Investigating the host-pathogen interaction in Tuberculosis by Bioelectrospray 3D cell culture modelling and label-free imaging

Abstract

Tuberculosis is a deadly infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb). It constitutes a major cause of death worldwide with approximately 1.5 million people dying from TB every year.1-6 Traditional animal models as well as conventional ‘two-dimensional’ cell cultures do not accurately mimic human tuberculosis infection in vivo, such as the formation of caseating granulomas and degradation of extracellular matrix.We study a bioelectrospray-generated 3D cell culture model of tuberculosis, using diverse imaging techniques. In the 3D culture, Mtb-infected PBMCs (strains: H37Rv, 0414B) are mixed with an alginate-collagen gel. We investigated the host-pathogen interaction at high resolution at various stages of infection using Transmission Electron Microscopy (TEM). We used Micro-Computed Tomography (μCT) to show distribution of PBMCs in 3D in comparison to a human tuberculous lung biopsy. This was then correlated to traditional H&E, and matrix staining of the sectioned tuberculous human lung block. Using label-free microscopy [Coherent Anti-Stokes Raman Scattering (CARS), Second Harmonic Generation (SHG), Two-Photon Excitation Fluorescence (TPEF)], PBMC aggregation and collagen fibres were imaged in the microspheres.More lipid bodies were detected by TEM in the Mtb-infected samples than the uninfected controls. TEM and SHG imaging revealed collagen fibres attached to the surface of PBMCs. CARS microscopy showed that infection with Mtb, as well as the presence of collagen in the 3D matrix, influence the number of PBMC aggregates forming within the 3D culture. Preliminary data from μCT indicate that this technique can provide quantitative data on PBMC aggregates in 3D that can be cross-correlated with human biopsies.A correlative imaging approach between traditional and emerging imaging modalities is necessary in order to gain new insight into the host-pathogen interaction in tuberculosis