Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content

Galectin-4 Controls Intestinal Inflammation by Selective Regulation of Peripheral and Mucosal T Cell Apoptosis and Cell Cycle

Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease

The role of Galectin-2 and Galektin-4 on the mucosal immune system

1\. Einleitung 4 1.1. Aufbau und Funktion der intestinalen Barriere 4 1.2. Chronisch entzündliche Darmerkrankungen 7 1.3. Galektine 8 1.4. Galektin-2 und Galektin-4 11 1.5. Zielsetzung der Arbeit 12 2\. Ergebnisse 13 2.1. Galectin-2 and -4, but not Galectin-1, promote intestinal epithelial wound healing in vitro through a TGF-beta-independent mechanism 13 2.1.1. Zusammenfassung 13 2.1.2. Publikation 14 2.2. Galectin-4 controls intestinal inflammation by selective regulation of peripheral and mucosal T cell apoptosis and cell cycle 15 2.2.1. Zusammenfassung 15 2.2.2. Publikation 16 2.3. Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice 17 2.3.1. Zusammenfassung 17 2.3.2. Publikation 18 3\. Diskussion 19 4\. Zusammenfassung 24 5\. Summary 26 Literaturverzeichnis 28 Publikationsliste 32 Anhang 33 Abkürzungen 33Bei chronisch entzündlichen Darmerkrankungen handelt es sich um Erkrankungen, die durch eine überschießende Immunantwort gekennzeichnet sind. Diese ist verbunden mit einer gestörten T-Zellapoptose. Das daraus resultierende Ungleichgewicht zwischen Proliferation und Apoptose sorgt für eine anhaltende Entzündungsreaktion in der intestinalen Mukosa. Im Verlauf der Erkrankung kommt es zu Schäden an der Mukosa, welche konsekutiv ihre Barrierefunktion verliert. Galektine sind zuckerbindende Lektine, die auf vielfältige Art Prozesse, wie z.B. Zellaktivierung, Proliferation, Zytokinsekretion, Migration und Apoptose, beeinflussen. Daher wurde in der vorliegenden Arbeit der Einfluss von Gal-2 und Gal-4 auf das mukosale Immunsystem in vivo und in vitro untersucht. Diese beiden Galektine wurden gewählt, da sie nahezu exklusiv im Gastrointestinaltrakt exprimiert werden. Anhand eines etablierten Wundheilungsmodells mit Epithelzellen konnte gezeigt werden, dass Gal-2 und Gal-4 die intestinale Epithelzellrestitution fördern. Dabei wurden die Migration und Proliferation intestinaler Epithelzellen gesteigert. Untersuchungen an T-Zellen aus dem peripheren Blut und aus der Lamina propria zeigten, dass Gal-2 an den β1-Integrin und Gal-4 an den CD3-Rezeptor auf der T -Zell-Oberfläche bindet. Die Interaktion beider Galektine mit T-Zellen initiiert eine gesteigerte Apoptose stimulierter, jedoch nicht ruhender T-Zellen. Die durch Gal-2 induzierte Apoptose verläuft über die Aktivierung von Caspase-3 und -9. Gal-4 induziert Apoptose caspase-unabhängig über einen calpain-abhängigen Signalweg. Weiterhin ergaben Untersuchungen der Zytokinsekretion eine Hemmung proinflammatorischer Zytokine, wie z.B. TNF-α, IL-6, IL-8 und IL-17 durch beide Galektine. Die Ergebnisse der Zellzyklusanalysen zeigten eine Hemmung des Zellzyklus von T-Zellen durch Gal-4, jedoch nicht durch Gal-2. In vivo Untersuchungen in einem DSS- induzierten Kolitis Modells in der Maus zeigten, dass es durch Gal-2 und Gal-4, im Vergleich zur Kontrollgruppe, zu einer signifikanten Verbesserung der klinischen und histologischen Entzündungsreaktion kommt. Dabei konnte eine verminderte Infiltration von inflammatorischer Zellen in den Darm sowie eine Abnahme der Mukosaschädigung gezeigt werden. Zusätzlich wurde durch die Gal-4-Behandlung die Anzahl proliferierender Zellen in der Mukosa, gesenkt. Untersuchungen der Lymphozytenfunktionalität durch Analyse der Zytokinsekretion ergaben eine verringerte Sekretion proinflammatorischer Zytokine. Gleichzeitig war das antiinflammatorische Zytokin IL-10 bei der Behandlung mit Gal-4 erhöht. Untersuchungen zur Apoptose zeigten eine signifikante Steigerung apoptotischer CD3-positiver Zellen in der Mukosa nach Behandlung mit Gal-2 und Gal-4. Diese Ergebnisse legen eine zentrale Rolle von Gal-2 und Gal-4 auf unterschiedliche Prozesse und Zellarten im mukosalen Immunsystem dar.Inflammatory bowel diseases are characterised by an uncontrolled immune reaction in common with an impaired T cell apoptosis. The resulting imbalance between proliferation and apoptosis induces a persistent inflammation in the intestinal mucosa. Furthermore, the persistant mucosal damage impairs the epithelial cell function and its barrier function. In our work we analysed the effect of galectin-2 and galectin-4 on the mucosal immune system. Galectins are a family of sugar-binding lectins which more and more emerged as major regulator of different signaling processes, including cell activation, proliferation, cytokine secretion, migration and apoptosis. Since galectin-2 and galectin-4 are mainly expressed in the gastrointestinal tract, we focussed on those two lectins. A wound healing assay with epithelial cells showed that galectin-2 and -4 promote epithelial cell restitution, with an increase in cell migration and cell proliferation. Analysis of the interaction of both galectins with peripheral blood T cells revealed that galectin-2 binds to the β1-integrin at the cell surface while galectin-4 binds at the CD3 Rezeptor of stimulated T cells. Both galectins induced apoptosis of stimulated, but not resting T cells. Galectin-2 induced apoptosis via caspsae-3 and caspase-9 dependent pathways. In contrast, apoptosis induced by galectin-4 was caspase- independent, but calpain-dependent. Determination of the cytokine secretion profile of T cells revealed a decrease in pro-inflammatory cytokines, like TNF-α, IL-6, IL-8 und IL-17, when incubated with galectin-2 or galectin-4. Cell cycle analyses showed an inhibitory effect of galectin-4 on cell cycle progression of T cells, while galectin-2 did not influence the cell cycle. In a model of DSS-induced colitis in mice, galectin-2 and -4 treatment significantly ameliorated clinical and histological signs of colitis compared to controls. Both lectins reduced the number of inflammatory cells migrated into the mucosa and diminished mucosal damage. In addition, galectin-4 decreased the number of proliferating cells in the intestinal mucosa. Analyses of the cytokine secretion profile showed a lowered secretion of pro- inflammatory cytokines. Furthermore, the anti-inflammatory cytokine IL-10 was increased in mice treated with galectin-4. Determination of apoptosis in mice treated with galectin-2 or galectin-4 showed a significant increase in apoptotic CD3 positive cells in the mucosa. Thus, our study provided for the first time evidence that galectin-2 and galectin-4 play a central and distinct role in the mucosal immune system

