In Inflammatory Bowel Disease (IBD), the colon and small intestine are chronically inflamed due, at least in part, to increased epithelial permeability and an inappropriate immune response to luminal antigens. IFN-gamma is a prominent pro-inflammatory cytokine that increases permeability of the intestinal epithelium and T-Cell Protein Tyrosine Phosphatase (TCPTP) is a negative regulator of IFN-gamma signaling. This research explores the effects of a probiotic preparation on TCPTP abundance, resolution of inflammatory signaling, and intestinal epithelial permeability by studying a probiotic blend called VSL#3. Research showed that in T84 colonic epithelial cells, VSL#3 was able to increase TCPTP protein levels by 150 ± 3% and enzymatic activity by 100 ± 5% (106 CFU/mL for 9 hours). Moreover, VSL#3 was able to decrease epithelial monolayer permeability by 25 ± 2% over 9 hours (106 CFU/mL). When IFN-gamma and VSL#3 were co-incubated, VSL#3 attenuated the increase in permeability that resulted from 24 hours pre-treatment of T84 cells with IFN-gamma by 120 ± 2%. Moreover, levels of phosphorylated STAT-1 (a downstream effect of IFN-gamma signaling) decreased when VSL#3 and IFN-gamma were co- incubated and TCPTP levels showed an increase as VSL#3 doses were increased. Results were inconclusive as to whether VSL#3's effects on transepithelial resistance in IFN-gamma pre-treated cells were TCPTP-mediated because the TCPTP inhibitor failed to display its expected action. Overall, the findings of this thesis indicate that VSL#3 can repair epithelial permeability defects caused by IFN- gamma and this may be due in part to upregulation of a negative regulator of IFN-gamma signaling, TCPTP

Background

VSL#3 is a probiotic compound that has been used in the treatment of inflammatory bowel disease. T-cell protein tyrosine phosphatase (TCPTP) is the protein product of the inflammatory bowel disease candidate gene, PTPN2, and we have previously shown that it protects epithelial barrier function. The aim of this study was to investigate whether VSL#3 improves intestinal epithelial barrier function against the effects of the inflammatory bowel disease-associated proinflammatory cytokine, interferon-gamma (IFN-γ) through activation of TCPTP.

Methods

Polarized monolayers of T84 intestinal epithelial cells were treated with increasing concentrations of VSL#3 to determine effects on TCPTP expression and enzymatic activity. Therapeutic effects of VSL#3 against barrier disruption by IFN-γ were measured by transepithelial electrical resistance and fluorescein isothiocyanate-dextran permeability. A novel TCPTP-deficient HT-29 intestinal epithelial cell line was generated to study the role of TCPTP in mediating the effects of VSL#3. Tight junction protein distribution was assessed with confocal microscopy.

Results

VSL#3 increased TCPTP protein levels and enzymatic activity, correlating with a VSL#3-induced decrease in IFN-γ signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ. Moreover, the restorative effect of VSL#3 against IFN-γ signaling, epithelial permeability defects, altered expression and localization of the tight junction proteins claudin-2, occludin, and zonula occludens-1, were not realized in stable TCPTP/(PTPN2)-deficient HT-29 intestinal epithelial cells.

Conclusions

VSL#3 reduces IFN-γ signaling and IFN-γ-induced epithelial barrier defects in a TCPTP-dependent manner. These data point to a key role for TCPTP as a therapeutic target for restoration of barrier function using probiotics.