Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation Gabriele Ho¨ rmannsperger1, Thomas Clavel1, Micha Hoffmann 1 , Caroline Reiff2, Denise Kelly2, Gunnar Loh 3 , Michael Blaut3, Gabriele Ho¨ lzlwimmer4, Melanie Laschinger5, Dirk Haller1* 1 Chair for Biofunctionality, ZIEL-Research Center for Nutrition and Food Science, Technische Universita¨t M u¨nchen, Freising-Weihenstephan, Germany, 2 Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom, 3 Gastrointestinale Mikrobiologie, Deutsches Institut fu¨ r Erna¨hrungsforschung, Potsdam- Rehbru¨ cke, Nuthetal, Germany, 4 Helmholz-Zentrum M u¨nchen, Institute for Pathology, Mu¨ nchen-Neuherberg, Germany, 5 Department of Surgery, Technische Universita¨t Mu¨ nchen, Munich, Germany Abstract Background: 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). Methodology/Principal Findings: VSL#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 TNFDARE and IL-102/2 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-102/2 mice, IP-10 protein expression was reduced in primary epithelial cells. Conclusions/Significance: We 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. Citation: H o¨rmannsperger G, Clavel T, Hoffmann M, Reiff C, Kelly D, et al. (2009) Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation. PLoS ONE 4(2): e4365. doi:10.1371/journal.pone.0004365 Editor: Baohong Zhang, East Carolina University, United States of America Received September 8, 2008; Accepted December 17, 2008; Published February 6, 2009 Copyright: 2009 Ho¨ rmannsperger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support for this study was obtained from the European Nutrigenomics Organisation, contract nr. FP6-506360 and the Bundesministerium fu¨ r Bildung und Forschung, AZ0313814. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: haller@wzw.tum.de Introduction Inflammatory bowel diseases (IBD) are spontanously relapsing, immunologically mediated disorders of the gastrointestinal tract. The incidence and prevalence of the two main idiopathic pathologies of IBD, Crohn’s disease (CD) and ulcerative colitis (UC), are rising globally in parallel to the progression of the industrialisation. More that 3.5 million people suffer from IBD in the United States and Europe [1], but although extensive research was performed in this field, the initial trigger is still unknown and efficient therapies are not available yet. Genetical susceptibility of the host [2] and the intestinal microbiota both were found to play a pivotal role in the onset and perpetuation of IBD. Enhanced numbers of mucosa associated bacteria and decreased microbiota diversity are associated with these diseases [3]. In contrast to the fact, that IBD is the result of an overreaction of the intestinal immune system towards intestinal microbes, clinical studies showed that oral uptake of specific probiotic bacterial strains like VSL#3 [4,5] or Escherichia coli Nissle 1917 [6,7] resulted in attenuation of IBD disease severity [8]. VSL#3, a mixture of eight different lactic acid bacteria (Lactobacillus (L.) acidophilus, L. bulgaricus, L. casei, L. plantarum, Streptococcus thermophilus, Bifidobacter- ium (B.) breve, B. infantis, B. longum) was effective in the prevention and in the maintenance treatment of pouchitis and UC [9–14]. Apart from its protective effect in clinical studies, VSL#3 was shown to reduce experimental colitis in IL-10-deficient (IL-102/2) mice. Treatment of IL-102/2 mice resulted in decreased histopathology, normalization of colonic function, barrier integrity and mucosal cytokine production [15]. Furthermore, VSL#3 was shown to induce protective heat-shock-proteins in intestinal PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4365