An alveolus lung on a chip model of Mycobacterium fortuitum lung infection

Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both 2D cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human lung microenvironment. Here we describe development of a humanized, 3D, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics Alveolar lung-on-a-chips (ALoCs) that were seeded with primary human alveolar epithelial cells & human monocyte-derived macrophages in the apical channel and human microvascular pulmonary endothelial cells in the basal channel were either infected with M. fortuitum at an MOI of ~1 to the epithelial cells or left uninfected. 24 hours post-infection, control and infected chips were lysed with TriZol and RNA was isolated using phenol & the Qiagen RNeasy Mini Kit. We performed bulk RNA-sequencing of the apical channel on uninfected and infected chips 24 hours after infection.