Pedro A. Ruiz

IL-10 Gene-Deficient Mice Lack TGF-β/Smad Signaling and Fail to Inhibit Proinflammatory Gene Expression in Intestinal Epithelial Cells after the Colonization with Colitogenic Enterococcus faecalis

Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10−/−). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10−/− mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10−/− mice. Accordingly, wild-type IEC expressed phosphorylated NF-κB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10−/− mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10−/− IEC. Of note, TGF-β1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-κB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10−/−, but not wild-type mice lack protective TGF-β/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-β in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.

Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats

Bifidobacteria comprise a dominant microbial population group in the human intestinal tract with purported beneficial health effects on the host. In this study, we characterized the molecular mechanisms for the initial interaction of probiotic Bifidobacterium lactis strain BB12 with native and intestinal epithelial cell (IEC) lines. We showed that B. lactis‐monoassociated Fisher F344 rats transiently induce phosphorylation/activation of the NF‐κB transcriptionally active subunit RelA and the mitogen‐activated protein kinase (MAPK) p38 in native IEC at day 5 after initial bacterial colonization. In addition, Interleukin 6 (IL‐6) gene expression was significantly increased at day 5, demonstrating the physiological relevance of transient transcription factor activation in IEC. In contrast, Bacteroides vulgatus‐monoassociated Fisher rats revealed RelA but not p38 MAPK phosphorylation and failed to trigger significant IL‐6 gene expression in native IEC. Moreover, we demonstrated that B. lactis triggers NF‐κB RelA and p38 MAPK phosphorylation in IEC lines. Adenoviral delivery of mutant IKK‐β (Ad5dnIKKβ) and inhibition of the p38 MAPK pathway through the pharmacological inhibitor SB203580 significantly blocked B. lactis‐induced IL‐6 gene expression in IEC, suggesting that B. lactis triggers NF‐κB and MAPK signaling to induce gene expression in the intestinal epithelium. Regarding the mechanisms of bacteria epithelial cell cross‐talk, B. lactis‐induced IL‐6 gene expression was completely inhibited in TLR2 deficient mouse embryogenic fibroblasts (MEF TLR2−/−) as well as TLR2ΔTIR transfected Mode‐K cells. In conclusion, we demonstrated that probiotic bacteria transiently trigger innate signal transduction and pro‐inflammatory gene expression in the intestinal epithelium at early stages of bacterial colonization.

IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.

Abstract:  Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in interleukin‐10 geneeficient mice (IL‐10‐/‐). Bacteria‐specific effects on the epithelium are difficult to distinguish because of the complex nature of the gut microflora. We showed that IL‐10‐/‐ mice compared to wild‐type mice fail to inhibit pro‐inflammatory gene expression in native intestinal epithelial cells after the colonization with colitogenic Gram‐positive Enterococcus faecalis. Of interest, pro‐inflammatory gene expression was transient after 1 week of E. faecalis monoassociation in IECs from wild‐type mice but persisted after 14 weeks of bacterial colonization in IL‐10‐/‐ mice. Accordingly, wild‐type IECs expressed phosphorylated NF‐kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis‐monoassociated IL‐10‐/‐ mice triggered persistent RelA but no Smad2 phosphorylation in IECs at days 3, 7, 14, and 28. Consistent with the induction of TLR2‐mediated RelA phosphorylation and pro‐inflammatory gene expression in E. faecalis‐stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis‐monoassociated wild‐type mice but persisted in IL‐10‐/‐ IECs. Of note, TGF‐beta‐activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF‐kappa Bependent gene expression in E. faecalis‐stimulated IEC lines. In conclusion, E. faecalis‐monoassociated IL‐10‐/‐ but not wild‐type mice lack protective TGF‐beta/Smad signaling and fail to inhibit TLR2‐mediated pro‐inflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL‐10 and TGF‐beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.

