Molecular Immunology 64 (2015) 183–189

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Lipoteichoic acid from Lactobacillus plantarum inhibits Pam2CSK4-induced IL-8 production in human intestinal epithelial cells Su Young Noh a,1 , Seok-Seong Kang a,1 , Cheol-Heui Yun b , Seung Hyun Han a,∗ a Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea b Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea

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Article history: Received 25 September 2014 Received in revised form 13 November 2014 Accepted 15 November 2014 Available online 4 December 2014 Keywords: Lactobacillus plantarum Pam2CSK4 Lipoteichoic acid Interleukin-8 Intestinal epithelial cells

a b s t r a c t Lactobacilli are probiotic bacteria that are considered to be beneficial in the gastrointestinal tract of humans. Although lactobacilli are well known to alleviate intestinal inflammation, the molecular basis of this phenomenon is poorly understood. In this study, we investigated the effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA), which is a major cell wall component of this species, on the production of interleukin (IL)-8 in human intestinal epithelial Caco-2 cells. Treatment with Pam2CSK4, a synthetic lipopeptide that is known to mimic Gram-positive bacterial lipoproteins as an important virulence factor, significantly induced IL-8 expression in Caco-2 cells. However, neither heat-inactivated L. plantarum nor L. plantarum peptidoglycan inhibited Pam2CSK4-induced IL-8 mRNA expression. In addition, both a deacylated form and a dealanylated form of Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, indicating that the lipid and D-alanine moieties are critical for Lp.LTA-mediated inhibition. Moreover, Lp.LTA inhibited Pam2CSK4-induced activation of p38 kinase, JNK, and NF-␬B transcription factor by suppressing toll-like receptor 2 activation. Collectively, these results suggest that Lp.LTA exerts antiinflammatory effects on human intestinal epithelial cells by blocking IL-8 production. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The gut microflora is composed of approximately 1014 bacteria, which are mainly distributed in the large intestine, and can be considered to be a functional human organ (O‘Hara and Shanahan, 2006). More than 400 different bacterial species are estimated to exist as commensal microorganisms that constantly interact with the intestinal epithelium, thereby influencing the health and physiology of the host (Mackie et al., 1999). Commensal bacteria actively promote epithelial barrier integrity by regulating tight junctions and also protect intestinal epithelial cells from injury by controlling their proliferation (O‘Hara and Shanahan, 2006). In addition, commensal bacteria are involved in diverse metabolic functions,

∗ Corresponding author at: Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. Tel.:+82 2 740 8641; fax: +82 2 743 0311. E-mail address: [email protected] (S.H. Han). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2014.11.014 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

including the synthesis and absorption of nutrients and metabolites such as vitamins, bile acids, and short-chain fatty acids (Brestoff and Artis, 2013; O‘Hara and Shanahan, 2006). An increasing body of evidence indicates that commensal bacteria, in particular Lactobacillus and other probiotic bacteria, play an important role in the development of the host immune system (Corr et al., 2007; Sheil et al., 2007). Bacteria of the Lactobacillus genus predominate in the gastrointestinal tract, where they exert a number of beneficial effects including maintenance of the microbial ecosystem and protection against microbial infection (Henderson et al., 2012; Hirano et al., 2003). Lactobacillus bacteria are well established to exert positive effects on the prevention and treatment of intestinal inflammatory diseases such as inflammatory bowel disease (Matsumoto et al., 2005; Sheil et al., 2007). In addition, certain Lactobacillus strains have been shown to prevent the adhesion and internalization of Shigella, and to inhibit the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-␣ and interleukin (IL)-8 in intestinal epithelial cells (Moorthy et al., 2010). More recently, treatment with Lactobacillus paracasei was reported to remarkably decrease the expression of TNF-␣ and

