Vadim Iablokov

Intrarectal Instillation of Clostridium difficile Toxin A Triggers Colonic Inflammation and Tissue Damage: Development of a Novel and Efficient Mouse Model of Clostridium difficile Toxin Exposure

ABSTRACT Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells

Proteinase-activated receptor (PAR)(2), a G protein-coupled receptor activated by serine proteinases, has been implicated in both intestinal inflammation and epithelial proliferation. Cyclooxygenase (COX)-2 is overexpressed in the gut during inflammation as well as in colon cancer. We hypothesized that PAR(2) drives COX-2 expression in intestinal epithelial cells. Treatment of Caco-2 colon cancer cells with the PAR(2)-activating peptide 2-furoyl-LIGRLO-NH(2) (2fLI), but not by its reverse-sequence PAR(2)-inactive peptide, for 3 h led to an increase in intracellular COX-2 protein expression accompanied by a COX-2-dependent increase in prostaglandin E(2) production. 2fLI treatment for 30 min significantly increased metalloproteinase activity in the culture supernatant. Increased epidermal growth factor receptor (EGFR) phosphorylation was observed in cell lysates following 40 min of treatment with 2fLI. The broad-spectrum metalloproteinase inhibitor marimastat inhibited both COX-2 expression and EGFR phosphorylation. The EGFR tyrosine kinase inhibitor PD153035 also abolished 2fLI-induced COX-2 expression. Although PAR(2) activation increased ERK MAPK phosphorylation, neither ERK pathway inhibitors nor a p38 MAPK inhibitor affected 2fLI-induced COX-2 expression. However, inhibition of either Src tyrosine kinase signaling by PP2, Rho kinase signaling by Y27632, or phosphatidylinositol 3 (PI3) kinase signaling by LY294002 prevented 2fLI-induced COX-2 expression. Trypsin increased COX-2 expression through PAR(2) in Caco-2 cells and in an EGFR-dependent manner in the noncancerous intestinal epithelial cell-6 cell line. In conclusion, PAR(2) activation drives COX-2 expression in Caco-2 cells via metalloproteinase-dependent EGFR transactivation and activation of Src, Rho, and PI3 kinase signaling. Our findings provide a mechanism whereby PAR(2) can participate in the progression from chronic inflammation to cancer in the intestine.

Proteinase-activated Receptor 2 (PAR2) Decreases Apoptosis in Colonic Epithelial Cells

Background: Inflammatory bowel disease management lacks therapies that heal the epithelial barrier. Results: PAR2 activation increases activities of MEK1/2 and PI3K in intestinal epithelial cells, which blocks apoptosis. Conclusion: Cytokine-induced apoptosis in colonic epithelial cells is inhibited by PAR2 signaling. Significance: PAR2 is important in maintaining intestinal epithelial homeostasis. Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser112 and Ser136 by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.

The Canadian MD/PhD training program needs reinstated support.

The Canadian Institutes of Health Research (CIHR) recently terminated its MD/PhD training program without clear alternative funding in place. This misguided decision must urgently be reversed, as it has the potential to diminish a unique pool of graduates at the forefront of translational research.

The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohns disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription

Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms involved are not known. We hypothesized that AQP3 expression in intestinal epithelial cells is altered in intestinal inflammation and that these changes are driven by tumor necrosis factor (TNF) α. Human colonic adenocarcinoma (HT‐29) cells were treated with TNFα to investigate signaling mechanisms in vitro. AQP3 expression was assessed by real‐time PCR and radiolabeled glycerol uptake, with select inhibitors and a luciferase reporter construct used to further elucidate intracellular signaling. AQP3 expression was downregulated in HT‐29 cells treated with TNFα. Luciferase reporter construct experiments revealed that TNFα downregulated constitutive transcriptional activity of the AQP3 promoter, and inhibition of MEK/ERK and nuclear factor κB (NF‐κB) signaling prevented the decrease in AQP3 mRNA expression. Constitutive AQP3 expression was suppressed by specificity protein (Sp) 3, and knockdown of this transcription factor bound to the AQP3 promoter was able to partially prevent the TNFα‐induced downregulation of AQP3. TNFα signals through MEK/ERK and NF‐κB to enhance the negative transcriptional control of AQP3 expression exerted by Sp3. Similar mechanisms regulate numerous ion channels, suggesting a common mechanism by which both ion and water transport are altered in inflammation.

Tu1825 Protease-Activated Receptor 2 Exerts a Tonic Anti-Apoptotic Effect in HT-29 Colonic Epithelial Cells

Backround: The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, which has been strongly associated with intestinal repair and the development of several human cancers. Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 10-14. 18Co cells were exposed to TNF-α (10 ng/ml) and EGF (5 ng/ ml) under varying conditions. EGFR protein expression, EGFR tyrosine phosphorylation at Y845 and Y1068, p42/44 MAPK phosphorylation, and COX-2 expression were assessed by Western blot analysis. Live cell imaging was performed using Quantum dot technology. Experiments were also performed using primary myofibroblasts harvested from human colon tissue. Results: Treatment of 18Co cells with TNF-α led to a time-dependent increase in cell surface EGFR expression, an effect that was completely prevented by inhibition of protein synthesis with cycloheximide. Stimulation of up-regulated EGFR with EGF or HBEGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a synergistic increase in COX-2 expression compared to 18Co cells treated with EGF andHB-EGF alone. Importantly, TNF-α also increased EGFR expression in primarymyofibroblasts isolated from human colon tissue. Conclusions: Our results imply that TNF-α induced a significant upregulation of EGFR in human colonic myofibroblasts that is associated with enhanced signaling and increased functional activity (ERK, COX-2). Induction of EGFR expression by TNF-α provides a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes IBD, and which may contribute to a microenvironment that predisposes to colitis-associated cancer.

S1694 Protease-Activated Receptor-2 Reduces TNFα-Induced Apoptosis in Colonic Epithelial Cells

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.