Using a human airway organoid-derived monolayer co-culture model to study Cryptococcus neoformans and other fungal infections

Introduction

Cryptococci are environmental fungi that cause localized or disseminated infections. The WHO considers C.neoformans to have the highest need for research among fungal pathogens. Studies on pathogenicity are mainly based on animal models or immune cells. However, to understand the pathomechanisms associated with the transition from pulmonary colonization to tissue infection, complementary models are required. We are using a human lung infection model to investigate parameters of early cryptococcal infection and epithelial transition.

Methods

We used well-characterized human airway-organoid derived monolayers (ODMs) from nasal and bronchial tissue and co-cultured them with C.neoformans (VNI) and C.gattii (VGII). Barrier function of ODMs was probed by measuring the transepithelial electrical resistance (TEER). We quantified internalization of fungi and fungal viability by immunofluorescence and colony forming unit assays. Fungal morphology was assessed using ink stainings and image analysis.

Results

Human ODMs could be co-cultured with different cryptococcal strains and TEER measurements revealed no breakdown of barrier function, even after prolonged time intervals. Rare events of fungal internalization by airway epithelial cells were detected. Fungi remained viable during co-culture and were able to further proliferate. Quantification of cryptococcal morphology revealed a shift from uniform cells towards distinct phenotypes, including micro- and giant cells.

Discussion

The established co-culture model provides a suitable in vitro model to study host-cryptococcal interactions. Quantification of TEER indicate no impact of different cryptococcal strains on the barrier function of airway epithelial cells, despite the fact that both fungal strains show different disease phenotypes in humans. However, the model revealed that fungi undergo morphological changes, indicating that they adapt different phenotypes as a consequence of epithelial cell presence. Changes in morphology have been observed in in vivo-studies and are associated with pathogenic potential. Thus, the co-culture model can be used in the future for risk assessment of different fungal pathogens.