ModelingMycobacterium tuberculosisearly granuloma formation in experimental human lung tissue

Abstract

The widely used animal models for tuberculosis (TB) display fundamental differences from human TB. Therefore, a validated model that recapitulates human lung TB is attractive for TB research. Here, we describe a unique method for establishment of TB infection in an experimental human lung tissue model. The model is based on cell lines derived from human lungs and primary macrophages from peripheral blood, and displays characteristics of human lung tissue, including evenly integrated macrophages throughout the epithelium, production of extracellular matrix, stratified epithelia and mucus secretion. Establishment of experimental infection in the model tissue with Mycobacterium tuberculosis, the bacterium that causes TB, resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma formation. We quantitated the extent of granuloma formation induced by different strains of mycobacteria and validated our model against findings in other TB models. We found that early granuloma formation is dependent on ESAT-6, which is secreted via the type VII secretion machinery of virulent mycobacteria. Our model, which can facilitate the discovery of the interactions between mycobacteria and host cells in a physiological environment, is the first lung tissue model described for TB.

A 3D Human Lung Tissue Model for Functional Studies on <em>Mycobacterium tuberculosis</em> Infection

“…We quantified the size and number of monocyte cell clusters and found that the size (volume) of cell clusters is enhanced (p<0.001), while the number of individual monocytes decreased (p<0.01) in M. tuberculosis infected tissues as compared to uninfected tissue models (Figure 3C and 3D). This data validates our previous finding of early granuloma formation in M. tuberculosis infection observed in lung tissue models analyzed in 2D tissue sections 9 .…”

“…For visualizing the M. tuberculosis-immune cell migration and interaction, we introduced macrophages infected with M. tuberculosis that express GFP (green) together with the freshly isolated PKH26-labelled monocytes (red) into the tissue model (blue, DAPI stained for nuclei). On day 7 post addition of M. tuberculosis-infected cells to the tissue model, confocal microscopy reveals clustering of red monocytes at the site of infection (green) (Figure 2), which mimics the hallmark lesions of human TB 9 .…”

“…Cell cultures of monolayers or co-cultures lack the 3D environment and tissue responses. Therefore, we have developed an innovative lung tissue model based on human primary immune cells and human lung-specific cell lines 8,9 . The model displays characteristic features of human lung tissue, including epithelia with evenly integrated macrophages, formation of extracellular matrix, stratified epithelia and mucus secretion 9 .…”

“…We have recently demonstrated that mutant strains defective in the ability to secrete the virulence factor ESAT-6 or Mycobacterium bovis BCG that lacks ESAT-6 did not induce the clustering of monocytes (no early granuloma), in contrast to the virulent M. tuberculosis 9 . These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12 .…”

“…These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12 . As there is no gold-standard model for TB, we took advantage of the surgically resected tissue biopsies from TB patients for validation of the method 9 . Our in vitro tissue model shares several characteristics with the lung and lymph node biopsies from TB patients, including the aggregation of macrophages in granuloma, the presence of both intra-and extracellular bacteria 18 and induction of necrosis 11 .…”