% SDS, 1% Triton X-100, phosphatase inhibitor and protease inhibitor cocktail). Thirty minutes after treatment, the lysate was centrifuged at 15 000 r/min for 15 min at 4°C. The protein content of the supernatant was determined using Bio-Rad DC reagents (Bio-Rad, Hercules, CA, USA). For western blotting, equal amounts of cellular lysate protein were mixed with Laemmli sample buffer and separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes, which were blocked and then immunoblotted with anti-phospho-ERK, anti-ERK and anti-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blot was then developed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence.

Statistical analysis

All data are expressed as means ± SD. Data comparisons were made with Student’s t test. Differences were considered significant at P < 0.05.

RESULTS

Transepithelial electrical resistance

To determine the effect of L. plantarum on TNF-α-induced epithelial barrier dysfunction, Caco-2 cells were basolaterally incubated with TNF-α alone or with TNF-α plus L. plantarum, which was administered apically. Transepithelial electrical resistance was monitored for 36 h. The monolayer resistance of TNF-α treated cells did not change until 12 h had elapsed. TNF-α caused a decline in transepithelial resistance 24 h after treatment. L. plantarum inhibited TNF-α-induced decrease in transepithelial electrical resistance at 24 h and 36 h after treatment (P < 0.05) (Figure 1). The epithelial barrier function of TNF-α-stimulated Caco-2 cells was thus preserved by L. plantarum.

Figure 1 Effect of L. plantarum on transepithelial resistance. TNF-α decreased Caco-2 monolayer resistance. L. plantarum reversed TNF-α-induced decreases in transepithelial resistance. ^aP < 0.05, compared with TNF-α. TER: transepithelial electrical resistance; LP: L. plantarum; TNF, TNF-α.

IL-8 induction

The secretion of IL-8 into culture medium was measured to determine the effect of L. plantarum on the inflammatory response of Caco-2 cells to TNF-α. IL-8 concentrations in media of Caco-2 cells cultured with L. plantarum were not significantly different from those of the controls. When TNF-α (10 ng/mL) was incubated with the cells for 5 h, IL-8 secretion was increased to 41.5 ± 7.2 pg/mL. IL-8 secretion was reduced to 9.5 ± 2.1 pg/mL (P < 0.05) when TNF-α was cocultured with L. plantarum (Figure 2). These data showed that L. plantarum inhibited TNF-α-induced IL-8 secretion.

Figure 2 Effect of L. plantarum on TNF-α-induced IL-8 secretion by Caco-2 cells. TNF-α-induced IL-8 secretion was significantly reduced by L. plantarum. ^aP < 0.05, compared with TNF-α. LP: L. plantarum; TNF, TNF-α.

Western blots of ERK and IκB-α

The effect of L. plantarum on TNF-α–induced ERK pathway activity was investigated. Treatment of Caco-2 cells with TNF-α induced phosphorylation of ERK-1 and ERK-2. The amount of p-ERK in L. plantarum-treated cells was not significantly different from that of the control. Phosphorylation of ERK-1 and ERK-2 in TNF-α-treated cells was decreased by L. plantarum. Nonphosphorylated forms of ERK showed the presence of same amounts of these proteins. L. plantarum thus inhibited TNF-α-induced activation of the ERK pathway (Figure 3).

Figure 3 Effect of L. plantarum on the ERK pathway. Caco-2 cells were incubated with L. plantarum, TNF-α or L. plantarum plus TNF-α. Cell lysates were immunoblotted with antibodies against phosphorylated ERK and total ERK. L. plantarum inhibited TNF-α-induced activation of ERK-1 and -2. LP: L. plantarum; TNF, TNF-α.

To study the effect of L. plantarum on the NF-κB pathway, the level of IκB-α was determined using western blotting. NF-κB activation involves the phosphorylation of IκB-α and subsequent degradation of IκB-α, resulting in the translocation of NF-κB to the nucleus. Treatment with TNF-α caused degradation of IκB-α. Coincubation with TNF-α and L. plantarum inhibited TNF-α-induced degradation of IκB-α (Figure 4).

