Jae Sung Ko, Hye Ran Yang, Ju Young Chang, Jeong Kee Seo, Department of Pediatrics, Seoul National University College of Medicine, Seoul 110-744, Korea

Supported by grant No. 0520050040 from the Seoul National University Hospital Research Fund and  by KT&G Reserach Fund

Correspondence to: Jeong Kee Seo, Department of Pediatrics, Seoul National University Children’s Hospital, 28 Yongon-dong, Chongro-gu, Seoul 110-744, Korea. jkseo@snu.ac.kr

Telephone: +82-2-20723627  Fax: +82-2-7433455

Received: 2007-02-05          Accepted: 2007-03-19

Abstract

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

METHODS: Caco-2 cells were incubated with TNF-a 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-IkB-a.

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

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

© 2007 The WJG Press. All rights reserved.

Key words: Lactobacillus plantarum; Tumor necrosis factor-a; Epithelial barrier; Interleukin-8; ERK; IkB-a

Ko JS, Yang HR, Chang JY, Seo JK. Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-a. World J Gastroenterol 2007; 13(13): 1962-1965

 http://www.wjgnet.com/1007-9327/13/1962.asp

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-a (TNF-a) is a proinflamma-tory cytokine and plays a central role in intestinal inflammation in Crohn disease. TNF-a 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-a^[7]. Treatment with anti-TNF-a 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-a. 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-a in human intestinal epithelial cells^[13-15]. TNF-a-stimulated IL-8 secretion by intestinal epithelial cells is mediated by extracellular signal-regulated kinase (ERK) and nuclear factor kB (NF-kB)^[16].

The aim of this study was to determine whether L. plantarum reverses the deleterious effects of TNF-a on intestinal epithelial cells. We performed an in vitro study in which Caco-2 cells were treated with TNF-a alone or with TNF-a plus L. plantarum. We investigated the effect of L. plantarum on TNF-a-induced alteration of epithelial barrier function, IL-8 production, and ERK/NF-kB 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 mg/mL streptomycin, 1% nonessential amino acids, and 4 mmol/L glutamine. Cultures were maintained at 37℃ 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℃ 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 mm pores; Corning Incorporated, Acton, MA, USA). Caco-2 monolayers with epithelial resistance greater than 500 Wcm² were used, and
L. plantarum was added apically to the polarized monolayers. TNF-a (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 Wcm² after subtracting mean values for resistance obtained from cell-free inserts.

ELISA for IL-8 measurement

TNF-a (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-a (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℃. 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-IkB-a (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-a-induced epithelial barrier dysfunction, Caco-2 cells were basolaterally incubated with TNF-a alone or with TNF-a plus L. plantarum, which was administered apically. Transepithelial electrical resistance was monitored for 36 h. The monolayer resistance of TNF-a treated cells did not change until 12 h had elapsed. TNF-a caused a decline in transepithelial resistance 24 h after treatment. L. plantarum inhibited TNF-a-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-a-stimulated Caco-2 cells was thus preserved by L. plantarum.

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-a. IL-8 concentrations in media of Caco-2 cells cultured with
L. plantarum were not significantly different from those of the controls. When TNF-a (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-a was cocultured with L. plantarum (Figure 2). These data showed that L. plantarum inhibited TNF-a-induced IL-8 secretion.

Western blots of ERK and IkB-a

The effect of L. plantarum on TNF-a–induced ERK pathway activity was investigated. Treatment of Caco-2 cells with TNF-a 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-a-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-a-induced activation of the ERK pathway (Figure 3).

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

DISCUSSION

Ma et al^[17] demonstrated that TNF-a 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-a-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-a-induced IL-8 secretion was inhibited by L. plantarum. This indicates that L. plantarum attenuates the epithelial inflammatory response to TNF-a. McCracken et al^[19] showed that L. plantarum decreased TNF-a-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-a-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-a-stimulated IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism^[16]. Yan et al showed that L. GG prevents cytokine-induced apoptosis in intestinal epithelial cells by inhibition of TNF-a-induced p38 MAP kinase activation^[21]. Jijon et al demonstrated that VSL3 inhibits IL-8 secretion and reduces p38 MAP kinase activation^[22]. The effect of L. plantarum on TNF-a-stimulated ERK activation had not been investigated. We demonstrated that L. plantarum inhibited ERK activation in TNF-a-treated intestinal epithelial cells. ERK signaling is involved in IL-8 production because ERK inhibitors attenuate IL-8 secretion induced by TNF-a^[23]. In our study,
L. plantarum inhibited TNF-a-induced ERK activation, suggesting that L. plantarum may inhibit IL-8 secretion, at least partially, through the ERK pathway. NF-kB regulates IL-8 transcription, and some lactobacilli have been shown to inhibit TNF-a-induced NF-kB translocation to the nucleus and IkB-a degradation^[13,14]. We also showed that
L. plantarum inhibited the degradation response of IkB-a to TNF-a. In contrast, L. GG did not affect TNF-a-induced ERK activation or IkB-a 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-kB and MAP kinase pathways. A TNF-a-induced increase in intestinal tight junction permeability was shown to be mediated by NF-kB 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-kB 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 IkB-a degradation in TNF-a-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.

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S- Editor  Liu Y    L- Editor  Alplini GD    E- Editor  Chin GJ