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Alveolar echinococcosis (AE) is a severe and life-threatening disease caused by the metacestode larva of the fox-tapeworm Echinococcus multilocularis. Parasite entry into the host evokes an early and potentially parasiticidal Th1 immune response that is gradually replaced by a permissive Th2 response. An immunoregulatory environment has also been reported in the host as the disease progresses. As a result of immunomodulation, E. multilocularis larvae persist in the host for decades without being expelled, and thus almost act like a perfect transplant. Very little is currently known on the molecular basis of the host immunomodulation by E. multilocularis. In this work, in vitro cultivation systems were used to assess the influence of metabolites released by the parasite larvae (E/S products) on host immune effector cells. E/S products of cultivated larvae that respresent the early (primary cells) and chronic (metacestode vesicles) phase of AE induced apoptosis and tolerogenic properties (poor responsiveness to LPS stimulation) in host dendritic cells (DC) whereas those of control larvae (protoscoleces) failed to do so. These findings show that the early infective stage of E. multilocularis induces tolerogenicity in host DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. Interestingly, metacestode E/S products promoted the conversion of naïve CD4+ T-cells into Foxp3+ regulatory T-cells in vitro, whereas primary cell and protoscolex E/S products failed to do it. Since Foxp3+ regulatory T-cells are generally known to mediate immunosuppression, the present finding indicates that Foxp3+ regulatory T-cells, expanded by E/S products of the metacestode larva, could play a role in the parasite-driven immunomodulation of the host observed during AE. Furthermore, a substantial increase in number and frequency of suppressive Foxp3+ regulatory T-cells could be observed within peritoneal exudates of mice following intraperitoneal injection of E. multilocularis metacestodes, indicating that Foxp3+ regulatory T-cells could also play an important role in E. multilocularis-driven immunomodulation in vivo. Interestingly, a parasite activin ortholog, EmACT, secreted by metacestodes, was shown to expand host regulatory T-cells in a TGF-β-dependent manner, similarly to mammalian activin A. This observation indicated that E. multilocularis utilizes evolutionarily conserved TGF-β superfamily ligands, like EmACT, to expand host regulatory T-cells. Taken together, the present findings suggest EmACT, a parasite activin secreted by the metacestode and capable of expanding host regulatory T-cells, as an important player in the host immunomodulation by E. multilocularis larvae. Another parasite factor EmTIP, homologous to mammalian T-cell immunomodulatory protein (TIP) was characterized in this work. EmTIP could be detected in the secretions of the parasite primary cells and localized to the intercellular space within the parasite larvae. EmTIP blockade inhibited the proliferation of E. multilocularis primary cells and the formation of metacestode vesicles indicating a major role for parasite development. Furthermore, EmTIP evoked a strong release of IFN-γ by CD4+ T-cells hence suggesting that the secretion of this factor as a result of its role in parasite development could “secondarily” induce a potentially protective Th1 response. In conclusion, this work identified two molecules, EmACT and EmTIP, with high immunomodulatory potential that are released by E. multilocularis larvae. The data presented do provide insights into the mechanisms of parasite-driven host immunomodulation during AE that are highly relevant for the development of anti-parasitic immune therapies.
Protein kinases as targets for the development of novel drugs against alveolar echinococcosis
(2015)
The metacestode larval stage of the fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most lethal zoonosis of the northern hemisphere. The development of metacestode vesicles by asexual multiplication and the almost unrestricted infiltrative growth within the host organs is ensured from a population of undifferentiated, proliferative cells, so-called germinative cells. AE treatment options include surgery, if possible, as well as Benzimidazole-based chemotherapy (BZ). Given that the cellular targets of BZs, the -tubulins, are highly conserved between cestodes and humans, the chemotherapy is associated with considerable side-effects. Therefore, BZ can only be applied in parasitostatic doses and has to be given lifelong. Furthermore, the current anti-AE chemotherapy is ineffective in eliminating the germinative cell population of the parasite, which leads to remission of parasite growth as soon as therapy is discontinued.
