Abstract
BackgroundClinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).Methodology/Principal FindingsVSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNFΔARE and IL-10−/− mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10−/− mice, IP-10 protein expression was reduced in primary epithelial cells.Conclusions/SignificanceWe demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.
Highlights
Inflammatory bowel diseases (IBD) are spontanously relapsing, immunologically mediated disorders of the gastrointestinal tract
The present study shows for the first time bacterial strainspecific effects of the clinically relevant probiotic mixture VSL#3 on the expression of a pro-inflammatory chemokine in primary mouse/day) or placebo for 15 weeks
Ileal intestinal epithelial cells (IEC) were isolated and pooled proteins (50 mg) of all mice in a group were analyzed for induced protein 10 (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
Summary
Inflammatory bowel diseases (IBD) are spontanously relapsing, immunologically mediated disorders of the gastrointestinal tract. Apart from its protective effect in clinical studies, VSL#3 was shown to reduce experimental colitis in IL-10-deficient (IL-102/2) mice. 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. Extensive 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. Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC)
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