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IL-8 and, instead, to increase the expression of anti-inflammatory cytokines such as transforming growth factor-␤ in human gut dendritic cells (Bermudez-Brito et al., 2012). Lipoteichoic acid (LTA) is a major cell wall component of lactobacilli and other Gram-positive bacteria (Wang et al., 2003). Recent studies have convincingly shown that Lactobacillus LTA is associated with immunomodulation in the intestine. For example, D-alanine-depleted LTA from Lactobacillus plantarum has been shown to reduce pro-inflammatory responses, thereby attenuating colitis (Grangette et al., 2005). In contrast, another study found that D-alanine-depleted L. rhamnosus GG LTA did not considerably affect cytokine production in intestinal epithelial cells (Perea Velez et al., 2007). Another major cell wall component of Lactobacillus, peptidoglycan, has been shown to mitigate inflammatory responses by upregulating IL-10 production in the colon, thereby conferring protection against colitis (Macho Fernandez et al., 2011). Although cell wall-associated molecules of Lactobacillus do not ubiquitously attenuate intestinal inflammatory responses, these molecules may exert immunomodulatory effects on intestinal inflammation. A number of Lactobacillus strains can attenuate disease and intestinal inflammation, making these strains good probiotic agents. However, the immunomodulatory activities of different lactobacilli strains and species vary considerably (Bron et al., 2013). Recently, cell wall-associated molecules have been proposed to be effector molecules that mediate the beneficial effects of lactobacilli (Lee et al., 2013). Nevertheless, the precise molecular mechanisms by which these lactobacilli cell wall-associated molecules exert their effects in the intestine have not yet been clearly elucidated. Therefore, in this study, we investigated the potential of Lactobacillus LTA to attenuate pro-inflammatory responses in human intestinal epithelial cells.

species, including L. sakei, L. delbrueckii, and L. rhamnosus GG, were kindly provided by Prof. Dae Kyun Chung (Kyung Hee University, Suwon, Korea).

2.3. Cell culture The human intestinal epithelial Caco-2 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in complete Dulbecco’s modified Eagle medium (DMEM) (HyClone, Logan, Utah, USA) containing 10% fetal bovine serum (GIBCO, Burlington, ON, Canada), 100 U/ml of penicillin, and 100 ␮g/ml of streptomycin (HyClone) at 37 ◦ C in a 5% CO2 atmosphere in a humidified incubator. Chinese hamster ovary (CHO) cells overexpressing human CD14 and TLR2 (CHO/CD14/TLR2 cells) (Medvedev et al., 2001) were maintained in complete Ham’s F-12 nutrient mixture (HyClone) containing 1 mg/ml of G418 (Invitrogen, Grand Island, NY, USA) and 400 U/ml of hygromycin (Invitrogen) at 37 ◦ C in a 5% CO2 , humidified incubator. To polarize Caco-2 cells, they were cultured on polycarbonate transwell-permeable filters (0.4 ␮m pore size; Costar, Corning, NY, USA) for 16 days. Cell polarization was confirmed by measuring the trans-epithelial electrical resistance (>450 /cm2 ) with an EVOM2 epithelial volt-ohm meter (World Precision Instruments, Sarasota, FL, USA). Since fully-differentiated Caco-2 monolayer cells exhibit tight junctions and brush border on the apical surface with similar physiological characteristics to human intestinal epithelium (Meunier et al., 1995), the cells have been widely used as a representative model for human primary intestinal enterocytes (Artursson et al., 2001; Hidalgo et al., 1989). Therefore, Caco-2 cells were cultured for 21 days until the cells were fully differentiated under the same conditions as described above.

2. Materials and methods 2.1. Bacteria and reagents L. plantarum KCTC 10887BP and Streptococcus mutans KCTC 3065 strains were obtained from the Korean Collection for Type Culture (Daejeon, Korea). Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 strains were purchased from the American Type Culture Collection (Manassas, VA, USA). Heat-inactivated L. plantarum was prepared as previously described (Ryu et al., 2009). Pam2CSK4 was purchased from EMC Microcollections (Tuebingen, Germany). Escherichia coli lipopolysaccharide (LPS), poly I:C, and muramyl dipeptide (MDP) were purchased from InvivoGen (San Diego, CA, USA). L-Ala-␥-D-Glu-meso-diaminopimelic acid (tri-DAP) was obtained from Anaspec (Fremont, CA, USA). Antibodies specific for p38 kinase, phospho-p38 kinase, JNK, phospho-JNK, ERK, phospho-ERK, and I␬B␣, in addition to HRP-conjugated anti-rabbit IgG antibodies, were obtained from Cell Signaling Technology (Beverly, MA, USA). FITC-conjugated anti-CD25 monoclonal antibodies were purchased from BioLegend (San Diego, CA, USA). 2.2. Preparation of LTA LTAs were prepared from L. plantarum, S. mutans, S. aureus, and E. faecalis. The presence of biologically-active molecules such as endotoxins, nucleic acids, and proteins in the LTA preparations was examined as previously described (Ryu et al., 2009). Dealanylated Lp.LTA, which is D-alanine-deficient in its repeating units, and deacylated Lp.LTA, which is acyl chain-deficient, were prepared by incubating native Lp.LTA in 0.1 M Tris-HCl at pH 8.5 for 24 h and in 0.5 N NaOH for 2 h, respectively. Thin-layer chromatography was conducted to confirm the complete depletion of alanines and lipids, respectively (data not shown). LTAs from other Lactobacillus

