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Crohn´s disease (CD) and Ulcerative colitis (UC) are idiopathic inflammatory disorders. Environmental factors, infectious microbes, ethnic origin, genetic susceptibility, and a dysregulated immune system can result in mucosal inflammation. However, the etiology of both CD and UC still remains largely unclear. Inflammatory bowel diseaserelated animal models suggest that a combination of genetic susceptibility factors and altered immune response driven by microbial factors in the enteric environment may contribute to the initiation and chronification of the disease. The intestinal immune system represents a complex network of different lymphoid and non-lymphoid cell populations as well as humoral factors. In inflammatory bowel disease, the controlled balance of the intestinal immune system is disturbed at all levels. In CD, naïve T cells preferably differentiate into Th1 or Th17 producing cells, while in UC, these cells differentiate into aberrant Th2 cells. Overall, in active inflammatory bowel disease effector T cell activity (Th1, Th17, Th2) predominates over regulatory T cells. Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of CD and UC. When chosen appropriately, these models proved to be a helpful tool to investigate pathophysiological mechanisms, as well as to test emerging therapeutic options in the preclinical phase. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) and oxazolone are the two major chemicals applied to induce Th1- and Th2-skewed intestinal inflammation, respectively. Colitis can be induced in susceptible strains of mice by intrarectal instillation of the haptenating substances TNBS or oxazolone in ethanol, which is necessary for an initial desintegration of the epithelial barrier. TNBS or oxazolone are believed to haptenize colonic autologous or microbiotic proteins rendering them immunogenic to the host immune system. While TNBS administration in the presence of ethanol results in a transmural infiltrative disease in the entire colon based on an IL-12/IL-23 driven, Th1-or Th17 mediated response, oxazolone instillation finally leads to a colitis caused by a polarized Th2 IL-13-dominated lymphocyte response. Rectal oxazolone instillation in ethanol produces a more superficial inflammation that affects the distal half of the colon rather than the whole colon. Therapeutic modulation of the disturbed immune response in patients with inflammatory bowel disease still represents a complex challenge in the clinic. Currently, none of the therapeutic measure are disease specific and they generally target the pathophysiology downstream of the driving immunpathology. So, there is still the need to develop a tailored approach to prevention of the initiation and perpetuation of the inflammatory cascade before tissue injury occurs. One important aspect of this approach might involve the induction or re-establishment of immunological tolerance. FTY720 following rapid phosphorylation to FTY-P by endogenous sphingosine kinases acts as a sphingosine-1-phosphate (S1P) receptor agonist and represents the prototype of a new generation of S1P receptor modulators. While changing currently its proposed mode of action still focus on the fact, that FTY720 effectively inhibits the egress of T-cells from lymph nodes, thereby reducing the number of antigen-primed/restimulated cells that re-circulate to peripheral inflammatory tissues. However, recent studies indicate, that its immunomodulatory properties might be more complex and exerted not only via interactions with other S1P receptor subtypes but also via a direct modulation of the inflammatory capacity of dendritic cells (DC) resulting in a modified regulation of T cell effector functions as well as in an induction of regulatory T cells and function. 1,25(OH)2D3, the active form of vitamin D, is a secosteroid hormone that has in addition to its central function in calcium and bone metabolism pronounced immune regulatory properties. The biological effects of calcitriol are mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear hormone receptors. A number of studies identified calcitriol/VDR as prominent negative regulators of Th1-type immune responses, whereas Th2 responses are not affected or even augmented. These effects have been mainly explained by direct activities on lymphocytes, subsequent studies clearly supported a role of calcitriol in modulating monocyte differentiation or DC maturation. However, to translate the immunosuppressive capacities of calcitriol into an effective immunointervention, a great challenge was the design of structural analogs of calcitriol that are devoid of adverse effects related to hypercalcemic activity. The intense study of the 25-oxa series generated a large number of calcitriol analogs exhibiting substantial dissociation between possible immunomodulatory capacities and undesired hypercalcemia. Especially, the combination of the 22-ene modification with the 25-oxa element as realized in ZK156979 yielded a very promising set of new analogs for further characterization in animal models resembling human autoimmune diseases. So, the overall aim of the studies presented here was to evaluate strategies of enhancing regulatory immunity in mouse models of Th1- and Th2-mediated colitis as a new therapeutic approach. To this end we used FTY720, 22-ene-25-oxa vitamin D (ZK156979), as well as the combination of calcitriol and dexamethasone to evaluate the respective pro-tolerogenic potential in intestinal inflammation models