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. 2014 Feb;7(2):281-8.
doi: 10.1242/dmm.013854. Epub 2013 Nov 7.

Modeling Mycobacterium tuberculosis early granuloma formation in experimental human lung tissue

Venkata Ramanarao Parasa  1 , Muhammad Jubayer Rahman, Anh Thu Ngyuen Hoang, Mattias Svensson, Susanna Brighenti, Maria Lerm
Affiliations

Modeling Mycobacterium tuberculosis early granuloma formation in experimental human lung tissue

Venkata Ramanarao Parasa et al. Dis Model Mech. 2014 Feb.

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.

Keywords: ESX-1; Granuloma; In vitro model; Lung tissue; M. tuberculosis; Tuberculosis.

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Figures

Fig. 1.
Fig. 1.
Optimization of parameters for Mtb (H37Rv) infection of the lung tissue model. (A) Immunohistochemistry of CD68-positive cells (brown) in tissue models without or with macrophages (MΦ). Cell nuclei are labeled with hematoxylin (HA, blue). (B) Apical Mtb infection of the tissue model without or with macrophages. Tissue sections were fixed and stained with HA (blue) and acid-fast staining (AFS, purple). (C) Mycobacterial infection of the tissue models using Mtb-infected macrophages as vehicles. Bright-field images of fixed sections stained with AFS (purple), immunohistochemistry (anti-human CD68, brown) and HA (blue). (D) Confocal images of Mtb-infected tissues. MΦ: anti-CD68 (red); Mtb: GFP (green); nuclei: DAPI (blue). The arrows in C and D indicate Mtb-infected macrophages. (E) CFSE-labeled monocytes (green) implanted in the model migrated towards the apical portion of the tissue in response to the monocyte-specific chemokine MCP1. Arrows in the image indicate green-labeled monocytes. (F) Quantitative analysis of the mean fluorescence intensity (MFI) in the apical versus basal layer of the tissues (means ± s.e.m., n=3). Statistical analysis was performed with a two-tailed unpaired t-test (F). Scale bars: 50 μm.
Fig. 2.
Fig. 2.
Monocytes/macrophages in the tissue model cluster around virulent Mtb. (A) Tissue models (nuclei stained with DAPI, blue) were implanted with PKH26-labeled monocytes (red) and macrophages infected with GFP-expressing (green) H37Rv, H37Ra or BCG, and sectioned. Fixed tissues were subjected to confocal microscopy at D7 or D10. Arrows indicate early granuloma structures. (B) Regions of interest (ROIs) with (ROIbact) and without (ROIcon) bacteria were selected in the green channel and the MFI in the red channel was determined to quantify the recruitment of PKH26-labeled cells to the site of infection (n=4). Statistical analysis was carried out using a two-tailed unpaired t-test. Scale bars: 50 μm.
Fig. 3.
Fig. 3.
RD1 and ESAT-6 are required for granuloma formation. (A) Analysis of clustering of PKH26-labeled cells in D7 tissue models infected with wild type (WT) Mtb H37Rv, ΔESAT-6 (GFP) and ΔRD1 (mCherry). Arrow indicates a nascent granuloma. (B) Quantification of MFI of PKH26 (WT and ΔESAT-6) and PKH67 (ΔRD1) at sites with bacteria (ROIbact) and without bacteria (ROIcon) (n=3). Statistical analysis was performed by using a two-tailed unpaired t-test. (C) Combined analysis of MFIbact:MFIcon ratios of the experiments with H37Rv, H37Ra, BCG, ΔESAT-6 and ΔRD1 (n=3–7). The solid lines represent the median ratios. Statistical analysis was performed with the Kruskal-Wallis test followed by Dunn’s multiple comparison test. Scale bars: 50 μm.
Fig. 4.
Fig. 4.
TB-infected human lymph node and lung tissue sections display macrophage aggregates at sites with Mtb. Sections of lymph node or lung biopsies obtained from TB patients were stained with anti-human CD68 (red), anti-Mtb (green) and DAPI (blue), and analyzed by confocal microscopy. Representative images from one of two analyzed lymph node (A) and lung tissue (B) biopsies from TB patients are shown. The area of the image enclosed by a solid line refers to a granulomatous region with macrophage aggregates and the arrows indicate macrophages harboring Mtb. Sections stained without primary antibodies (Abs) were used as controls. Scale bars: 50 μm.
Fig. 5.
Fig. 5.
Necrosis occurs in tissue models infected with virulent Mtb. (A) The tissue models were infected with H37Rv, BCG or H37Rv–ΔESAT-6 and sectioned, fixed and stained for HMGB-1 (brown) and HA (blue) at D7. (B) In situ quantification of the HMGB-1 strain in A. Data are given as percentage of stained area out of total cell area and given as medians with interquartile range (n=3). Statistical analysis was performed with one-way ANOVA followed by Tukey’s multiple comparison test. Scale bars: 50 μm.
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