An optimized in vitro blood–brain barrier model reveals bidirectional transmigration of African trypanosome strains

Abstract

The transmigration of African trypanosomes across the human blood-brain barrier (BBB) is the critical step during the course of human African trypanosomiasis. The parasites Trypanosoma brucei gambiense and T. b. rhodesiense are transmitted to humans during the bite of tsetse flies. Trypanosomes multiply within the bloodstream and finally invade the central nervous system (CNS), which leads to the death of untreated patients. This project focused on the mechanisms of trypanosomal traversal across the BBB. In order to establish a suitable in vitro BBB model for parasite transmigration, different human cell lines were used, including ECV304, HBMEC and HUVEC, as well as C6 rat astrocytes. Validation of the BBB models with Escherichia coli HB101 and E. coli K1 revealed that a combination of ECV304 cells seeded on Matrigel as a semisynthetic basement membrane and C6 astrocytes resulted in an optimal BBB model system. The BBB model showed selective permeability for the pathogenic E. coli K1 strain, and African trypanosomes were able to traverse the optimized ECV304-C6 BBB efficiently. Furthermore, coincubation indicated that paracellular macrophage transmigration does not facilitate trypanosomal BBB traversal. An inverse assembly of the BBB model demonstrated that trypanosomes were also able to transmigrate the optimized ECV304-C6 BBB backwards, indicating the relevance of the CNS as a possible reservoir of a relapsing parasitaemia. INTRODUCTIONHuman African trypanosomiasis is a vector-borne parasitic disease, which currently causes about 10 000 deaths each year. At the end of the last century the number of lethal cases was estimated as up to 300 000, according to WHO data. The disease threatens over 60 million people of 36 African nations, reaching lethality of 100 % without treatment (WHO, 2010). The parasites, called trypanosomes, are transmitted during a blood meal of tsetse flies of the Glossina genus and can infect humans as well as cattle. Trypanosoma brucei brucei is one of the causative agents of Nagana, a severe cattle disease, which hampers intensive cattle farming in endemic areas (Steverding, 2008). T. b. gambiense and T. b. rhodesiense are human pathogens and multiply within the blood circulation system. Therein, the parasites evade the host immune system by different strategies, for instance by switching their surface-coat antigens (Dubois et al., 2005). As the disease progresses, the parasites infect the central nervous system (CNS), leading to the severe outcome of the disease. Once inside the CNS, parasites are hardly reached by drugs or by the immune system. Depending on the trypanosome subspecies the parasites transmigrate through the human blood-brain barrier (BBB) within a few weeks (T. b. rhodesiense), some months or even years (T. b. gambiense). So far, the invasion into the CNS of African trypanosomes is poorly understood (Grab & Kennedy, 2008). It has been shown that secreted proteases (Nikolskaia et al., 2006(Nikolskaia et al., , 2008, in the case of T. b. rhodesiense, and the composition of ...

Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

“…C6 cells have the properties of astrocytes and maintain the functionality of primary BMECs in vitro [21,22]. It has also been shown to increase in the barrier functions of primary BMECs in vitro by treatment of C6CM [23].…”

Capsule Contributes to Transmigration of Streptococcus pneumoniae Serotype 7F Meningitis Isolates through Complex Blood Brain Barrier Models

“…Our barrier model systems employed in this study combined the advantage of multicellular complexity with functional specificity. The ECV304-C6 system has already successfully established as an experimental model for analysis of BBB penetration by African trypanosomes causing human trypanosomiasis and has been confirmed as reliable analytical method for the quantification of drug penetration properties [25,26]. In addition to this multicellular model we employed a second system based on a Brain Derived Endothelial Cells (HBMEC) in our assays in order to elucidate transmigration efficiencies with this brain-tissue specific barrier system.…”

“…After 5 days of cultivation the two BBB models were used for transmigration analyses. The barrier integrity of both barrier systems was controlled with the non-invasive E. coli strain HB101 [25].…”

“…The BBB models were prepared as described in [25]. In brief, human endothelial cells ECV304 (ECACC 92090409), murine astrocytes C6 (ECACC 92091712, [32]) or HBMEC cells (human brain derived microvascular endothelial cells [33]) were cultivated at 37°C, 5% CO 2 .…”

“…Our recently designed and evaluated BBB models designated as ECV304-C6 BBB and HBMEC-BBB allow the analysis of transmigration mechanisms of bacterial and parasitical pathogens including streptococci and African trypanosomes [25,26] causing inflammatory CNS diseases like bacterial meningitis [27] and human African trypanosomiasis [28]. In this study we have used these models to investigate the role of pneumococcal polysaccharide capsule in bacterial transmigration of BBB using a highly invasive serotype 7F isolate from a meningitis patient.…”