2.4. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized by mixing total RNA (1 ␮g) with random hexamers (Promega, Madison, WI, USA) and reverse transcriptase (Promega). Amplification of cDNA was performed by PCR in a total volume of 20 ␮l. Reactions contained rTaq DNA polymerase (0.5 units) and 10 picomoles of primers specific for IL-8 (forward primer: 5 -TCT GCA GCT CTG TGT GAA GG-3 , reverse primer: 5 TGA ATT CTC AGC CCT CTT CAA-3 ) and ␤-actin (forward primer: 5 -GTG GGG CGC CCC AGG CAC CA-3 , reverse primer: 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 ). PCR amplification of IL-8 was conducted as follows: an initial denaturation step at 94 ◦ C for 5 min; 32 cycles of 94 ◦ C for 40 s, 60 ◦ C for 40 s, and 72 ◦ C for 40 s; and a final extension step at 72 ◦ C for 7 min. PCR amplification of ␤-actin was conducted as follows: an initial denaturation step at 95 ◦ C for 5 min; 25 cycles of 95 ◦ C for 1 min, 55 ◦ C for 1 min, and 72 ◦ C for 1 min; and a final extension step at 72 ◦ C for 10 min. PCR products were resolved by electrophoresis on 1.5% agarose gels and visualized by staining with ethidium bromide.

2.5. Enzyme-linked immunosorbent assay (ELISA) Caco-2 cells (4 × 105 cells/ml) were seeded in 96-well culture plates and grown until fully confluent. The cells were incubated in serum-free DMEM for 18 h and then treated with the indicated stimuli for an additional 24 h. After treatment, the cell culture supernatants were collected and the production of IL-8 was determined using a commercial IL-8 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

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Fig. 1. Lp.LTA inhibits Pam2CSK4-induced IL-8 expression in Caco-2 cells. (A) Caco-2 cells were treated with Lp.LTA (30 ␮g/ml), E. coli LPS (0.1 ␮g/ml), Pam2CSK4 (0.1 ␮g/ml), poly I:C (10 ␮g/ml), tri-DAP (1 ␮g/ml), or MDP (1 ␮g/ml) for 1 h. (B) The cells were treated with Pam2CSK4 (0.1 ␮g/ml) in the absence or presence of Lp.LTA (1, 3, 10, or 30 ␮g/ml) for 1 h. Then, total RNA was extracted and IL-8 mRNA expression was determined by RT-PCR. Results shown are representative of three independent experiments. (C) The cells were treated with Pam2CSK4 (0.1 ␮g/ml) in the absence or presence of Lp.LTA (10 or 30 ␮g/ml) for 24 h. The culture supernatants were then collected and the amounts of secreted IL-8 were measured by ELISA. (D) Polarized Caco-2 cells were apically treated with Lp.LTA (30 ␮g/ml) and/or Pam2CSK4 (0.1 ␮g/ml) for 24 h. Then, the culture supernatants from the apical compartment were collected, and the amounts of secreted IL-8 were determined by ELISA. Data are expressed as mean values ± standard deviations of triplicate samples. Asterisk (*) and (**) indicate statistically significant differences (P < 0.05 and P < 0.01, respectively) compared with nontreated cells or Pam2CSK4-treated cells. NT; non-treated.

