Ex vivo expansion of alveolar macrophages with Mycobacterium tuberculosis from the resected lungs of patients with pulmonary tuberculosis

Abstract

Tuberculosis (TB), with the Mycobacterium tuberculosis (Mtb) as the causative agent, remains to be a serious world health problem. Traditional methods used for the study of Mtb in the lungs of TB patients do not provide information about the number and functional status of Mtb, especially if Mtb are located in alveolar macrophages. We have developed a technique to produce ex vivo cultures of cells from different parts of lung tissues surgically removed from patients with pulmonary TB and compared data on the number of cells with Mtb inferred by the proposed technique to the results of bacteriological and histological analyses used for examination of the resected lungs. The ex vivo cultures of cells obtained from the resected lungs of all patients were largely composed of CD14-positive alveolar macrophages, foamy or not, with or without Mtb. Lymphocytes, fibroblasts, neutrophils, and multinucleate Langhans giant cells were also observed. We found alveolar macrophages with Mtb in the ex vivo cultures of cells from the resected lungs of even those TB patients, whose sputum smears and lung tissues did not contain acid-fast Mtb or reveal growing Mtb colonies on dense medium. The detection of alveolar macrophages with Mtb in ex vivo culture as soon as 16-18 h after isolation of cells from the resected lungs of all TB patients suggests that the technique proposed for assessing the level of infection in alveolar macrophages of TB patients has higher sensitivity than do prolonged bacteriological or pathomorphological methods. The proposed technique allowed us to rapidly (in two days after surgery) determine the level of infection with Mtb in the cells of the resected lungs of TB patients and, by the presence or absence of Mtb colonies, including those with cording morphology, the functional status of the TB agent at the time of surgery.

Fig 1. Lung tissue used for ex vivo expansion of alveolar macrophages.

(A) The upper lobe of the left lung surgically removed from patient 8. (B) The tissue specimen of the part of the resected lung (A) distant from macroscopic lesions. The diameter of the Petri dish is 10 cm. (C) Alveolar macrophages produced from the lung tissue specimen (B) and stained by the ZN method after ex vivo culture for 18 hours. The black arrow points to an alveolar macrophage with acid-fast Mtb. The scale bar is 10 μm.

Fig 2. Different cell types obtained from the cavity wall in the resected lung of patient 6 and stained by the ZN method after ex vivo culture for 16 hours.

(A, B) A Langhans giant cell and a neutrophil containing acid-fast Mtb are indicated by the red and green arrows, respectively. (C) A lymphocyte interplaying with alveolar macrophages is indicated by the black snowflake. The other cells (A, B, C) are alveolar macrophages. Those with acid-fast Mtb are indicated by the black arrows, others are uninfected. The scale bars are 10 μm each.

Fig 3. CD14 staining of alveolar macrophages with Mtb performed after ex vivo culture for 18 hours.

(A, B, C) Representative confocal fluorescent 3D images show alveolar macrophages stained by human CD14-specific (green signal) and Mtb Ag38-specific antibodies (red signal). Nuclei are stained by DAPI (blue signal). Alveolar macrophages were obtained from the resected lungs of patients 16 (A, B) and 18 (C). Black or white arrows indicate a single Mtb (A) and mycobacterial colonies (B, C) residing within alveolar macrophages.

Fig 4. Histological examination of the resected lung from patient 7 shows the absence of cells with Mtb. Ex vivo analysis of alveolar macrophages disagrees.

(A) A surgically removed part of the lung. (B-F) Multiple TB lesions detected by histological analysis of tissue from the resected lung (A): N, caseous necrosis; M, a ring of macrophages; F, fibrous capsule; L, multinucleate Langhans giant cell. The scale bars are (B, D, E) 20 μm each, (C) 50 μm, and (F) 10 μm. (B, C, and E, F) Representative histological images show enclosed necrotizing granulomas stained by hematoxylin/eosin, a mixture of picric acid and fuchsin acid to detect collagen, and after the ZN method, respectively. (D) Multinucleate Langhans giant cells determined in an early non-necrotizing granuloma by ZN staining. (F) Scanty Mtb revealed in the caseous matter. Enlarged view of the part of this image with Mtb indicated by black arrows is shown in the upper right corner. (G) The tissue specimen of the resected lung (A) was cut into small pieces for producing alveolar macrophages. The diameter of the Petri dish is 10 cm. (H, I) Alveolar macrophages stained by the ZN method after ex vivo culture for 18 hours contain Mtb in isolation and as a colony, respectively. The black arrows point to alveolar macrophages with acid-fast Mtb. The scale bars are 10 μm each.

Fig 5. Foamy alveolar macrophages with Mtb determined in ex vivo cultures.

(A, C, D) Representative confocal fluorescent 3D images show lipid-rich foamy alveolar macrophages stained by Mtb LAM-specific antibodies (green signal) and Nile red (red signal). Nuclei are stained by DAPI (blue signal). Foamy alveolar macrophages obtained from the resected lungs of patients 4 (A, B), 9 (C), and 15 (C). White arrows point to Mtb residing in foamy alveolar macrophages. (B) In the cell profile (A), a foamy alveolar macrophage demonstrates lack of colocalization of Mtb and host lipid bodies (lack of yellow signal). However, the markers studied show colocalization for numerous intracellular lipid-laden vesicles (A, B, C, D).

Fig 6. Alveolar macrophages with Mtb in colonies are detected both in cavity walls and in the distant parts of lung tissue.

(A-C and D-F) Lung tissues and alveolar macrophages obtained from patients 6 and 8, respectively. (A, B, D, E) Representative histological images of the cavity walls (A, D) and the distant parts of lung tissue (B, E) stained by the ZN method demonstrate a massive load of replicating Mtb in the cavities and single Mtb within alveolar macrophages in other lung regions, respectively. (B, E) Enlarged views of the parts of these images with alveolar macrophages containing single Mtb (black arrows) are shown in the upper right corners. (C) Alveolar macrophages obtained from the cavity wall and after ex vivo culture for 16 hours contain colonies of replicating Mtb. (F) Alveolar macrophages obtained from the distant part of lung tissue and after ex vivo culture for 18 hours contain replicating Mtb in colonies with cording morphology. Alveolar macrophages (C, F) were stained by the ZN method. The black arrows point to alveolar macrophages with acid-fast Mtb. The scale bars are 10 μm each.

Fig 7. TB patients with the “advanced” extent of disease and “high-activation" status of TB inflammation have a larger population of alveolar macrophages with Mtb and mycobacterial colonies in the distant parts of lung tissue.

(A, B) The number of alveolar macrophages with Mtb (in isolation or as colonies) expressed as the percentage of the total number of alveolar macrophages analyzed. (C, D) The number of alveolar macrophages with Mtb in colonies expressed as the percentage of the total number of alveolar macrophages with any Mtb. Data are expressed as the means ± SEM. *P < 0.05; **P < 0.01, Student’s t-test.

Fig 8. A significant increase in the number of alveolar macrophages with Mtb in the distant parts of lung tissue in patients with cavities.

(A) The number of alveolar macrophages with Mtb (in isolation or as colonies) expressed as the percentage of the total number of alveolar macrophages analyzed. (B) The number of alveolar macrophages with Mtb in colonies expressed as the percentage of the total number of alveolar macrophages with any Mtb. Data are expressed as the means ± SEM. *P < 0.05, Student’s t-test.