Human in vitro tissue and single cell approaches to model tuberculosis
Ronde 2012 Module Proefdiervrije Technieken: Ons innovatieve longweefselmodel is uniek in vele aspecten en kan bijzonder belangrijk worden voor tuberculoseonderzoek, vooral vanwege het feite dat er nog geen goed in vitro weefselmodel is beschreven.In ons model vormen macrofagen gezamenlijk een cluster dat gelijkenis vertoont met het voor TB specifieke granuloma. Meerdere epitheelcellagen vormen een matrix waarin ook immune cellen kunnen migreren. Onze voorlopige resultaten laten zien dat het model gebruikt kan worden bij studies naar TB-infecties in fysiologische omstandigheden. Een tweede model maakt het mogelijk dat we de condities waarin TB latent wordt kunnen bestuderen. Voor beide modellen geldt dat de subcellulaire lokalisatie van TB in cellen bestudeerd zal worden.Een belangrijk verschil tussen ziekteverwekkende mycobacteriën zoals TB en niet ziekteverwekkende mycobacteriën is de lokalisatie in de gastheercel. Na valorisatie van de modellen zal de lokalisatie bestudeerd worden in de twee door ons ontwikkelde model systemen
Samenvatting van de aanvraag
An urgent task of today’s medical research is to find new ways to treat infections caused by Mycobacterium tuberculosis. There are numerous reports on multidrug-resistant strains of this pathogen, which causes tuberculosis (TB). Most experimental studies of TB rely on animal experiments and there are no existing alternative models such as in vitro tissue models, with the exception of infected single cell cultures. Replacing existing animal models with human in vitro systems is also important because of the questioned validity of these models for TB. The aim of the presented project is to further develop two novel human in vitro models for TB, validate them against tissues from TB patients and to use them instead of animals for testing anti-TB combination regimens based on existing drugs. The models are based on human primary cells and cell lines and the investigation of these models as substitutes for animal models for preclinical drug testing. The first model is based on macrophages obtained from human blood that are infected with virulent M. tuberculosis. Similar models have been described, but the unique feature of the presented model that the model displays is that under the right conditions, it can mimic clinical latent TB. Understanding latent TB is of importance, since the latent infection remaining in patients on TB treatment is very tolerant towards antibiotics and is causative of multidrug resistance. The so far confirmed results include absence of net growth of the bacteria inside the macrophages over two weeks, alteration of characteristics of the bacteria identical to observations in other studies, suppression of immune activation of the infected cells, tolerance towards antibiotics and most importantly, regrowth of the bacteria during immunosuppression, which is a well-known clinical problem and has so far only been modeled in animals. The second model is based on the establishment of co-cultures of human cell lines and primary immune cells on a filter with a collagen matrix. Human blood monocyte-derived macrophages infected with M. tuberculosis are introduced into the system. The tissues are exposed to air on the apical side, causing the epithelial cells to secrete mucus, further mimicking the microenvironment in the lung. This unique TB model will be characterized within the project During the present project, the models will be compared with and validated against human TB-infected tissues on an ultrastructural level using advanced electron microscopy (Delft University of Technology). The proposed project is a translational academic project linking experimental research to clinical testing and including Dutch and Swedish research groups. The models will be used for screening combinations of approved drugs together with TB-antibiotics and for achieving proof-of-principle of adjuvant treatments for a more effective TB treatment. Together, the two models may prove to serve as good substitutes for animal models for TB. If the project is successful, the models can be further developed to mimic different kinds of bacterial and viral infections.