Year: 2007
Type: dissertation
Abstract: In this study we used IL-10 gene deficient mice monoassociated with Gram-positive Enterococcus faecalis in order to characterize bacteria-epithelial interactions and host-derived mechanisms involved in the regulation of chronic intestinal inflammation in genetically susceptible host. We showed that intestinal epithelial cells (IEC) of IL-10-/- mice lack protective TGFSmad signalling and fail to inhibit expression of NF-B-dependent genes of pro-inflammatory cytokines. In addition, the molecular mechanism for negative regulation of TLR2 expression by TGF/Smad signalling was described in IEC. Protein expression profiling was performed in native IEC isolated from colonic tissue of wild type and IL-10-/- mice after 2 and 14 weeks of monoassociation with E. faecalis. Consistent with the presence of histopathology and persistent TLR2-mediated NF-B activation in IEC of IL-10-/- mice, proteome analysis revealed divergent protein expression changes between WT and IL-10-/- mice at early and late colonization time points. Down-regulation of important metabolic, detoxification and immunregulatory proteins was associated with induction of endoplasmic reticulum stress (ER) in IEC of IL-10-/- mice under pathologic conditions. We performed functional analysis of galectin-3 and molecular chaperone grp-78 regulated in IL-10-/- mice after 14 weeks of bacterial colonization. Using specific siRNA knock-down in intestinal epithelial cell line Mode-K we investigated functions of galectin-3 and molecular chaperone grp-78 in order to explain their contribution and physiologic relevance for disease development. We described a novel molecular mechanism of ER-stress response regulation mediated by IL-10 and grp-78. Increased expression of grp-78 was found in IEC of IL-10-/- mice after 14 weeks of bacterial colonization. Grp-78 gene targeting in Mode-K cell line showed that the absence of grp-78 modulated NF-B signalling. We presented an evidence that grp-78 is recruited to the IKK complex upon TNF-stimulation. In addition, we found that IL-10 was able to down-regulate transcriptional activity of grp-78 via the inhibition of ATF6 nuclear translocation and binding to the grp-78 promoter. Pharmacological p-38 MAPK inhibitor blocked IL-10-mediated inhibition of ATF6 recruitment to grp-78 promoter in TNF-stimulated IL-10-receptor reconstituted Mode-K cells suggesting the involvement of p-38 MAPK signalling in regulation of ER-stress response. Specific gene targeting of another protein galectin-3 in Mode-K cell line resulted in the induction of cleaved caspase-3, suggesting anti-apoptotic functions of this protein in IEC. Consistent with this finding galectin-3 down-regulation in native IEC of IL-10-/-mice correlated with increased caspase-3 expression and down-regulation of E-cadherin. Thus, protein expression profiling in native IEC revealed involvement of ER-stress, energy depletion and disturbance of mitochondrial homeostasis in the pathogenesis of experimental colitis in IL-10-/- mice. Finally, we performed a pilot study with a limited number of IBD patients to apply proteome analysis in human disease. We used native IEC isolated from inflamed mucosal tissue of patients with Crohn’s disease (CD) and ulcerative colitis (UC). We identified Rho GDIprotein to be significantly up-regulated in inflamed mucosa of CD and UC patients in comparison to control non-inflamed samples from colon carcinoma patients. We also conducted proteome analysis in inflamed versus non-inflamed mucosal tissue separately obtained from CD and UC patients. This study is aimed to identify potential protein markers involved in etiopathogenesis of human IBD.
Author: Anna Shkoda
Language: English
Topic: Inflammatory Bowel Disease
Subfield: Gastroenterology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2752067945