Secretional Blockade by VSL#3 Figure 5. Degradation of IP-10 protein is the result of an IP-10 specific secretional blockade. (A) Mode-K cells were stimulated for 1, 3 or 24 h with TNF (10 ng/ml) alone or together with L. casei (moi 20) and intracellular as well as secreted levels of IP-10 were analyzed by Western Blot or ELISA analysis. Bars in A represent mean values (+/2 SD) of triplicate samples. The shown figure is representative for three independent experiments. (B) Mode-K cells were stimulated with TNF (10 ng/ml) during a 3h pulse period (S35-labelled cysteine/methionine, 25 mCi) followed by a 3 h chase period in DMEM supplemented with L. casei (moi 20) or not. Subsequent immunoprecipitation for IP-10 was followed by protein separation on a 15% SDS gel. Intracellular levels of S35-labelled IP-10 were then made visible by a Phosphoimager plate. The shown figure is representative for two independent experiments. (C) Mode-K cells were stimulated with TNF (10 ng/ml) alone or together with L. casei in the presence or absence of brefeldin A (0,5 mM). The amount of intracellular IP-10 after 6 h of stimulation was analyzed by Western blot. (D) Mode-K cells were stimulated for 6 h with TNF (10 ng/ml) alone or with L. casei (moi 20) before lysis and subsequent immunoprecipitation using an anti-IP-10 antibody was performed. Western blot was performed to investigate the presence of IP-10 and ubiquitine in the precipitated fractions (single experiment). (E) Mode-K cells were stimulated with IFNc (100 ng/ml) or TNF (10 ng/ml) alone or together with L. casei (moi 20) in the presence of lactacystin or NH4Cl for 24h and intracellular IP-10 was analyzed by Western blot. The shown figures are representative for three independent experiments. doi:10.1371/journal.pone.0004365.g005 To analyze whether the observed inhibition of IP-10 has any functional consequence, we performed a T cell transmigration assay with Mode-K culture supernatants after a 24h stimulation experiment. Figure 1C shows that TNF-conditioned media increases the transmigration of activated T cells whereas supernatants of IEC that were costimulated with L. casei did not exert this chemotactic effect. To show that the reduced transmigration rate in the TNF/L. casei–conditioned media is due to the inhibition of IP-10 secretion by the probiotic bacteria, we used an IP-10 neutralizing antibody in the TNF-conditioned media. We demonstrated that the neutralization of IP-10 in this supernatant was sufficient to reduce the transmigration of T cells analogous to the effect of L. casei. These results clearly support the functional importance of the observed inhibition of IP-10 secretion in IEC by L. casei. Inhibition of TNF-induced IP-10 expression in IEC is mediated by a cell surface protein of L. casei Further analysis of other clinically relevant probiotic strains, Lactobacillus plantarum 299v and E. coli Nissle 1917, revealed that the inhibition of TNF-induced IP-10 secretion is dose-dependent and a unique feature of L. casei (Figure 2A). To investigate whether the inhibition of TNF-induced IP-10 expression was due to an active interaction between live L. casei and IEC, we inactivated L. casei by formaldehyde fixation, lysozyme lysis or heat treatment and subsequently stimulated Mode-K cells. Fig. 2B shows that fixed bacteria significantly inhibited TNF-induced IP-10 expression. However, lysis as well as heat-inactivation of L. casei resulted in complete loss of the observed inhibitory effect, suggesting a heat- labile surface structure of L. casei as the active bacterial component. To characterize this component in more detail, we treated L. casei with phospholipase A or the peptidases trypsine or proteinase K. Subsequent bacterial stimulation of Mode-K cells revealed that both peptidases completely reversed the effect of L. casei on TNF- induced IP-10 expression. In contrast, phospholipase A treatment did not affect the inhibitory effects of L. casei (Figure 2C). These results demonstrated that the inhibition of TNF-activated IP-10 expression by L. casei is likely mediated through a cell surface protein. Consistently, the inhibition of TNF-induced IP-10 expression was not dependent on the pattern recognition receptor TLR2 (Figure 2D). Of note, the inhibition of IP-10 was dependent upon the presence of L. casei during TNF stimulation, since the preincubation of IEC with L. casei did not result in an inhibition of subsequently TNF-induced IP-10 expression (data not shown). L. casei induces post-transcriptional inhibition of TNF- induced IP-10 expression in IEC We were next interested in the molecular mechanism underlying the observed inhibitory effect of L. casei on IP-10 expression. Since the transcription factor NFkB plays an important role in the induction of IP-10 expression [31], we investigated whether NFkB signaling might be impaired by stimulation of IEC with L. casei. As shown in Figure 3A, TNF- induced IkB degradation as well as TNF-induced RelA phos- phorylation (Figure 3B) was not inhibited by L. casei. In addition, ChIP analysis revealed that TNF-induced recruitment of RelA to the IP-10 promoter was not affected by the probiotic bacteria (Figure 3C). To investigate whether the activation of any other relevant transcription factor was blocked by L. casei, we transfected Mode-K cells with an IP-10-promoter reporter gene construct and observed that TNF-induced IP-10 promoter-dependent luciferase expression was not inhibited by L. casei (Figure 3D). Consistent with unaffected IP-10 promoter activity, TNF-induced increase of IP-10 mRNA levels was not inhibited by L. casei (Figure 3E). In contrast to the elevated IP10 transcript level, Western blot analysis revealed that intracellular IP-10 protein was almost completely lost after 24 hours of costimulation with L. casei (Figure 3F). Inhibition of IP-10 secretion is independent of the signal- specific activation of IEC We next performed a stimulation experiment with IFNc, another potent inducer of IP-10 expression. As shown in Figure 4, IFNc-induced IP-10 secretion (Figure 4A, ELISA analysis) and intracellular IP-10 protein expression (Figure 4B, Western blot analysis) was completely inhibited by L. casei. These results were analogous to the above described results observed under TNF-stimulation, suggesting that the inhibitory effects of L. casei were TNF-independent. To further characterize the signal- specificity of the inhibitory effect, we next performed a stimulation experiment with HEK293 cells that were transfected with a murine IP-10 overexpression plasmid resulting in constitutive expression of the chemokine. As shown in Figure 4C, L. casei significantly reduced intracellular and secreted levels of IP-10 in this experimental setup, whereas the production of the Ds-Red- control protein was not affected by the probiotic treatment. This result showed that the inhibitory effect of L. casei is indeed independent of the signal-specific activation pathways for IP-10, supporting our finding that the observed inhibition is mediated by a post-transcriptional mechanism. L. casei inhibits IP-10 secretion and triggers subsequent intracellular degradation of the chemokine We next investigated whether L. casei had an inhibitory effect on IP-10 protein translation. As shown in Figure 1, general protein translation and secretion was not impaired by the probiotic bacteria as indicated by normal or even increased secretion of other cytokines/chemokines like IL-6 (Figure 1A/B) or MIP-2 (data not shown). Western blot analysis of intracellular IP-10 protein levels revealed that TNF-induced IP-10 protein expression was not inhibited by L. casei at early time points (3h after costimulation). In contrast, the loss of intracellular IP-10 protein was detectable after 6 to 24 h of costimulation although it was not PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4365