in mice. First, to induce Th1-mediated colitis a rectal enema of TNBS was given to Balb/c mice. FTY720 was administered i.p. from day 0-3 or 3-5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFβ and CTLA4. Moreover, we observed a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4+ T cells of FTY720-treated mice. The impact of FTY720 on regulatory T cell induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. Thus, our data provide new and strong evidence that besides its well-established migratory properties FTY720 down-regulates proinflammatory signals while simultaneously inducing the functional activity of CD4+CD25+ regulatory T cells. In a second approach, the rectal instillation of oxazolone yielded a Th2-mediated colitis. Treatment with FTY720 prominently reduced the clinical and histopathologic severity of oxazolone-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of FTY720 were associated with a prominent reduction of the key Th2 effector cytokines IL-13, IL-4 and IL-5. Moreover, FTY720 inhibited GATA3 and T1/ST2 expression, which represent distinct markers for Th2 differentiation and Th2 effector function. Thus, our data are supportive for the view that FTY720 exhibits beneficial prophylactic as well as therapeutic effects in Th2-mediated experimental colitis by directly affecting Th2 cytokine profiles, probably by reducing GATA3 and T1/ST2. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs showing prominent immunomodulative capacities. Here, we used the Th1-mediated TNBS colitis to test its anti-inflammatory properties in vivo. We found that treatment with ZK156979 clearly inhibited the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated all the clinical macroscopic and microscopic parameters of colitis severity; in addition we observed a clear down-regulation of the relevant Th1 cytokine pattern including the T-box transcription factor, T-bet. On the other hand, application of ZK156979 increased local tissue IL-10 and IL-4. Finally, as a new approach we evaluated the pro-tolerogenic potential of calcitriol and dexamethasone in acute Th1-mediated colitis. Calcitriol and/or dexamethasone were administered i.p. from day 0-3 or from day 3-5 following the instillation of the haptenating agent. The combination of these steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down- while Th2 markers like IL-4 and GATA3 were up-regulated. Clearly distinguishable from known steroid effects calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFß, FoxP3 and CTLA4. Furthermore, analysis of DC mediators responsible for a pro-inflammatory differentiation of T cells revealed a clear reduction of IL-12p70, and IL23p19 as well as IL-6 and IL-17. Thus, our data suggest the concept of a steroid-sparing application of calcitriol derivatives in inflammatory bowel disease. Furthermore, the data presented suggest that early markers of inflammatory DC and Th17 differentiation might qualify as new target molecules for both calcitriol as well as for selective immune modulating vitamin D analogs. In conclusion the data of these published investigations added to the substantial progress in understanding the biology of tolerogenic DC and regulatory T cells with respect to their roles in health and disease achieved in the past years. This has led to an increasing interest in the possibility of using DC and regulatory T cells as biological therapeutics to preserve and restore tolerance to self antigens and alloantigens. Especially DC may be helpful to exert their important roles in directing tolerance and immunity by modulation of subpopulations of effector T cells and regulatory T cells. The data demonstrated in the present studies may assist to define the divergent implications of new therapeutic concepts for the treatment of inflammatory bowel disease, especially with regard to a possible auspicious impact on pro-tolerogenic DC and regulatory T cell functions. However, further studies are needed to fulfil our understanding of the complex immunomodulatory profiles of FTY720 as well as of calcitriol and its low calcemic analog ZK156979, thus accelerating their entry into the clinic as new therapeutic options for the cure of inflammatory bowel disease.
Bei endogenen Retroviren handelt es sich um feste Bestandteile des Genoms. Im Fall von PERV (porzine endogene Retroviren) existieren zusätzlich infektiöse, xenotrope Vertreter. Aufgrund dieser Tatsache ist es notwendig, diese replikationskompetenten Proviren aus dem Genom potentieller Donortiere für die Xenotransplantation zu entfernen. Mit dem Wissen um die chromosomale Lage und der damit verbundenen Möglichkeit des Nachweises per PCR wurde im Rahmen dieser Arbeit gezeigt, dass aufgrund einer polymorphen Verteilung ein Ausschluss dieser funktionellen Proviren, mittels konventioneller Züchtung, möglich ist. Allerdings stellen sowohl die deletierten und mutierten proviralen Sequenzen durch eine Rekombination oder eine Komplementation, als auch ekotrope PERV-C ein Restrisiko im Falle einer Xenotransplantation dar. Es ist eine PERV-A/C Rekombinante beschrieben ex vivo worden, welche eine höhere Infektiösität aufweist als alle bisher untersuchten PERV. Bis auf die Rezeptor-Bindedomäne stellt dieses Virus ein PERV-C dar. Deshalb sollten chromosomal PERV-C identifiziert werden, um bei polymorpher Verteilung im Schweinegenom durch entsprechende Züchtung diese aus dem Genom heraushalten zu können. Im Rahmen dieser Arbeit ist es mit Hilfe einer speziellen PCR gelungen sieben Integrationsorte von PERV-C zu