2.6. Flow cytometric analysis

2.8. Statistical analysis

To examine whether Lp.LTA affects Pam2CSK4-induced TLR2 activation, CHO/CD14/TLR2 cells (4 × 105 cells/ml) were incubated with Pam2CSK4 (0.1 ␮g/ml) in the absence or presence of Lp.LTA (30 ␮g/ml) for 24 h. The cells were then stained with FITCconjugated anti-CD25 monoclonal antibodies. TLR2 activation was determined using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

All data are expressed as mean values ± standard deviations from triplicate samples. Statistical analysis was performed using Student’s t-test by comparing treatment groups with an appropriate control. 3. Results 3.1. Lp.LTA inhibits Pam2CSK4-induced IL-8 expression in Caco-2 cells

2.7. Western blot analysis Caco-2 cells (4 × 105 cells/ml) were seeded in 6-well culture plates and grown until fully confluent. The cells were incubated in serum-free DMEM for 18 h and then treated with Pam2CSK4 (0.1 ␮g/ml) in either the absence or presence of Lp.LTA (30 ␮g/ml) for 30 min. Cells were then lysed in PRO-PREPTM buffer (iNtRON Biotechnology, Seongnam, Korea). Equal amounts of protein were resolved on 10% SDS-PAGE gels and then electrotransferred onto PVDF membranes (Millipore; Bedford, MA, USA). After blocking with 5% skim milk, membranes were probed with primary antibodies against MAP kinases, phosphorylated MAP kinases, or I␬B␣. After washing, membranes were incubated with HRPconjugated secondary antibodies. Immunoreactive bands were visualized using a Luminescent Image Analyzer (Fuji Film, Japan).

Intestinal epithelial cells respond to microbial components not as strongly as macrophages or dendritic cells (Philpott et al., 2001). To initially identify the microbial components and derivatives thereof that induce IL-8 expression in Caco-2 cells, the cells were treated with E. coli LPS, Lp.LTA, Pam2CSK4, poly I:C, triDAP, or MDP. Among all microbial components and derivatives tested, only Pam2CSK4 significantly induced IL-8 gene expression in Caco-2 cells (Fig. 1A). Thus, Pam2CSK4 was used in all subsequent experiments. Next, we investigated whether Lp.LTA inhibits Pam2CSK4-induced IL-8 gene expression in Caco-2 cells. As shown in Fig. 1B, Lp.LTA inhibited Pam2CSK4-induced IL-8 gene expression in a dose-dependent manner. To confirm the reduction of IL-8 production on the protein level, Caco-2 cells were treated with Pam2CSK4 in the presence of Lp.LTA and the amount of secreted

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Fig. 2. Lp.LTA is the most effective bacterial LTA for inhibiting Pam2CSK4-induced IL-8 expression in Caco-2 cells. (A) Caco-2 cells were treated with 30 ␮g/ml of various Lactobacillus LTAs, including L. plantarum LTA (Lp.LTA), L. sakei LTA (Ls.LTA), L. delbrueckii LTA (Ld.LTA), or L. rhamnosus GG LTA (Lg.LTA) in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 1 h. Then, total RNA was extracted and IL-8 mRNA expression was quantitated by RT-PCR. The results shown are representative of experiments performed in triplicate. (B) The cells were treated with 30 ␮g/ml of S. aureus LTA (Sa.LTA), E. faecalis LTA (Ef.LTA), S. mutans LTA (Sm.LTA), or Lp.LTA in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 24 h. Then, the cell culture supernatants were collected and the amounts of secreted IL-8 were determined by ELISA. Data are expressed as mean values ± standard deviations of triplicate samples. Asterisk (*) and (**) indicate statistically significant differences (P < 0.05 and P < 0.01, respectively) compared with control cells. NT; non-treated.

IL-8 was measured by ELISA. Consistent with RT-PCR results, Lp.LTA inhibited Pam2CSK4-induced IL-8 secretion (Fig. 1C). Since 30 ␮g/ml of Lp.LTA was the most effective concentration for inhibiting Pam2CSK4-induced IL-8 expression, this concentration was used in all subsequent experiments. Polarized Caco-2 cells provide a more physiologically and morphologically relevant in vitro model of mature intestinal enterocytes (Sambuy et al., 2005). Thus, Caco2 cells were cultured in transwell-permeable filters for 16 days to achieve polarization. As shown in Fig. 1D, Lp.LTA also significantly inhibited Pam2CSK4-induced IL-8 production in polarized Caco-2 cells (Fig. 1D). 3.2. Lp.LTA is a more potent inhibitor of Pam2CSK4-induced IL-8 expression in Caco-2 cells than LTAs from other bacteria We next determined whether inhibition of Pam2CSK4-induced IL-8 expression in Caco-2 cells is a common feature of LTAs from various Lactobacillus strains. As shown in Fig. 2A, LTAs from L. delbrueckii and L. rhamnosus GG, but not from L. sakei, slightly inhibited Pam2CSK4-induced IL-8 expression; however, this inhibition was not statistically significant. In contrast, Lp.LTA strongly suppressed

