epithelial cells (IEC) [16] or proliferation of IL-10-dependent TGFb-bearing regulatory T-cells in Th1-dependent murine colitis [17]. However, there is no study showing protective effects of VSL#3 in the context of CD or experimental ileitis, suggesting disease-and intestinal segment-specific effects of VSL#3. Exten- sive progress has been made in understanding probiotic effects of VSL#3 in the context of IBD but the molecular mechanisms as well as strain-specificity remain to be elucidated. IEC are crucial for maintaining intestinal homeostasis [18] and failure to control inflammatory processes at the epithelial cell level may critically contribute to the disease pathogenesis. The intestinal epithelium must interact with and adapt to the constant changing environment by processing the combined biological information from luminal enteric bacteria and host-derived signals [19]. IEC react on bacterial as well as immune-derived pro-inflammatory signals by secreting cytokines and chemokines like interleukin 6 (IL-6) and interferon c-induced protein 10 (IP-10) to activate and attract Th1-immune cells and phagocytic cells to the site of infection. Cytokine and chemokine levels were shown to be strongly elevated in inflamed intestinal regions of IBD patients [20]. Experimental studies confirmed that IP-10 plays an outstanding role in uncontrolled disease development as the blockade of IP-10 by an anti-IP-10 antibody was sufficient to decrease disease severity in IL-102/2 mice. This effect was shown to be due to reduced Th1 cell generation in inductive sites and reduced recruitment of Th1 effector cells to the colon [21]. Another experimental study revealed that anti-IP-10 therapy attenuates murine acquired immunodeficiency syndrome (MAIDS) colitis through the blockade of Th1-cell trafficking and the reduction of IEC apoptosis [22]. Although the importance of IP-10 as a pro-inflammatory mediator in IBD has been clearly demonstrated, the molecular mechanisms underlying the regula- tion of IP-10 expression in IEC during disease development are not well understood. Interestingly, recent studies revealed that probiotics are able to regulate chemokine expression in IEC. Although E. coli strain Nissle 1917 and VSL#3 were shown to reduce TNF-or Salmonella dublin-induced IL-8 chemokine expres- sion in IEC [23,24], the molecular mechanisms of target-specific inhibition of IEC activation through probiotic bacteria under conditions of chronic intestinal inflammation are not understood. The aim of our study was to analyze the molecular mechanisms of strain-specific effects of VSL#3 on IP-10 production in activated IEC. Furthermore, the effect of VSL#3 on TNF-induced ileitis in heterozygous TNFDARE mice and on colitis in IL-102/2 mice was investigated in the context of IP-10 expression in IEC. Materials and Methods Cell culture The small intestinal epithelial cell line Mode-K [25] (passage 10–25) as well as the human embryonic kidney epithelial cell line HEK293 [26] was grown in a humidified 5% CO2 atmosphere at 37uC to confluency in 6, 12 or 24 well tissue culture plates (Cell Star, Greiner bio-one, Frickenhausen, Germany). TLR2 deficient HEK293 cells were bought from InvivoGen (Cat.Nr.: 293-null) and HEK293 cells expressing TLR2 were also bought from InvivoGen (Cat.Nr.: 293-mtlr2). The Mode-K cell culture media was Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 1,0% Glutamine and 0,8% antibiotic antimycotic (Invitrogen, Carlsbad, USA). Glutamine was omitted for the cultivation of HEK293 cells and Blasticidin was added for the cultivation of the stably transfected HEK293 cells. Cell culture media was changed prior to stimulation. Secretional Blockade by VSL#3 Cell culture stimulation and bacterial treatment Confluent Mode-K cell monolayers were stimulated with TNF (10 ng/ml) (R&D Systems Europe), brefeldin A (0,5 mM) (Calbio- chem), lactacystin (22 mM) (Biomol), NH4Cl (20 mM) (Sigma), 3- methyladenine (5 mM) and/or VSL#3 bacteria (VSL#3 mixture or single strains, generous gift from Dr. DeSimone, L’Aquila, Italy) (moi 20) for 24 h if not otherwise indicated. Viability of cells after brefeldin A treatment (6h) (93% of viable cells, data not shown) was analysed by a WST assay (Roche). VSL#3 derived L. casei and L. plantarum 299v were grown anaerobically (Anaerogen, Oxoid) at 37uC in MRS Broth (Fluka, Heidelberg) containing 0,05% L- cysteine (Roth). E. coli strain Nissle 1917 (a generous gift from Dr. Sonnenborn, Ardeypharm GmbH, Herdecke, Germany) was grown aerobically in LB-media. Bacteria were centrifuged (4500 g, 10 min) and resuspended in DMEM. L. casei was heat- killed (30 min, 90uC), fixed (fL.c) (3 hours, 5% formaldehyde, 4uC) or lysed by lysozyme (Sigma, 50 mg/ml) in filter sterilized 10 mM Tris buffer (pH 8). Fixed bacteria were washed three times with sterile PBS before IEC stimulation. For cell surface treatment, bacteria were incubated with Phospholipase A (2 mg/ml) (Fluka), Trypsin (2 mg/ml) (Roth) or Proteinase K (50 mg/ml) (Roth) in filter-sterilized Tris buffer (50 mM Tris, 0,1 M NaCl, pH 8, 1h, 37uC) in a shaker. Western blot Purified primary IEC or Mode-K cells were lysed in Laemmli buffer and 50 mg of protein were subjected to electrophoresis on 10% or 15% SDS-PAGE gels. Anti IP-10 (R&D Systems Europe, Arlington), anti-IkB, anti-phospho-RelA (Cell Signaling, Beverly, MA), anti-ubiquitine (Cell Signaling, Beverly, MA), anti-DsRed (clontech) and anti-ß-actin-antibody (ICN, Costa Mesa, CA) were used to detect immunoreactive IP-10, IkB, phospho-RelA, DsRed, ubiquitine and ß-actin, using an enhanced chemoluminescence light-detecting kit (GE, Arlington Heights, IL). ELISA IP-10 and IL-6 concentrations were determined in IEC supernatants using the appropriate ELISA kits (R&D Systems Europe) according to the manufacturer’s instructions. Chromatin Immunoprecipitation (ChIP) Mode-K cells in 75 cm2 flasks were pre-incubated with L. casei for 1 h and stimulated with TNF (10 ng/ml) for 2 h. Cells were fixed by formaldehyde fixation (1%) and nuclear extraction as well as chromatin immunoprecipitation were performed using a ChIP- kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Immunoprecipitation was carried out over night at 4uC with an anti-NFkappaB p65 antibody (Cell Signaling, Beverly, MA). DNA/protein/antibody-complexes were collected with salmon sperm saturated protein A/G agarose for 30 min and washed three times in high salt buffer followed by three more washes in no salt buffer. DNA was released from the immune complex by heating and subsequent proteinase K treatment. DNA was then extracted by phenol-chloroform and eluted in water. The input control for the PCR was DNA from total nuclear extract. PCR was performed with total DNA (input control, 1 ml) and immunoprecipitated DNA (1 ml) using the following IP-10 promoter-specific primers 59-AACAGCT- CACGCTTTG, 59-GTCCTGATTGGCTGACT. The length of the amplified product was 186 bp. PCR products (10 ml) were subjected to electrophoresis on 2% agarose gels. PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4365