identifizieren. Da das Genom des Schweins bisher noch nicht komplett sequenziert ist, war es noch nicht möglich die gefundenen chromosomalen Bereiche zu kartieren. Dies wäre wiederum die Basis für eine Durchmusterung von Schweinen auf die Anwesenheit der gefundenen Proviren. Des Weiteren ist noch nicht bekannt, ob es sich bei diesen PERV-C um vollständige Proviren handelt, da aufgrund der verwendeten PCR und der sehr hohen Homologie verschiedener PERV untereinander nur provirale 3'Enden mit entsprechenden Flanken identifiziert werden konnten. Zusätzlich wurden in den Proben transgener Schweine PERV-C env spezifische Anteile nachgewiesen. Die Verteilung dieser Sequenzen, welche ebenfalls polymorph ist, gibt zwar keinen Aufschluss über die Anwesenheit eines Volllängen Provirus, jedoch ist aufgrund dieser Verteilung gleichfalls ein Ausschluss dieses Virus durch herkömmliche Züchtung möglich. Auf der anderen Seite besteht ein weiteres Risiko nach einer Xenotransplantation, wenn ein infektiöses PERV durch Komplementation gebildet wird, welches als Erbinformation ein env-deletiertes Provirus trägt. Das komplementierte PERV könnte potentiell, nach erfolgter Xenotransplantation, menschliche Zellen infizieren. Daraufhin wäre es zwar nicht mehr in der Lage infektiöse Partikel zu bilden, jedoch besteht noch das Risiko einer Retrotransposition, welche an sich schon mutagen wirkt. Zusätzlich könnten durch diesen Vorgang Gene zerstört oder Onkogene angeschaltet werden. Um dieses Risiko abschätzen zu können, wurden im Rahmen dieser Arbeit modifizierte Proviren von PERV-B(33) und MoMLV (Positivkontrolle) hergestellt und in einem Retrotranspositions Assay getestet. Die Modifikation der Proviren beinhaltete die Deletion des für die Retrotransposition nicht notwendigen env-Leserahmens, im Austausch gegen eine inserierte Indikatorkassette für die Retrotransposition (neoint). Im Rahmen der in dieser Arbeit durchgeführten Experimente konnte im Fall des Molekularklons PERV-B(33) eine Frequenz der Retrotransposition von maximal 1,2*10-6 pro Zelle und Generation ermittelt werden. Demzufolge stellt eine Retrotransposition von env-deletierten proviralen porzinen Sequenzen nach erfolgter Xenotransplantation ein minimales Risiko dar.
Disruption of the complex gastrointestinal ecosystem between the resident microflora and the colonic epithelial cells has been associated with increased inflammation and altered cell growth. Possible endpoints of this disturbance are IBD and CRC. The data presented in this thesis, entitled "PPARgamma as molecular target of epithelial functions in the gastrointestinal tract", shed further light on the underlying molecular mechanisms contributing to the well ordered homeostasis of this gastrointestinal ecosystem. Except for elucidating important roles for mesalazine and the dietary HDAC inhibitors butyrate and SFN in a) the modulation of cellular growth, b) the induction of APs, and c) the control of NFkappaB signalling in CRC cells, the involvement of the nuclear hormone receptors PPARgamma und VDR as "gatekeepers" in these intricate regulatory mechanisms were established. Future work will be engaged in analysing whether these in vitro findings are also physiologically relevant in regard to prevention and therapy of gastrointestinal diseases. Within the scope of this work, in Paper I and II it could be demonstrated that butyrate and mesalazine act via PPARgamma to induce their anti-proliferative and pro-apoptotic actions along the caspase signalling pathway. Activation of the intrinsic and extrinsic signalling trail and the down-regulation of anti-apoptotic proteins are responsible for increased caspase-3 activity caused by butyrate. In contrast, mesalazine merely activates this cascade via the extrinsic trail and the IAPs. Moreover, a signal transduction pathway leading to increased cell death via p38 MAPK - PPARgamma - caspase-3 in response to butyrate was unveiled. In addition, there is strong evidence that mesalazine-mediated pro-apoptotic and growth-inhibitory abilities are controlled by PPARgamma-dependent and -independent mechanisms which appear to be triggered at least in part by the modulation of the tumor suppressor gene PTEN and the oncoprotein c-myc, respectively. In Paper III and IV the induction of the APs HBD-2 and LL-37 in response to the dietary HDAC inhibitors butyrate and SFN was pinpointed. Regarding the molecular events of this regulation, the data presented in this thesis provide strong evidence for the involvement of VDR in HBD-2- and LL-37-induced gene expression, while the participation of PPARgamma was excluded. Moreover, the role for p38 MAPK and TGF-beta1 in the up-regulation of LL-37 caused by butyrate was established. In contrast, SFN-mediated induction of HBD-2 is modulated via ERK1/2 signalling. The findings in Paper V clearly refer to the involvement of the nuclear hormone receptors PPARgamma and VDR in butyrate-mediated suppression of inducible NFkappaB activation dependent on the stimulated signalling pathway caused by LPS or TNFalpha. Moreover, an inhibitory role for VDR in the regulation of basal NFkappaB activation was revealed. On the contrary, a modulating role for PPARgamma on basal NFkappaB could be debarred. Altogether the data presented in this thesis not only provide new insights in the understanding of the fundamental gastrointestinal physiology regulated by nuclear hormone receptors, but also may offer opportunities for the development of potential drug targets and therapeutic strategies in the treatment of IBD and CRC.