Extracellular Matrix Components as Diagnostic Tools in Inflammatory Bowel Disease

Work from the last years indicates that the extracellular matrix (ECM) plays a direct role in various cellular processes, including proliferation, migration and differentiation. Besides homeostatic processes, its regulatory function in inflammation becomes more and more evident. In inflammation, such as inflammatory bowel disease, the ECM composition is constantly remodeled, and this can result in a structuring of fistulizing disease course. Thus, tracking early ECM changes might bear the potential to predict the disease course. In this review, we provide an overview of relevant diagnostic methods, focusing on ECM changes

Galectin-4 induces T cell apoptosis caspase-independently via calpain-mediated pathways.

<p>(A) PBT and LPT were stimulated with anti-CD3/CD28 or -CD2, respectively, in the presence of 0, 50, 100 or 200 µg/ml Gal-4 and apoptosis was determined by annexin-V staining. Data represent mean±SEM of eight individual experiments. *p≤0.05 for increase vs. baseline. (B) PBT were stimulated in the presence of 0, 25, 50, 100 or 200 µg/ml Gal-4. Apoptosis was detected by annexin V/PI staining, necrosis by PI staining followed by flow cytometry. Data represent mean±SEM of six individual experiments. *p≤0.05 for increase vs. baseline. (C) PBT were transfected with Gal-4 siRNA or scrambled control, activated by anti-CD3/CD28 and cultured in the presence of the TNF-α blocker adalimumab for three days. Apoptosis was detected by annexin V/PI staining followed by flow cytometry. Data are representative for three individual experiments. (D) Caspase-3, -8, and -9 activity in anti-CD3/CD28 stimulated T cells cultured in the presence or absence of 100 µg/ml Gal-4. Data are representative for three individual experiments. (E) PBT were activated by anti-CD3/CD28 and cultured for 24 h in the presence or absence of 100 µg/ml Gal-4 and 50 mM Calpain inhibitor z-LLY-fmk, 4 mM EGTA or 30 mM BAPTA-AM. Data are representative for three individual experiments. (F) Disruption of the mitochondrial membrane potential was detected by rhodamine123 staining followed by flow cytometric analysis. T cells were stimulated with anti-CD3/CD28 and incubated for 3 h in the presence or absence of 100 µg/ml Gal-4. Afterwards cells were analysed by flow cytometry. All data are representative for three individual experiments. (G) Disruption of the mitochondrial membrane potential was detected after 12 h by rhodamine123 staining followed by flow cytometric analysis. All data are representative for three individual experiments.</p

Galectin-4 binds to stimulated, but not resting T cells.

<p>(A) Flow cytometric analysis of Gal-4 binding to resting and anti-CD3/CD28 stimulated T cells using Gal-4 detected by an anti-Gal-4 Ab and PE-labeled secondary Ab. Carbohydrate-dependence of the binding was analysed by addition of 50 mM lactose as a pan-galectin inhibitor and 50 mM sucrose as control. Data are representative of three individual experiments. (B) Immunoprecipitation of Gal-4 binding complexes. BSA served as negative, Gal-1 as positive control. Data are representative for four individual experiments.</p

Identification of Galectin-4 expression.

<p>(A) Gal-4 expression was detected in cryosections of the GI tract of healthy volunteers. The results are representative for four individuals (original magnifications: ×100). (B) PCR analysis of Gal-4 mRNA expression in resting and anti-CD3/CD28 stimulated PBT. (C) Flow cytometric analysis of intra- and extracellular Gal-4 content in resting and anti-CD3/CD28 stimulated PBT. Data are representative of three individual experiments.</p