Secretional Blockade by VSL#3 Figure 6. Blockade of 3-MA-dependent vesicular transport by L. casei might induce the degradation of IP-10. (A) Mode-K cells were stimulated with TNF (10 ng/ml) alone or together with L. casei (moi 20) in the presence or absence of 3-MA for 24 h and the amount of IP-10 in the cell culture supernatant as well as intracellularly was analyzed by ELISA and Western blot. Figure A is representative for three independent experiments and bars represent mean values (+/2 SD) of triplicate samples. (B) The scheme illustrates our hypothesis that L. casei might target 3-MA dependent vesicular transport of IP-10 resulting in a secretional blockade and subsequent degradation of the chemokine. doi:10.1371/journal.pone.0004365.g006 secreted into the cell culture supernatant (Figure 5A). This result demonstrated that initial TNF-induced IP-10 translation is not targeted by the probiotic bacteria suggesting that the observed loss of IP-10 protein is due to post-translational degradation. To further analyse the fate of initially produced IP-10 protein in more detail, we performed a S35 pulse-chase labelling experiment. Interestingly, IP-10 immunoprecipitation experiments showed that TNF-induced S35-IP-10 protein, that was produced during the initial 3h pulse period, was absent in Mode-K cells after the subsequent 3h chase period, demonstrating the functionality of the secretion system. In contrast, TNF-induced S35-IP-10 was still detectable intracellularly after the chase period in Mode-K cells that were costimulated during the chase period (Figure 5B). This result clearly showed that L. casei prevents the secretion of IP-10 protein. However, this finding was in sharp contrast to the observation that IP-10 protein is lost intracellularly at later time points instead of intracellular accumulation as it would be expected to be the result of a secretional blockade. To further analyze this discrepancy, we performed TNF and L. casei c(o)stimulation experiments in the presence of brefeldin A, a known inhibitor of vesicular transport from the endoplasmic reticulum to the golgi network. Surprisingly, we found that the inhibition of the protein export machinery by brefeldin A resulted in complete loss of intracellular IP-10 protein analogous to the results described above in the costimulation experiments with L. casei (Figure 5C). This result confirms that indeed, a secretional blockade of IP-10 results in subsequent loss of intracellular IP-10 protein, suggesting the initiation of degradative mechanisms as a consequence of initial IP-10 accumulation. We next addressed the question which degradation pathway might be responsible for the observed loss of IP-10 protein in IEC. Co-immunoprecipitation analysis revealed that IP-10 was ubiqui- tinated in the presence of L. casei (Figure 5D) indicating proteasomal degradation. Since TNF-induced IP-10 expression depends on proteasomal IkB degradation, we stimulated cells with IFNc to investigate the effect of proteasomal inhibition on IP-10 degradation. Figure 5E shows that stimulation with the proteaso- mal inhibitor lactacystin did not rescue IFNc-induced IP-10 protein from degradation. Next, we investigated whether IP-10 protein was degraded through lysosomal pathways. The inhibition of lysosomal degradation by NH4Cl (Figure 5E) did not prevent the loss of IP-10 protein, suggesting an additional proteolytic mechanism for IP-10 degradation. L. casei mimicks the effects of the inhibition of autophagic-lysosomal pathways on cytokine/chemokine expression To investigate whether autophagic-lysosomal degradation pathways might play a role in the observed loss of IP-10 protein, we applied TNF and L. casei to the epithelial cell culture in the presence of 3-methyladenine (3-MA). Surprisingly, we demon- strated that the stimulation of IEC with 3-MA, which is a known inhibitor of vesicle formation in the context of autophagy- lysosomal degradation, did not prevent the loss of intracellular IP-10 (Figure 6A, Western blot analysis), but rather mimicked the effects of L. casei on the TNF-induced cytokine secretion pattern (Figure 6A, ELISA analysis). Figure 6A shows that 3-MA decreased the level of IP-10 protein intracellularly as well as in the cell culture supernatant. In contrast, TNF-induced IL-6 secretion was not inhibited in the presence of 3-MA. These observations indicated that IP-10 but not IL-6 secretion is dependent on the formation of secretory vesicles that can be inhibited by 3-MA. We therefore hypothesize that L. casei impairs 3-MA-dependent vesicular trafficking resulting in disturbed IP-10 secretion and subsequent degradation of IP-10 protein (Figure 6B). The protective activity of VSL#3 on experimental colitis is intestinal segment specific and correlates with IP-10 expression in IEC To evaluate the physiological impact of VSL#3 on experimen- tal intestinal inflammation, we performed probiotic feeding studies with heterozygous TNFDARE mice, an animal model for TNF- induced experimental ileitis [32], and IL-102/2 mice, an animal model for experimental colitis [33]. TNFDARE mice, IL-102/2 mice and wildtype mice were fed VSL#3 (1.36109 cfu per mouse/day) for 15 and 21 weeks post-weaning. Histopathological analysis of distal ileal sections revealed that VSL#3 did not inhibit the development of severe ileal inflammation in TNFDARE mice (Figure 7A). As expected, we found that TNF-induced ileitis correlates with enhanced IP-10 protein expression in primary ileal epithelial cells from TNFDARE mice. The absence of T-cell contaminations in the purified IEC from inflamed mice confirmed the purity of the epithelial cell isolation (data not shown). Consistent with the tissue pathology, VSL#3 did not reduce IP- 10 protein expression in primary ileal IEC (Figure 7B). The presence of VSL#3 bacteria in the intestine was proven by the use of bacteria-specific PCR on DNA isolated from intestinal contents (Figure 7C). In contrast to the unresponsiveness of heterozygous TNFDARE mice to VSL#3 treatment, we found that cecal (Figure 8A) but not colonic (Figure 8B) inflammation in IL-102/2 mice was significantly reduced by the probiotic mixture, suggesting intestinal segment specific effects of VSL#3. In consistence with the results derived from TNFDARE mice, we detected elevated levels of IP-10 in isolated intestinal epithelial cells of IL-102/2 mice compared to wildtype mice. Although we were not able to separate cecal and colonic epithelial cells during the isolation procedure (for the sake of yield), Western blot analysis showed that VSL#3 clearly reduced the expression of IP-10 even in the pooled cell population. Real-time PCR analysis of IP-10 mRNA levels in these primary IECs showed that the observed reduction of IP-10 protein is not due to reduced levels of IP-10 mRNA (Figure 8C), supporting our finding that the reduction of IP-10 protein in IEC is the result of a post-transcriptional mechanism induced by VSL#3. Most importantly, immunohistochemical staining of IP-10 in cecal tissue sections confirmed that VSL#3 strongly reduced IEC-specific IP-10 expression in the cecum of IL102/2 mice (Figure 8D). Discussion The present study shows for the first time bacterial strain- specific effects of the clinically relevant probiotic mixture VSL#3 on the expression of a pro-inflammatory chemokine in primary PLoS ONE | www.plosone.org 12 February 2009 | Volume 4 | Issue 2 | e4365