Un nouveau modèle de peau sur puce révèle les adaptations biologiques des Trypanosoma brucei extravasculaires dans le microenvironnement du derme

Trypanosoma brucei sp. is an extracellular parasite causing human African trypanosomiases. It is transmitted to the mammalian host by tsetse flies during their blood meals. The fly deposits metacyclic trypomastigotes into the skin dermis. From there, the parasites enter the blood and lymphatic systems. Recent studies revealed that T. brucei maintains a population in the extravascular dermis of unconfirmed seropositive suspects and patients with undetectable parasitemia in blood. Experimental infections in mice demonstrated the presence of substantial quantities of parasites in the dermal niche that remain transmissible to tsetse flies, highlighting the skin as an important reservoir. However, the characteristic traits and functions of these skin-dwelling forms are not yet fully understood. To address this knowledge gap, we characterized these dermal trypanosomes to understand their role in transmission. Specifically, we focused on understanding parasite proliferation, differentiation, and motility between the dermal and vascular compartments. To achieve this, we developed an in-house skin-on-chip (SoC) model optimized for quantitative imaging using confocal microscopy. The skin-on-chip model consists of a microfluidic chamber containing three channels, incorporating elements from both vascularized (Zerventonakis et.al, 2012) and non-vascularized full-thickness skin systems (Sriram et.al, 2018). The model comprises three distinct layers: 1) human umbilical vein endothelial cells forming the vasculature, 2) human dermal fibroblasts embedded in a fibrinogen-based hydrogel populating the dermis, and 3) keratinocytes structuring the epidermis. We validated this model using various assays, including live/dead tests for cell viability, dextran diffusion tests for endothelial integrity, and immunofluorescence with cell-specific antibodies for layer formation. First, we investigated parasite motility, which is essential for T. brucei to establish infection in both tsetse flies and humans. To understand the motility of T. brucei in the dense microenvironment of the dermis, the mean speeds of the parasites were determined in the vascular and dermal channels. Parasites in the dermal channel moved at a significantly lower mean speed compared to those in the vascular channel. Interestingly, removing fibroblasts from the dermal channel slightly increased parasite speed, while adding collagen significantly enhanced it. These results highlight the dual role of fibroblasts in T. brucei motility: they may act as physical barriers reducing parasite movement while also producing collagen, which paradoxically increases parasite speed. Furthermore, we examined the differentiation of T. brucei parasites, which exist as dividing slender and cell cycle arrested stumpy forms in the mammalian host. The stumpy forms, adapted for transmission, are primarily responsible for establishing infection in the tsetse fly's midgut. Our SoC model revealed a variation in number of parasites expressing a stumpy form marker in the vascular channel compared to the dermal channel with notable changes occuring at the dermo-vascular interface, underscoring the complex interplay between parasite biology in the dermal tissue microenvironment. In conclusion, we have successfully engineered a 3D skin model that enables both live and fixed imaging of fluorescently-tagged trypanosome parasites, allowing quantification of key biological parameters with high reproducibility. This model has provided critical insights into fundamental parasitological traits, specifically trypanosome motility and differentiation. Our future investigations will further elucidate these two aspects, along with parasite proliferation dynamics. By understanding these key aspects of parasite biology, we aim to unravel the complex host-parasite interactions that govern T. brucei survival and propagation in dermal tissue.