Role of the adipocyte-specific NF-κB activity in the regulation of IP-10 and T cell migration

Infiltration of immune cells into adipose tissue plays a central role in the pathophysiology of obesity-associated low-grade inflammation. The aim of this study was to analyze the role of adipocyte NF-κB signaling in the regulation of the chemokine/adipokine interferon-γ-induced protein 10 kDa (IP-10) and adipocyte-mediated T cell migration. Therefore, the regulation of IP-10 was investigated in adipose tissue of male C57BL/6J mice, primary human and 3T3-L1 preadipocytes/adipocytes. To specifically block the NF-κB pathway, 3T3-L1 cells stably overexpressing a transdominant mutant of IκBα were generated, and the chemical NF-κB inhibitor Bay117082 was used. Adipocyte-mediated T cell migration was assessed by a migration assay. It could be shown that IP-10 expression was higher in mature adipocytes compared with preadipocytes. Induced IP-10 expression and secretion were completely blocked by an NF-κB inhibitor in 3T3-L1 and primary human adipocytes. Stable overexpression of a transdominant mutant of IκBα in 3T3-L1 adipocytes led to an inhibition of basal and stimulated IP-10 expression and secretion. T cell migration was induced by 3T3-L1 adipocyte-conditioned medium, and both basal and induced T cell migration was strongly inhibited by stable overexpression of a transdominant IκBα mutant. In addition, with the use of an anti-IP-10 antibody, a significant decrease of adipocyte-induced T cell migration was shown. In conclusion, in this study, we could demonstrate that the NF-κB pathway is essential for the regulation of IP-10 in 3T3-L1 and primary human adipocytes. Adipocytes rather than preadipocytes contribute to NF-κB-dependent IP-10 expression and secretion. Furthermore, NF-κB-dependent factors and especially IP-10 represent novel signals from adipocytes to induce T cell migration.

Gp96 deficiency affects TLR4 functionality and impairs ERK and p38 phosphorylation

Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn’s disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.

Iron prevents hypoxia-associated inflammation through the regulation of nuclear factor κB in the intestinal epithelium

Background & Aims Hypoxia-associated pathways influence the development of inflammatory bowel disease. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are regulated by iron-dependent hydroxylases. Signals reflecting oxygen tension and iron levels in enterocytes regulate iron metabolism. Conversely, iron availability modulates responses to hypoxia. In the present study we sought to elucidate how iron influences the responses to hypoxia in the intestinal epithelium. Methods Human subjects were exposed to hypoxia, and colonic biopsy specimens and serum samples were collected. HT-29, Caco-2, and T84 cells were subjected to normoxia or hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signaling were assessed by quantitative polymerase chain reaction and Western blot. Chromatin immunoprecipitation was performed using antibodies against nuclear factor (NF)-κB and primers for the promoter of tumor necrosis factor (TNF) and interleukin (IL)1β. Results Human subjects presented reduced levels of ferritin in the intestinal epithelium after hypoxia. Hypoxia reduced iron deprivation–associated TNF and IL1β expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL1β expression, and NF-κB activation in Caco-2 and T84 cells. Iron blocked autophagy in Caco-2 cells, while reducing hypoxia-associated TNF and IL1β expression through the inhibition of NF-κB binding to the promoter of TNF and IL1β. Conclusions Hypoxia promotes iron mobilization from the intestinal epithelium. Hypoxia-associated autophagy reduces inflammatory processes in HT-29 cells. In Caco-2 cells, iron uptake is essential to counteract hypoxia-induced inflammation. Iron mobilization into enterocytes may be a vital protective mechanism in the hypoxic inflamed mucosa.

P102 Hypoxia reduces inflammation through the downregulation of NLRP3/mTOR signaling and the activation of autophagy

P102 Hypoxia reduces inflammation through the downregulation of NLRP3/mTOR signaling and the activation of autophagy J. Cosin-Roger*1, S. Simmen1, H. Melhem1, K. Atrott1, I. Frey-Wagner1, M. Hausmann1, C. de Vallière1, M.R. Spalinger1, P. Spielmann2,3, R.H. Wagner2,3, J. Zeitz1, S.R. Vavricka1, G. Rogler1, P.A. Ruiz1 1University of Zurich, Gastroenterology, Zurich, Switzerland; 2Institute of Physiology, University of Zurich, Zurich, Switzerland; 3Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland