Inhibitor of Apoptosis Proteins as Novel Targets in Inflammatory Processes

Objective: Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NFκB signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. Methods and Results: In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNFα-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte– endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NFκB signaling but decreased the TNFα-induced activation of the TGF-β–activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. Conclusion: Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells

Regulatory T cells control the Th1 immune response in murine crescentic glomerulonephritis

Crescentic glomerulonephritis is mediated by inappropriate humoral and cellular immune responses toward self-antigens that may result from defects in central and peripheral tolerance. Evidence now suggests that regulatory T cells (Tregs) may be of pathophysiological importance in proliferative and crescentic forms of glomerulonephritis. To analyze the role of endogenous Tregs in a T cell-dependent glomerulonephritis model of nephrotoxic nephritis, we used ‘depletion of regulatory T cell' (DEREG) mice that express the diphtheria toxin receptor under control of the FoxP3 (forkhead box P3) gene promoter. Toxin injection into these mice efficiently depleted renal and splenic FoxP3+ Treg cells as determined by fluorescent-activated cell sorting (FACS) and immunohistochemical analyses. Treg depletion exacerbated systemic and renal interferon-γ (IFNγ) expression and increased recruitment of IFNγ-producing Th1 cells into the kidney without an effect on the Th17 immune response. The enhanced Th1 response, following Treg cell depletion, was associated with an aggravated course of glomerulonephritis as measured by glomerular crescent formation. Thus, our results establish the functional importance of endogenous Tregs in the control of a significantly enhanced systemic and renal Th1 immune response in experimental glomerulonephritis

Sharpin Contributes to TNFα Dependent NFκB Activation and Anti-Apoptotic Signalling in Hepatocytes

TNFα stimulates both pro- and anti-apoptotic signalling in hepatocytes. Anti-apoptotic signalling depends on a cascade of ubiquitylation steps leading to NFκB activation. Using Sharpin-deficient mice, we show that the ubiquitin binding protein Sharpin interacts with Hoip, an E3 ligase which generates linear ubiquitin chains. Sharpin-deficiency sensitized hepatocytes to induction of apoptosis by TNFα even in the absence of transcriptional inhibition. TNFα induced activation of NFκB was strongly reduced in hepatocytes from Sharpin-deficient mice, due to reduced and delayed phosphorylation and degradation of IκBα. Injection of TNFα-inducing lipopolysaccharides led to strongly exacerbated liver damage and premature death in Sharpin-deficient mice. Our findings point to an essential role of Sharpin in linear ubiquitin chain formation, NFκB activation, and protection of the liver against inflammatory damaging signals

Toleranzinduktion in der Leber nach T- und NKT-Zellaktivierung

Concanavalin A (Con A)-induced liver failure serves as model for immune-mediated hepatic injury, in particular for human autoimmune hepatitis, since the murine model and the human disease have some features in common such as good responsiveness to immunosuppressive drugs, genetic prevalence with respect to susceptibility, prevalence of CD4+ T cells, and immunosuppression in state of remission. This study describes new aspects of mechanisms of immunosuppression following Con A restimulation and especially elucidates the role of IL-10-producing CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells in development of Con A tolerance. 1. Con A restimulation induced tolerance against Con A within one week. The typical pro-inflammatory Th1/Th17 cytokine response detected in Con A hepatitis shifted to a Th2 response with downregulation of IFNg, TNFa, IL-2, IL-6 and IL-17 and a simultaneous augmentation of IL-10 expression. Moreover, transaminase activities and liver necrosis were clearly diminished indicating an attenuated liver damage. 2. Con A tolerance developed within 8 days after the first Con A administration and persisted for several weeks suggesting a long-lasting process. 3. In vivo depletion of both CD4+CD25+ Tregs by anti-CD25 mAb and KCs by clodronate-liposomes significantly reduced the tolerization-induced IL-10 release suggesting these two populations as main source of IL-10. Con A pretreatment appears to convert CD4+ T cells into IL-10-producing FoxP3+ regulatory T cells and KCs from type I macrophages into IL-10-secreting type II macrophages, respectively. 4. CD4+CD25+ Tregs from Con A-tolerized mice significantly ameliorated Con A induced hepatitis in wt mice in an IL-10-dependent manner, since tolerized Tregs from IL10-/- mice failed to reduce Con A liver injury. Interestingly, the adoptively transferred CD4+CD25+ Tregs were FoxP3-positive in contrast to peripherally induced ‘IL-10 Tregs’, which have recently been identified to exert therapeutic effects in other mouse models of autoimmune diseases. 5. The necessity of IL-10 during establishment of Con A tolerance seems to depend on gender, since female IL10-/- mice were still able to develop Con A tolerance in contrast to male IL10-/- mice, correlating with gender-related differences in humans regarding the incidence of autoimmune diseases. 6. CD4+CD25+ regulatory T cells from tolerized mice also exhibited a higher suppressive activity in vitro, since they inhibited cytokine-production of cocultured CD4+CD25- responder cells more efficiently than naïve Tregs. Moreover, in vitro neutralization or lack of IL-10 failed to reverse the immunosuppressive capacity of Tregs whereas IL-10 was indispensable in vivo in the present study. 7. NKT cells are essential for Con A hepatitis due to their pronounced IFNg release. However, NKT-cell deficient CD1d-/- mice developed Con A tolerance, thereby excluding NKT cells as tolerance-mediating cell population, although the frequency of intrahepatic NKT cells is significantly elevated in Con A-treated mice at day 8 following intervention. In summary, Con A pretreatment caused an immunosuppressive milieu followed by induction of Con A tolerance upon restimulation. The tolerogenic state was characterized by an anti-inflammatory cytokine profile with elevated IL-10 release, which was mainly produced by Kupffer cells and CD4+CD25+FoxP3+ Tregs. The latter population primed by the first Con A challenge ameliorated fulminant Con A-induced hepatitis in an IL-10-dependent manner. In contrast, in vitro Treg-mediated suppression was mediated cytokine-independent.Die Concanavalin A (Con A)-induzierbare, T- und NKT-Zell-vermittelte Leberschädigung bei der Maus dient als Modell für immunvermittelte Lebererkrankungen beim Menschen. Es spiegelt vor allem das Krankheitsbild der Autoimmunhepatitis sehr gut wider, da das Mausmodell und die humane Erkrankung einige Gemeinsamkeiten aufweisen wie zum Beispiel die gute Ansprechbarkeit auf Immunsuppressiva, die genetische Prävalenz, die Abhängigkeit von CD4+ T-Zellen und Immunsuppression in der Remissionsphase. Diese Arbeit beschreibt neue Aspekte immunsuppressiver Mechanismen nach Con A-Restimulation und untersucht im Besonderen die Rolle von IL-10- produzierenden CD4+CD25+FoxP3+ regulatorischen T-Zellen und Kupffer-Zellen bei der Entwicklung der Con A-Toleranz. 1. Con A-Injektion induzierte innerhalb einer Woche Toleranz gegenüber Con A-Restimulation. Die bei der Con A-vermittelten Hepatitis ausgeprägte proinflammatorische Th1/Th17-Antwort verschob sich zugunsten einer Th2-Antwort. Eine verminderte IFNg−, TNFa−, IL-2-, IL-6- und IL-17-Produktion ging mit einem Anstieg des immunosuppressiven Zytokins IL-10 einher. 2. Con A-Toleranz entwickelte sich ab Tag 8 nach der ersten Con A-Gabe und hielt über mehrere Wochen an, was für einen langfristigen Prozess spricht. 3. In vivo-Depletion regulatorischer T-Zellen mittels anti-CD25-Antikörpern und Depletion der Kupffer-Zellen mit Hilfe von Clodronat-Liposomen führte zu einem signifikanten Rückgang der IL-10-Freisetzung in Con Avorbehandelten Tieren. Somit stellten diese beiden Zellpopulationen die Hauptproduzenten von IL-10 dar. Es wird postuliert, dass die Con A-Vorbehandlung eine FoxP3-Expression und gesteigerte IL-10-Freisetzung in herkömmlichen CD4+ T-Zellen induziert hat. Zusätzlich veränderten Kupffer-Zellen ihren Phänotyp von Typ I-Makrophagen zu IL-10-produzierenden Typ II-Makrophagen. 4. CD4+CD25+ regulatorische T-Zellen aus Con A-toleranten Mäusen schützten signifikant vor einem Con A-induzierten Leberschaden. Der therapeutische Effekt hing von IL-10 ab, da regulatorische T-Zellen aus Con A-vorbehandelten IL-10 KO-Mäusen nicht in der Lage waren, den Leberschaden zu reduzieren. Interessanterweise exprimierten die injizierten Zellen den Transkriptionsfaktor FoxP3, was den spezifischen Marker natürlich-vorkommender, im Thymus gereifter regulatorischer T-Zellen darstellt. Normalerweise sind in der Peripherie induzierte regulatorische T-Zellen, die in anderen Mausmodellen für Autoimmunerkrankungen therapeutische Effekte mit Hilfe von IL-10 erzielen, FoxP3-negativ. 5. Die Notwendigkeit von IL-10 für die Ausprägung der Con A Toleranz hängt möglicherweise vom Geschlecht ab, da weibliche IL-10 KO-Mäuse trotz fehlendem IL-10 Toleranz entwickelten im Gegensatz zu männlichen IL-10 KO-Mäusen. Solche geschlechtsspezifischen Unterschiede kann man auch bei humanen Autoimmunerkrankungen beobachten, da in vielen Fällen Frauen eine stärkere Prävalenz aufweisen. 6. Zusätzlich zeichneten sich CD4+CD25+ regulatorische T-Zellen aus toleranten Mäusen durch erhöhte Suppressionskapazität in vitro aus. Sie inhibierten die Zytokinantwort von CD4+CD25- Effektor-T-Zellen stärker als naive regulatorische T-Zellen. In vitro-Neutralisation von IL-10 hob die Suppressionseigenschaften der regulatorischen T-Zellen nicht auf, wohingegen IL-10 für immunmodulatorische Effekte in vivo unabdinglich war. 7. NKT-Zellen sind für einen Con A-vermittelten Leberschaden aufgrund der IFNg-Freisetzung notwendig. NKT-Zell-defiziente CD1d KO-Mäuse waren jedoch in der Lage, Con A-Toleranz zu entwickeln. Deshalb konnte man NKT-Zellen als eine mögliche Toleranz-vermittelnde Zellpopulation ausschließen, obwohl sich die Anzahl der NKT-Zellen in Con Abehandelten Mäusen an Tag 8 nahezu verdoppelt hatte. Zusammenfassend kann man feststellen, dass eine Vorbehandlung mit Con A eine Immunaktivierung hervorrief, die bei Restimulation Toleranz gegenüber Con A induziert hat. Das Toleranzstadium war durch ein anti-inflammatorisches Zytokinprofil und erhöhte IL-10-Produktion gekennzeichnet. IL-10 wurde größtenteils von Kupffer-Zellen und CD4+CD25+FoxP3+ regulatorischen T-Zellen freigesetzt. Die durch die erste Con A-Gabe polarisierten regulatorischen T-Zellen zeigten in vivo therapeutische Effekte, da sie einen Con A-vermittelten Leberschaden mit Hilfe von IL-10 verbessern konnten, wohingegen die suppressive Aktivität dieser Zellpopulation in vitro nicht von IL-10 abhing