Development and Validation of a Human Lung Tissue Model (3D) to Study Histoplasma capsulatum Biofilm Host-Pathogen Interaction

Abstract

Histoplasmosis, an Invasive Fungal Infection (IFI), is caused by the dimorphic fungus Histoplasma capsulatum, which has a worldwide distribution. It is the fourth IFI with the highest number of annual cases, reaching more than 600 thousand people every year. In the last two decades, IFIs have increased worldwide and the number of patients at risk has grown dramatically. Recently, a correlation was established between the mode of infection of H. capsulatum and the formation of biofilms, characterized by being complex cell agglomerates, where there is the production of an extracellular matrix that by itself confers protection to the external environment, which induces, among others, antifungal resistance. The host-pathogen interaction of H. capsulatum biofilms is essential for the establishment of the disease and requires studies with credible models as observed in humans. Thus, this research project aims to develop and validate a three-dimensional model of human lung tissue to study the pathogen-host interaction of H. capsulatum biofilm, in order to characterize the virulence mechanisms involved in the infection in the future. This model is closer to the real than the methodology of mammalian animals in vivo, contributing to faster research on a large scale, to evaluate the pathogen-host interaction of H. capsulatum biofilm in the future. For this, we will develop a functional human lung tissue composed of the triculture of hSAEC cells differentiated by ALI, MRC-5, and THP-1 macrophage-like cells for the construction of stratified tissue in vitro in order to evaluate the infection by planktonic cells and H. capsulatum biofilms. The validation of the three-dimensional model will be performed by High-Speed Video Microscopy Analysis (HSVA) to visualize the cilia beating and the analysis of the differentiation of hSAEC cells by qRT-PCR of genes characteristic of each cell subtype. In addition, standardization of the model infection by H. capsulatum biofilms will be performed by quantifying the expression of differentially expressed genes already characterized by our group both by the fungus and by the host cells.