This work focuses on protein kinases involved in the proliferation and development of the parasite with the intention of developing novel anti-AE therapies. Polo-like kinases (Plks) are important regulators of the eukaryotic cell cycle and are involved in the regulation and formation of the mitotic spindles during the M-phase of the cell cycle. Plks have already been shown to be associated with deregulated cellular growth in human cancers and have been investigated as novel drug targets in the flatworm parasite Schistosoma mansoni. In the first part of this work, the characterisation of a novel and druggable parasite enzyme, EmPlk1, which is homologous to the polo-like kinase 1 (Plk1) of humans and S. mansoni (SmPlk1), is presented. Through in situ hybridisation, it could be demonstrated that emplk1 is specifically expressed in the Echinococcus germinative cells. Upon heterologous expression in the Xenopus oocyte system, EmPlk1 induced germinal vesicle breakdown, thus indicating that it is an active kinase. Furthermore, BI 2536, a compound originally designed to inhibit the human ortholog of EmPlk1, inhibited the EmPlk1 activity at a concentration of 25 nM. In vitro treatment of parasite vesicles with similar concentrations of BI 2536 led to the elimination of the germinative cells from Echinococcus larvae, thus preventing the growth and further development of the parasite. In in vitro cultivation systems for parasite primary cells, BI 2536 effectively inhibited the formation of new metacestode vesicles from germinative cells. Thus, BI 2536 has profound anti-parasitic activities in vitro at concentrations well within the range of plasma levels measured after the administration of safe dosages to patients (50 nM after 24 h). This implies that EmPlk1 is a promising new drug target for the development of novel anti-AE drugs that would specifically affect the parasite’s stem cell population, namely the only parasite cells capable of proliferation. In addition to the chemotherapeutic aspects of this work, the inhibitor BI 2536 could be further used to study the function of stem cells in this model organism, utilising a method of injection of parasite stem cells into metacestode vesicles, for instance, as has been developed in this work.
In the second part of this work, a novel receptor tyrosine kinase, the Venus flytrap kinase receptor (EmVKR) of E. multilocularis has been characterised. Members of this class of single-pass transmembrane receptors have recently been discovered in the related trematode S. mansoni and are associated with the growth and differentiation of sporocyst germinal cells and ovocytes. The ortholog receptor in EmVKR is characterised by an unusual domain composition of an extracellular Venus flytrap module (VFT), which shows significant similarity to GABA receptors, such as the GABAB receptor (γ-amino butyric acid type B) and is linked through a single transmembrane domain to an intracellular tyrosine kinase domain with similarities to the kinase domains of human insulin receptors. Based upon the size (5112bp) of emvkr and nucleotide sequence specificities, efforts have been made to isolate the gene from cell culture samples to study the ligand for the activation of this receptor type in Xenopus oocytes. To date, this type of receptor has only been described in invertebrates, thus making it an attractive target for drug screening. In a first trial, the ATP competitive inhibitor AG 1024 was tested in our in vitro cell culture.
In conclusion, the EmVKR represents a novel receptor tyrosine kinase in E. multilocularis. Further efforts have to be made to identify the activating ligand of the receptor and its cellular function, which might strengthen the case for EmVKR as a potential drug target. The successful depletion of stem cells in the metacestode vesicle by the Plk1 inhibitor BI 2536 gives rise to optimising the chemical component for EmPlk1 as a new potential drug target. Furthermore, this inhibitor opens a new cell culture technique with high potential to study the cellular behaviour and influencing factors of stem cells in vitro.
Die alveoläre Echinokokkose (AE) ist eine lebensbedrohliche Erkrankung des Menschen, welche durch das infiltrative Wachstum des Metazestoden-Larvenstadiums des Fuchsbandwurms (Echinococcus multilocularis) in der Leber verursacht wird. Das tumorartige Wachstum des Metazestoden beruht auf einer Echinococcus-spezifischen Modifikation der anterior-posterioren-Körperachse (AP Achse). Es wird vermutet, dass dabei der anteriore Pol der invadierenden Oncospären-Larve zunächst abgeschaltet wird und sich der Metazestode anschließend asexuell als vesikuläres, posteriorisiertes Gewebes im Wirt vermehrt. Nach massiver Proliferation wird der anteriore Pol reetabliert und führt zur Bildung zahlreicher Bandwurm-Kopfanlagen (Protoskolizes). Da die Ausbildung der AP Körperachse evolutionsgeschichtlich konserviert über den wingless-related (Wnt)-Signalweg gesteuert wird, wurde in dieser Arbeit die Rolle von Wnt-Signaling bei der Musterbildung von E. multilocularis über molekular- und zellbiologische Studien näher beleuchtet.
Zentraler methodischer Ansatz der vorliegenden Arbeit war ein E. multilocularis Stammzell-Kultursystem, das Primärzellsystem, welches die in vitro-Generierung von Metazestoden-Vesikeln durch Proliferation und Differenzierung von germinativen Zellen (Stammzellen) erlaubt. Über RNA-Sequenzierung wurde zunächst gezeigt, dass in Primärzellkulturen sowohl Markergene für posteriore Entwicklung in Richtung Metazestode wie auch für Anterior-und Protoskolexmarker exprimiert werden. Unter Verwendung von RNA-Interferenz (RNAi) wurde anschließend ein erfolgreicher Knockdown des vermuteten Hauptregulators des kanonischen Wnt-Signalwegs, β Catenin (em-bcat1), erreicht und führte zu einem charakteristischen, sogenannten ‚red dot‘ Phänotyp, dem ersten jemals beschriebenen RNAi Phänotyp für E. multilocularis-Primärzellen. Primärzellkulturen nach em-bcat1 RNAi zeigten eine stark verminderte Fähigkeit, Metazestoden-Vesikel zu bilden sowie eine Überproliferation von germinativen Zellen. Zusätzliche RNA-Seq-Analysen des Transkriptoms von RNAi(em-bcat1)-Kulturen zeigten eine signifikant verringerte Expression von Posterior- und Metazestodenmarkern, während Anterior- und Protoskolexmarker deutlich überexprimiert wurden. Durch umfangreiche Whole-mount-in-situ-Hybridisierung (WMISH)-Experimente wurden diese Daten für eine Reihe ausgewählter Markergene für posteriore (Metazestode; em-wnt1, em-wnt11b, em-muc1) und für anteriore Entwicklung (Protoskolex; em sfrp, em-nou-darake, em npp36, em-frizzled10) verifiziert. In allen genannten Fällen zeigte sich durch Änderung der Polarität eine verminderte Genexpression von Posteriormarkern, während Anteriormarker deutlich erhöht exprimiert wurden. Ähnlich wie bei den verwandten, freilebenden Planarien, führt demnach ein Knockdown des zentralen Wnt-Regulators β-Catenin bei E. multilocularis zu einer anteriorisierten, Anterior- und Protoskolexmarker dominierte Genexpression, welche der posteriorisierten Entwicklung zum Metazestoden entgegenwirkt.
Neben Markergenen für die Ausbildung der AP-Achse wurden in dieser Arbeit auch solche für die medio-laterale (ML)-Körperachse bei Zestoden erstmals beschrieben. So zeigte sich, dass ein Slit-Ortholog (em slit) im E. multilocularis Protoskolex im Bereich der Körper-Mittellinie exprimiert wird und lieferte Hinweise darauf, dass, ähnlich zur Situation bei Planarien, die ML Achse von E. multilocularis durch Morphogengradienten aus slit (Mittellinie) und wnt5 (lateral) definiert wird. Im Metazestoden wird hingegen nur em-slit exprimiert. Der Metazestode besitzt damit als posterior-medianisiertes Gewebe Anlagen zur Polarität zur AP- und ML-Achse, welche erst mit Bildung von Protoskolizes vollständig etabliert werden. Schließlich deuten die Ergebnisse dieser Arbeit darauf hin, dass bei der Wiederherstellung der Körperachsen während der Entwicklung von Protoskolizes Hedgehog (Hh)-Signale entscheidend mitwirken.
Zusammenfassend wurde in dieser Arbeit der zentrale Faktor des kanonischen Wnt Signalwegs, β-Catenin, als Hauptregulator der Entwicklung des tumorartig wachsenden E. multilocularis-Metazestoden identifiziert. Zudem wurde gezeigt, dass zur Metazestodenbildung neben einer Echinococcus-spezifischen Modifikation der AP Körperachse auch eine solche der ML Achse beiträgt. In humanen malignen Tumoren sind der Wnt-, Slit-Robo- und Hh-Signalweg gut erforschte Wirkstofftargets und könnten in Zukunft in ähnlicher Weise für eine zielgerichtete Therapie von AE dienen.