Secretional Blockade by VSL#3 Figure 7. VSL#3 does not reduce experimental ileitis in TNFDARE mice. TNFDARE (n = 6) and wt mice (n = 5) were fed VSL#3 (1,36109 cfu/ mouse/day) or placebo for 15 weeks. Mice were sacrificed at 18 weeks of age and histopathological scoring was performed by blindly assessing the inflammation between 0 (not inflamed) and 12 (highly inflamed). Figure A shows the median histopathologic score of TNFDARE mice in the study. (B) Ileal IEC were isolated and pooled proteins (50 mg) of all mice in a group were analyzed for IP-10 expression by Western blot. (C) The presence of VSL#3 derived S. thermophilus (S.t) in the gut of the TNFDARE mice was examined by bacteria-specific PCR analysis. doi:10.1371/journal.pone.0004365.g007 IEC and IEC lines. VSL#3-derived L. casei was identified to inhibit TNF-induced IP-10 expression to the same extent as the complex probiotic mixture. This effect was found to be a unique feature of VSL#3-derived L. casei as other clinically relevant probiotic bacteria like L. plantarum 299 and E. coli Nissle 1917 did not reduce IP-10 secretion. Since formaldehyde fixed L. casei had the same inhibitory effect on IP-10 secretion as live bacteria, active interaction between bacteria and IEC is not necessary for the inhibitory effect, suggesting a bacterial cell surface component as the effective probiotic structure. In contrast to L. rhamnosus GG, a probiotic bacteria that was shown to have protective effects on IEC survival and growth via two secreted proteins [34], we demonstrated that the inhibition of TNF-induced IP-10 secretion was due to a heat-labile surface protein of L. casei. The proteinasous nature of the bacterial structure responsible for the inhibition of IP-10 was supported by the finding that the effect was independent of TLR2-dependent signalling mechanisms. Stimu- lation experiments with IFNc and IP-10 overexpression experi- ments revealed stimuli-independent mechanisms to underly the inhibition of IP-10 expression by L. casei. Given the high physiological relevance of elevated IP-10 expression in inflamed intestinal tissue regions of IBD patients and animal models of experimental colitis [20–22], the inhibitory effect on this chemokine may play an important role in the anti-inflammatory effects of the probiotic mixture VSL#3 on inflammatory bowel diseases [9]. We were able to show that IP-10 expression in IEC correlates with inflammation, as IP-10 expression was elevated in primary ileal IEC from heterozygous TNFDARE mice as well as in colonic IEC from IL-102/2 mice compared to healthy wildtype mice. In a healthy host, pathogen-induced secretion of IP-10 triggers enhanced recruitment of effector Th1 cells and monocytes into the mucosa, resulting in subsequent deletion of the infectious agent. In the case of IBD, chronically activated IEC steadily produce high amounts of IP-10 in the absence of any pathogenical stimuli resulting in constant Th1 and monocyte recruitment. The presence and activity of these effector cells may lead to the loss of epithelial cell integrity and tissue damage. In consequence, the expression of IP-10 in IEC has to be tightly regulated to prevent infections on the one hand and the development of pathological conditions like chronic intestinal inflammations on the other hand. Consistently, IP-10 expression has been shown to be modulated at various levels. The activation of PPARc by PPARc ligands inhibited IP-10 expression by reducing IP-10 promoter activity [35]. In addition, butyrate inhibited IFNc and TNF-induced IP-10 transcription in colonic subepithelial myofibroblasts [36] and interleukin 10 suppressed LPS-induced IP-10 gene transcription in peritoneal macrophages [37]. Many studies have analyzed the transcriptional regulation of cytokines and chemokines like IP-10 but although post-transcriptional and post-translational regulation of chemokines is suggested to be very important [38], exact mechanisms are mostly unknown. The pro-inflammatory chemo- kine interleukin 8 was shown to be upregulated by a TLR5- mediated, p38-dependent post-transcriptional mechanism [39] and furthermore, to be processed at the amino terminal end by a matrix metallo proteinase [40]. In addition, the biosynthesis of IFNc was shown to be suppressed post-transcriptionally by the stimulation with lead [41]. Concerning IP-10, complex post-transcriptional regulatory mechanisms seem to play an important role. Recent studies revealed regulatory mechanisms like IP-10 mRNA half-life modulation in monocytes [42] or C- terminal truncation through furin, a pro-protein convertase, resulting in another active form of the chemokine [43]. Furthermore, IP-10 was found to be processed C-terminally by gelatinase B and neutrophil collagenase [40]. In addition, the activity of IP-10 was shown to be regulated by its oligomerization status [44]. Our study revealed for the first time that probiotic bacteria are able to induce post-translational regulation of IP-10 protein in IEC. Decreased IP-10 protein stability was identified as a new post-translational regulatory mechanism. L. casei-induced ubiquitination of IP-10 is very likely involved in the observed degradation of the chemokine although it does not target the protein for proteasomal degradation. It has already been shown that stimulation with L. casei has multiple effects on the expression of genes involved in proteasome and ubiquitination pathways in IEC [45], but the exact mechanisms are not known yet. In contrast to lysosomal degradation of LPS-induced TNF under hypoxia [46], lysosomal pathways were not involved in L. casei-induced loss of IP-10 protein, suggesting alternative degra- dation pathways to be involved. The inhibition of protein secretion through brefeldin A was found to induce degradation of IP-10 instead of intracellular accumulation, as it was shown for other secretory proteins like makrophage inflammatory protein-2 [47]. This result led to the hypothesis that L. casei might selectively impair secretion of IP-10 followed by subsequent degradation as a protective mechanism to prevent protein accumulation. In contrast to interleukin-6, the secretion of IP-10 was found to be dependent on tightly regulated vesicular transport. L. casei mimicks the effect of 3-methyladenine, an inhibitor of vesicular trafficking and exosome secretion [48], which specifically inhibits IP-10 secretion and induces loss of intracellular IP-10 protein. Considering the extensive post-translational processing of IP-10, this inhibitory mechanism might be due to the necessity for chemokine maturation processes in endo-lysosomal compartments prior to exocytosis as it has been shown for several other secretory proteins [49,50]. We therefore hypothesize that L. casei inhibits vesicle trafficking similar to 3-methyladenine, resulting in impaired maturation and secretion of IP-10 followed by subsequent degradation of the chemokine (Figure 6B). Our in vivo feeding studies revealed that VSL#3 has no effect on the development of ileal inflammation in heterozygous TNFDARE mice. This result may indicate that the constant overexpression of TNF in these mice and the resulting T-cell driven immunopa- thology constitute such a strong pro-inflammatory stimulus that probiotic therapy is not sufficient to reduce tissue inflammation. Apart from that, one could raise the hypothesis that probiotic therapy is generally less effective in the ileum than in the colon due to high luminal content passage rates in this intestinal segment. The failure of VSL#3 in the context of ileitis together with the observed reduction of cecal inflammation in IL-102/2 mice is consistent with clinical data showing effective probiotic treatment mostly in the context of UC [9] and pouchitis patients [51]. The finding that VSL#3 has protective effects on cecal but not on colonic inflammation clearly reveals intestinal segment specific effects of the probiotic mixture. The differences in response to PLoS ONE | www.plosone.org 14 February 2009 | Volume 4 | Issue 2 | e4365