Figure 4 Effect of L. plantarum on IκB-α degradation. Caco-2 cells were incubated with L. plantarum, TNF-α or L. plantarum plus TNF-α. Cell lysates were immunoblotted with antibodies against IκB-α. TNF-α caused degradation of IκB-α. L. plantarum inhibited TNF-α-induced IκB-α degradation. LP: L. plantarum; TNF, TNF-α.

DISCUSSION

Ma et al[17] demonstrated that TNF-α decreases transepi-thelial electrical resistance of Caco-2 cells after 24 and 48 h. We also observed a decrease in transepithelial electrical resistance after 24 h. We showed that the TNF-α-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. Saccharomyces boulardii prevented a decrease in transepithelial electrical resistance in enteropathogenic E. coli-infected T84 cells[18]. Intestinal mucosal permeability is decreased by VSL3 in IL-10 gene-deficient mice[12]. All these findings support the contention that probiotics enhance epithelial barrier function.

In our study, TNF-α-induced IL-8 secretion was inhibited by L. plantarum. This indicates that L. plantarum attenuates the epithelial inflammatory response to TNF-α. McCracken et al[19] showed that L. plantarum decreased TNF-α-induced IL-8 secretion in HT-29 cells in which IL-8 mRNA levels were elevated. In contrast, Lactobacillus reuteri and L. GG inhibited TNF-α-induced IL-8 secretion and IL-8 mRNA expression[14,15]. The level of IL-8 expression is correlated with disease activity in patients with inflammatory bowel disease. A number of Lactobacillus and Bifidobacterium species, including L. plantarum[10], L. reuteri[19], VSL3[12], L. salivarius and B. infantis[20], attenuate experimental colitis in IL-10 knockout mice.

ERK and p38 mitogen-activated protein (MAP) kinase contribute to TNF-α-stimulated IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism[16]. Yan et al[21] showed that L. GG prevents cytokine-induced apoptosis in intestinal epithelial cells by inhibition of TNF-α-induced p38 MAP kinase activation. Jijon et al[22] demonstrated that VSL3 inhibits IL-8 secretion and reduces p38 MAP kinase activation. The effect of L. plantarum on TNF-α-stimulated ERK activation had not been investigated. We demonstrated that L. plantarum inhibited ERK activation in TNF-α-treated intestinal epithelial cells. ERK signaling is involved in IL-8 production because ERK inhibitors attenuate IL-8 secretion induced by TNF-α[23]. In our study, L. plantarum inhibited TNF-α-induced ERK activation, suggesting that L. plantarum may inhibit IL-8 secretion, at least partially, through the ERK pathway. NF-κB regulates IL-8 transcription, and some lactobacilli have been shown to inhibit TNF-α-induced NF-κB translocation to the nucleus and IκB-α degradation[13,14]. We also showed that L. plantarum inhibited the degradation response of IκB-α to TNF-α. In contrast, L. GG did not affect TNF-α-induced ERK activation or IκB-α degradation[21]. Probiotics may exert anti-inflammatory responses by modifying the signal transduction pathway. The mechanisms involved may depend on the species of probiotics.

Epithelial barrier functions are modulated by the NF-κB and MAP kinase pathways. A TNF-α-induced increase in intestinal tight junction permeability was shown to be mediated by NF-κB activation[17]. The increase in transepithelial resistance induced by VSL3 is mediated in part via the ERK pathway[24]. The effect of L. plantarum on monolayer resistance appears to be mediated by NF-κB and the ERK pathway. Although in vitro models are useful for evaluating mechanisms by which probiotics exert beneficial effects and provide a rationale for the therapeutic use of probiotics, the beneficial health effects of probiotics should also be determined by double- blinded placebo-controlled trials.

In summary, L. plantarum inhibits epithelial barrier dysfunction, IL-8 secretion, ERK activation, and IκB-α degradation in TNF-α-stimulated Caco-2 cells. Our findings suggest that probiotics may preserve epithelial barrier function and inhibit the inflammatory response by affecting the signal transduction pathway in human intestinal epithelium.