CONICET | Buscador de Institutos y Recursos Humanos

The bronchial tissue contains epithelial cells and fibroblasts in close contact that interact with each other. Therefore, infection studies performed on cultures of a single cell type (epithelial cells or fibroblasts) fail to reproduce what happens in vivo in the lung. This problem can be solved by using a 3-dimensional (3D) air-exposed organotypic human lung tissue model. This model consists of a monolayer of bronchial epithelial cells on top of a collagen matrix with embedded fibroblasts. The 3D model was constructed on Transwell inserts and apically infected with Brucella abortus for 4h and then incubated with gentamicin for 1h to kill extracellular bacteria. At different days post-infection (p.i.) cells were lysed to determine CFU of intracellular bacteria (CFUi) and conditioned media (CM) from the basolateral side were collected to determine CFU and to measure cytokines. Results showed that B. abortus infects and replicates in the 3D model since day 1 p.i. until day 16 p.i. (end of follow-up). Whereas no viable bacteria were detected in CM during the first days p.i., a progressive increase of CFU was detected between days 5 and 16 p.i. The presence of viable bacteria in the CMs would not be due to a cytotoxic effect of the infection, since no significant increase in lactate dehydrogenase activity was detected in CMs of the infected models as compared to non-infected controls. In concordance with the presence of bacteria in CM, at day 5 p.i. the highest levels of interleukin-6 (IL-6) (p˂0.001), IL-8 (p˂0.0001) and monocyte chemoattractant protein-1 (MCP-1) (p˂0.0001) were achieved. These results suggest that B. abortus persists in the lung, but part of the bacterial progeny leaves the tissue to colonize other organs. In response to the infection the bronchial mucosa secretes cytokines and chemokines that may attract neutrophils and monocytes to the site of infection.