Lp.LTA 1 10 (30 µg/ml) Lp.PGN without WTA (µg/ml)

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Fig. 3. Neither L. plantarum whole cell nor its peptidoglycan inhibits Pam2CSK4induced IL-8 expression in Caco-2 cells. (A) Caco-2 cells were treated with heat-inactivated L. plantarum (105 to 108 CFU/ml) in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 1 h. (B) The cells were treated with Pam2CSK4 (0.1 ␮g/ml), Lp.LTA (30 ␮g/ml), or L. plantarum peptidoglycan (Lp.PGN), either with or without wall teichoic acid (WTA) (1 or 10 ␮g/ml) for 1 h. Then, total RNA was extracted and IL-8 mRNA expression was quantitated by RT-PCR. The results shown are representative of experiments performed in triplicate. Asterisk (**) indicates a statistically significant difference (P < 0.01) compared with the control. NT; non-treated.

IL-8 expression in Caco-2 cells, indicating that inhibition of IL-8 expression in Caco-2 cells is not a general feature of all Lactobacillus LTAs. To examine whether LTAs from pathogenic Gram-positive bacteria inhibit Pam2CSK4-induced IL-8 expression, Caco-2 cells were co-treated with Pam2CSK4 and LTAs from S. aureus, E. faecalis, or S. mutans. In contrast to the Lactobacillus LTAs, the LTAs from pathogenic bacteria inhibited Pam2CSK4-induced IL-8 expression. However, Lp.LTA was a more potent inhibitor of IL-8 expression compared with the LTAs from pathogenic bacteria (Fig. 2B).

3.3. Both whole cells and peptidoglycans of L. plantarum fail to inhibit Pam2CSK4-induced IL-8 expression in Caco-2 cells Certain Lactobacillus strains have been reported to attenuate intestinal inflammation (Madsen et al., 1999). Accordingly, we examined whether whole L. plantarum cells attenuated Pam2CSK4induced IL-8 expression in Caco-2 cells. Various concentrations (105 to 108 CFU/ml) of heat-inactivated L. plantarum bacteria did not inhibit Pam2CSK4-induced IL-8 expression in Caco-2 cells (Fig. 3A). To examine whether peptidoglycan, another major cell wall component of L. plantarum, is involved in the attenuation of Pam2CSK4-induced IL-8 expression, Caco-2 cells were treated with Pam2CSK4 in either the presence or absence of L. plantarum peptidoglycan. L. plantarum peptidoglycan did not inhibit Pam2CSK4-induced IL-8 expression (Fig. 3B). These data indicate

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Both Pam2CSK4 and LTA are known to be TLR2 agonists (Kang et al., 2009). Therefore, we examined whether Lp.LTA regulates Pam2CSK-induced TLR2 activation. Since Caco-2 cells express relatively low levels of TLR2 compared with CHO/CD14/TLR2 cells (data not shown), we assessed TLR2 activation in the presence of Lp.LTA and/or Pam2CSK4 in CHO/CD14/TLR2 cells. As shown in Fig. 5A, the cells treated with either Lp.LTA or Pam2CSK4 exhibited increased CD25 expression. However, Lp.LTA remarkably decreased the extent of Pam2CSK4-induced CD25 expression in co-treated cells, suggesting that Lp.LTA attenuates Pam2CSK4-induced TLR2 activation. To determine whether Lp.LTA inhibits Pam2CSK4-mediated activation of MAPK and NF␬B, Caco-2 cells were treated with Pam2CSK4 in the presence or absence of Lp.LTA. Pam2CSK4 enhanced the phosphorylation of both p38 kinase and JNK; in contrast, treatment with Lp.LTA abrogated the phosphorylation of both p38 kinase and JNK. However, Pam2CSK4 did not enhance the phosphorylation of ERK. Moreover, Lp.LTA did not inhibit ERK phosphorylation (Fig. 5B), indicating that ERK phosphorylation is not required for Pam2CSK4-induced IL-8 expression in Caco-2 cells. Furthermore, Pam2CSK4 strongly induced I␬B␣ degradation, whereas Lp.LTA inhibited Pam2CSK4-induced I␬B␣ degradation (Fig. 5C). These results indicate that Lp.LTA suppresses Pam2CSK4-mediated activation of p38 kinase, JNK, and NF-␬B by suppressing TLR2 activation, thereby dampening IL-8 expression in Caco-2 cells.

