Host-pathogen interactions in Mycobacterium tuberculosis infection of airway epithelial cells

Although tuberculosis is transmitted via the respiratory route, little is known of the interaction between Mycobacterium tuberculosis (Mtb) and the airway epithelium: the first line of defence against inhaled pathogens. As non-professional phagocytes, airway epithelial cells (AEpC) internalise bacteria, including Mtb, and contribute to the immune response by producing cytokines and interacting with immune cells. While immortalised AEpC cell lines can be productively infected by Mtb, they fail to replicate many structural and cellular features of the natural airway epithelium. In this thesis, we investigated the host-pathogen interactions between Mtb and the human pseudostratified tracheobronchial epithelium, using an in vitro 3D air-liquid interface (ALI) culture system of primary human AEpC (ALI-AEpC). We showed that under ALI conditions, primary AEpC were able to differentiate to all major cell types in a pseudostratified epithelial layer exhibiting functional mucociliary clearance by 28 days post-air exposure. The presence of both major and rare cell types corresponding to the tracheobronchial epithelium in vivo, as well as the annotation of two novel rare clusters, was confirmed by single-cell RNA-Seq (scRNA-Seq). For this, a complete workflow for single-cell RNA-Seq (scRNA-Seq) of ALI-AEpC was set up, optimised and used to perform a technical comparison of two scRNA-Seq platforms. Using this model, we showed that while undifferentiated primary AEpC were productively infected by Mtb, infection of ALI-AEpC was minimal up to 48 hours post-infection (hpi). Both undifferentiated and ALI-AEpC were transcriptionally and immunologically unresponsive to Mtb infection within 48 hpi. However, over 14 days post-infection (dpi), Mtb entered and replicated within apical cells in the ALI model. Over that period, bacteria were released apically without widespread cell death or loss of epithelial integrity. Altogether, we suggest that the tracheobronchial epithelium acts as a reservoir for persistent Mtb replication and shedding within the respiratory tract