Secretional Blockade by VSL#3 Figure 4. The inhibition of IP-10 secretion is independent of the signal-specific activation of IEC. (A) Mode-K cells were stimulated for 24 h with IFNc (100 ng/ml) and L. casei (moi 20). Cell culture supernatants were analyzed by ELISA and (B) intracellular IP-10 protein levels by Western blot. (C) Mode-K cells were transfected with an IP-10 overexpression (pIP-10-DsRed)- or a ctr-DsRed-plasmid (transfection control) for 6 h and were then treated with L. casei for 24 h. IP-10 levels in the supernatants were determined by ELISA and intracellular DsRed-tagged IP-10 (IP-10-DsRed) or DsRed levels (transfection control) were determined by Western blot. Bars in A/C represent mean values (+/2 SD) of triplicate samples and all figures are representative for three independent experiments. doi:10.1371/journal.pone.0004365.g004 either lysed in 200 ml of 1xlysis buffer (Cell Signaling, Beverly, MA) supplemented with PMSF (1 mM) or they underwent a chase period of 3h with or without stimulation with L. casei. After the chase period, cells were washed three times with 16PBS and lysed. Co-immunoprecipitation for IP-10 was performed as described and beads were resuspended in 20 ml of Laemmli buffer for subsequent electrophoresis on a 15% SDS gel. After drying the gel it was placed on a Kodak Storage Phosphor screen (Amersham bioscience) in a cassette (Amersham Bioscience) over night. The Storage Phosphor screen was then scanned by a Typhoon TRIO+ scanner (Amersham, Bioscience). Animals Conventionally raised TNFDARE mice (a generous gift from Kollias G., Institute for Immunology, Biomedical Sciences Research Center ‘‘Al. Fleming’’, Greece) on C57BL/6 background and SPF-raised IL-102/2 mice on 129 SvEv background as well as wildtype C57BL/6 and 129 SvEv mice were fed 1,36109 cfu of VSL#3 bacteria (a generous gift from Dr. DeSimone, VSL#3 pharma, Italy) in 13,2% (w/v) gelatine, 20% (w/v) glucose in water every weekday for 15 and 21 weeks starting at the age of three weeks. Placebo fed mice were used as controls, respectively. The gelatine was prepared freshly every third day. Mice were killed at the age of 18 and 24 weeks followed by sampling of gut content and IEC isolation. Sections of the distal ileum, cecal tip and distal colon were fixed in 10% neutral buffered formalin (Sigma Aldrich). Fixed tissues were hematoxylin- and eosin-stained and embedded in paraffin. Histology scoring was performed by blindly assessing the degree of lamina propria mononuclear cell infiltration, crypt hyperplasia, goblet cell depletion and architectural distortion, resulting in a score from 0 (not inflamed) to 12 (inflamed), as previously described [29]. Animal use was approved by the institution in charge (approval no. 55.2-1-S4-2531-74-06 and 32-2347/4+63). Isolation of primary mouse intestinal epithelial cells Primary IEC were purified as previously described [27]. Briefly, either ileal or cecal and colonic tissue was cut into pieces and incubated (37uC, 15 min) in Mode-K cell culture media supplemented with 1 mM dithiothreitol (Roth, Karlsruhe, Ger- many). The tissue/IEC suspensions were filtered, centrifuged (7 min, 300 g, RT) and cell pellets were resuspended in DMEM containing 5% fetal calf serum. The remaining tissue was incubated in 30 ml PBS (10 min, 37uC) containing 1.5 mM EDTA (Roth, Karlsruhe, Germany). After filtration, the tissue was discarded and the cell suspension from this step was centrifuged as above. Finally, primary IEC were purified by centrifugation through a 20%/40% discontinuous Percoll gradient (GE Health- care, Uppsala, Sweden) at 600 g for 30 min. Primary IEC were collected in lysis buffer for subsequent Western blot analysis or in trizol for subsequent RNA isolation. Purity of IEC was confirmed by anti-CD3+-Western blot analysis. DNA isolation from gut content and bacteria-specific PCR Gut content DNA was extracted from 200 mg of content using the QIAamp DNA Stool Mini Kit (Qiagen) according to the manfacturer’s instructions. Streptococcus thermophilus (S. thermophilus) specific PCR of the 16S–23S rRNA gene spacer region was performed as described previously [30]. Immunohistochemical labeling The cecum was dissected and immediately immersed in Tissue Tek OCT compound (Agar Scientific) and frozen in liquid nitrogen. Samples were stored under liquid nitrogen until analysed. 8 mm sections were cut using a Leica CM1950 cryostat, picked up onto polylysine coated slides and air dried at room temperature for 45 minutes. Sections were fixed in 4%parafor- maldehyde 0.1M phosphate buffer (pH7.4) for 5 minutes before being washed with 6 changes of PBS (pH 7.4) over 20 minutes. Slides were then incubated with 0.1% Triton6100 (Sigma) in PBS for 3 minutes before being washed as above. Fc receptors were blocked (Fcc III/II receptor from BD Pharminogen) and slides incubated in 10% BSA 5% normal donkey serum in PBS for 2 hrs at room temperature. Blocking buffer was removed by capillary action and the slides incubated over night at 4uC in either IP-10 goat polyclonal IgG (Santa Cruz G-15 sc-14641) or control goat IgG (Santa Cruz sc-2028) in 2% BSA. The slides were washed with 6 changes of PBS over 1 hour. Alexa Fluor 488 donkey anti goat IgG (Invitrogen Molecular Probes) was applied to the sections and incubated for 30 minutes at room temperature. The slides were washed as above, counter stained with DAPI and mounted in vectashield (Vector laboratories). Sections were viewed on a Zeiss Axioskop microscope using a FITC and DAPI filter set and imaged using an QIMAGING camera. Statistical analysis Data were expressed as mean of triplicates +/2 standard deviation. Statistical tests were performed using two-tailed Student test for the in vitro experiments. The in vivo feeding studies were analysed using rank sum test. Differences were considered significant if values were ,0.05 (*) or ,0.01 (**). Results VSL#3-derived L. casei specifically inhibits TNF-induced secretion of chemotactically functional IP-10 To investigate the effects of VSL#3 on pro-inflammatory IP-10 expression in IEC, we stimulated Mode-K cells with VSL#3 bacteria (Moi 20) in the presence or absence of TNF, a potent inducer of IP-10 expression. The concentration of secreted IP-10 in the cell culture supernatant was analyzed by ELISA. VSL#3 did not induce IP-10 expression. In contrast, VSL#3 significantly reduced TNF-induced IP-10 but not IL-6 expression (Figure 1A, suggesting an IP-10 specific mechanism for the inhibitory function of IP-10. Besides, the stimulation of IEC with VSL#3 bacteria alone induced IL-6 expression. A series of additional experiments with the eight VSL#3 single bacterial strains (Moi 20) revealed that one of the eight strains, L. casei, exhibits analogous effects on IP-10 and IL-6 expression as the complex mixture VSL#3 (Figure 1B). L. casei was therefore identified as the effective bacterial strain in the probiotic mixture concerning the observed cytokine expression profile. PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4365