4. Discussion Fig. 4. The lipid moiety and D-alanine groups of Lp.LTA are required to inhibit Pam2CSK4-induced IL-8 expression in Caco-2 cells. (A) Caco-2 cells were treated with intact, dealanylated or deacylated Lp.LTA (30 ␮g/ml) in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 24 h. (B) Caco-2 cells were cultured for 21 days until fully differentiated. Then, the cells were treated with Lp.LTA, dealnylated Lp.LTA or deacylated Lp.LTA (30 ␮g/ml) in the presence of Pam2CSK4 (0.1 ␮g/ml) for 24 h. Then, the cell culture supernatants were collected and the amounts of secreted IL-8 were determined by ELISA. Data are expressed as mean values ± standard deviations of triplicate samples. Asterisk (**) indicates a statistically significant difference (P < 0.01) compared with the control. NT; non-treated.

that neither whole L. plantarum bacteria nor L. plantarum peptidoglycan inhibit Pam2CSK4-induced IL-8 expression. 3.4. The lipid moiety and D-alanine groups of Lp.LTA are essential for the inhibition of Pam2CSK4-induced IL-8 expression in Caco-2 cells The LTA lipid moiety and D-alanine groups have been reported to be important for its immunomodulatory effects (Morath et al., 2001, 2002). To determine whether the lipid moiety and D-alanine groups play a role in Lp.LTA-mediated inhibition of IL-8 expression, Caco-2 cells were treated with dealanylated or deacylated Lp.LTA in the presence of Pam2CSK4. As shown in Fig. 4A, both lipid moiety-deficient and D-alanine-deficient forms of Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 production, indicating that the lipid moiety and D-alanine groups of Lp.LTA are both essential for the inhibition of Pam2CSK4-induced IL-8 expression. In addition, to verify the physiological relevance, the fully-differentiated Caco-2 cells were treated with native Lp.LTA, dealanylated Lp.LTA or deacylated Lp.LTA in the presence of Pam2CSK4. Similarly, Lp.LTA dramatically inhibited Pam2CSK4-induced IL-8 production whereas lipid moiety-deficient and D-alanine-deficient forms of Lp.LTA did not inhibit Pam2CSK4-induced IL-8 production (Fig. 4B).

As a probiotic agent, Lactobacillus plays an important role in the attenuation of acute and chronic inflammation in the gastrointestinal tract (Macho Fernandez et al., 2011; Tien et al., 2006). Certain Lactobacillus strains can even reduce pathogen-induced production of pro-inflammatory mediators, such as IL-8 (Moorthy et al., 2010). However, it has not been clearly understood the components of Lactobacillus responsible for the anti-inflammatory effect and the underlying mechanisms. Here, we demonstrated that Pam2CSK4, a synthetic bacterial lipopeptide, remarkably induced IL-8 expression in human intestinal epithelial cells and Lp.LTA attenuated Pam2CSK4-induced IL-8 expression. IL-8 is implicated in the recruitment of leukocytes, such as neutrophils, during inflammation (Singer and Sansonetti, 2004) further to promote tissue damage which in turn contributes to chronic inflammation (Simonet et al., 1994; Wright et al., 2010). In addition, in light of the fact that LTA is actively secreted from the bacteria (Wicken and Knox, 1975), the attenuation of IL-8 expression by Lp.LTA in the human intestinal cells might be an important anti-inflammatory mechanism induced by Lactobacillus. In general, LTA is known to enhance bacterial biofilm formation, adhesion to the host, and inflammatory responses (Fabretti et al., 2006; Ginsburg, 2002). Although S. aureus LTA itself does not strongly induce inflammatory responses (Ahn et al., 2014), it can cause inflammatory disease in cooperation with other cell wall components such as peptidoglycan (De Kimpe et al., 1995). Conversely, L. plantarum LTA suppresses LPS-induced production of inflammatory mediators in human monocytes (Kim et al., 2008). Concordantly, our current study also showed that Lp.LTA effectively inhibited Pam2CSK4-induced IL-8 expression but LTAs from other Lactobacilli and even pathogenic bacteria hardly or weakly inhibited it. Therefore, LTAs do not ubiquitously exert antiinflammatory functions but the effects are species-specific and

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Fig. 5. Lp.LTA attenuates Pam2CSK4-induced activation of p38 kinase, JNK, and NF-␬B by suppressing TLR2 activation. (A) CHO/CD14/TLR2 cells were treated with Lp.LTA (30 ␮g/ml) in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 24 h. Then, the cells were stained with FITC-conjugated anti-CD25 monoclonal antibodies and the levels of CD25 expression were determined by flow cytometry. Data are expressed as mean values ± standard deviations of triplicate samples. Asterisk (*) indicates a statistically significant difference (P < 0.05) compared with the control. (B–C) Caco-2 cells were treated with Lp.LTA (30 ␮g/ml) in the absence or presence of Pam2CSK4 (0.1 ␮g/ml) for 30 min. Then, the cells were lysed and equal amounts of protein were subjected to Western blot analysis using antibodies specific to phosphorylated or nonphosphorylated p38 kinase, JNK, or ERK (B), or with antibodies against I␬B␣ (C).

strain-specific. The higher potency of Lp.LTA compared with LTAs of other bacteria for inhibiting Pam2CSK-induced IL-8 expression seems to be a result of the unique structure of Lp.LTA. Interestingly, the glycolipid moiety of Lp.LTA contains two unsaturated fatty acids and an additional acyl chain on the trihexosyl-diacyl ceramide structure (Jang et al., 2011), whereas the L. rhamnosus GG LTA has only two unsaturated fatty acids (Lebeer et al., 2012). Unlike Lactobacillus LTA, S. aureus LTA has only saturated fatty acids on the dihexosyl-diacyl glycerol group (Jang et al., 2011). We demonstrated that the removal of D-alanine groups from Lp.LTA prevented it from inhibiting Pam2CSK4-induced IL-8 expression in Caco-2 cells. Similar to our results, native S. aureus LTA significantly induced the production of TNF-␣, whereas the loss of D-alanine groups from S. aureus LTA prevented this effect (Morath et al., 2001). These data indicate that D-alanine is a determinant for the biological activity of LTA. In addition to D-alanine groups, lipid moieties are also known to be important for the immunological functions of LTA since removal of the lipid moiety from Lp.LTA abolished its inhibitory effect on Pam2CSK4-induced IL-8 expression. Consistent with the results, the loss of LTA activity by deacylation has been also observed in LTAs of other bacteria such as S. aureus, B. subtilis, E. faecalis, and S. mutans (Baik et al., 2011; Hong et al., 2014; Ryu et al., 2009). Thus, it is a common feature of LTAs that the lipid moiety is essential for LTA-mediated immunological functions. One of the most probable explanations for this phenomenon is that LTA structurally interacts with TLR2 through the lipid moiety as demonstrated by X-ray crystallography (Kang et al., 2009). In this study, we demonstrated that Lp.LTA inhibits Pam2CSK4induced IL-8 production in human intestinal epithelial cells. Lp.LTA could be highly efficient in attenuating pathogenic bacteriainduced inflammatory responses in intestinal epithelial cells.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgements This work was supported by grants from the National Research Foundation of Korea, which is funded by the Korean government (MISIP) (2010-0029116, 2008-0062421, and NRF2012R1A1A2039022). This work was also supported by the R&D Convergence Center Support Program of the Ministry for Food, Agriculture, Forestry and Fisheries, and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which is funded by the Ministry of Health & Welfare (HI14C0469), Republic of Korea.

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Lipoteichoic acid from Lactobacillus plantarum inhibits Pam2CSK4-induced IL-8 production in human intestinal epithelial cells.

Lactobacilli are probiotic bacteria that are considered to be beneficial in the gastrointestinal tract of humans. Although lactobacilli are well known...
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