Mycobacteria infect different cell types in the human lung and cause species dependent cellular changes in infected cells

Abstract

Background: Mycobacterial infections remain a significant cause of morbidity and mortality worldwide. Due to limitations of the currently available model systems, there are still comparably large gaps in the knowledge about the pathogenesis of these chronic inflammatory diseases in particular with regard to the human host. Therefore, we aimed to characterize the initial phase of mycobacterial infections utilizing a human ex vivo lung tissue culture model designated STST (Short-Term Stimulation of Tissues).

Methods: Human lung tissues from 65 donors with a size of 0.5-1 cm(3) were infected each with two strains of three different mycobacterial species (M. tuberculosis, M. avium, and M. abscessus), respectively. In order to preserve both morphology and nucleic acids, the HOPE® fixation technique was used. The infected tissues were analyzed using histo- and molecular-pathological methods. Immunohistochemistry was applied to identify the infected cell types.

Results: Morphologic comparisons between ex vivo incubated and non-incubated lung specimens revealed no noticeable differences. Viability of ex vivo stimulated tissues demonstrated by TUNEL-assay was acceptable. Serial sections verified sufficient diffusion of the infectious agents deep into the tissues. Infection was confirmed by Ziel Neelsen-staining and PCR to detect mycobacterial DNA. We observed the infection of different cell types, including macrophages, neutrophils, monocytes, and pneumocytes-II, which were critically dependent on the mycobacterial species used. Furthermore, different forms of nuclear alterations (karyopyknosis, karyorrhexis, karyolysis) resulting in cell death were detected in the infected cells, again with characteristic species-dependent differences.

Conclusion: We show the application of a human ex vivo tissue culture model for mycobacterial infections. The immediate primary infection of a set of different cell types and the characteristic morphologic changes observed in these infected human tissues significantly adds to the current understanding of the initial phase of human pulmonary tuberculosis. Further studies are ongoing to elucidate the molecular mechanisms involved in the early onset of mycobacterial infections in the human lung.

Fig. 1

Results of the TUNEL assays. PC = Positive Control (fresh lung preparations without ex vivo cultivation, N = 17), ev: Mean of all different ex vivo cultivated lung specimens, including Medium control (MED, N = 12), lungs infected with M. abscessus (AB, N = 17), M. avium (AV, N = 7) and M. tuberculosis (TB, N = 18). NC = Negative Controls (treated by DNase I, N = 8). Left panel: No significant increase of DNA-double strand breaks upon tissue culture. Right panel: Differences between the respective mycobacterial species. These results related with multiple factors analyzed by a factorial ANOVA [2×6 factorial design: subject factors were cell type (1-alive and 2-apoptotic cells) and groups (1-PC, 2-MED, 3-AB, 4-AV, 5-TB, 6-NC)]

Fig. 2

a Observation of serial sections of a whole paraffin block. Its 3D trapezoid size was approximately 0.7×0.5×0.4×0.4 mm. Serial cuts resulted in 250 sections with a thickness of 2 μm. The 1st -50th and 201st -250th sections were considered to be identical (same marginal condition), as well as the 51st -100th and 151st -200th sections (same para-peripheral condition). To compare incidents of infected cells among tissue, every odd numbered slides from 1st-50th (peripheral region, N = 25) and 101st-150 (central region, N = 25) were utilized. b Distribution of infected cells in different layers of the tissue. The x-axis shows the number of sections, the y-axis the numbers of infected cells. Descriptive statistics showed that the peripheral slides were associated with a number of infected cells 47.87 ± 38.9, whereas, central slides had 126.92 ± 59.47 of infected cells with high variances, followed by inferential statistics (non-parametric Mann–Whitney test)

Fig. 3

Mycobacteria are taken up by different cell types in the ex vivo infected human lung tissues a. Infected cell with labels, at × 640 magnification, intracellular bacilli were visualized by Ziel Neelsen staining in pink color, scale bar =100 μm. b. Infected cell types were shown separetely with labels, the images were acquired at × 1000 magnification, scale bars = 30 μm

Fig. 4

Cellular populations infected by 6 mycobacterial strains. a The frequencies of the infected cell types shown in the graph and occurrences of absolute numbers given in the table. b 30 infected cells were counted among 2 diagonal lines in each slide and in order to involve whole tissue equivalently as illustrated. c Correspondence analysis map for mycobacterial strains and cell types. Dispersions of all profile points produced an asymmetric map. The centroid was labeled by a star. Around the centroid, the profile points of the various mycobacterial species (blue square) and of the different cell types (black dot) were plotted. A bar plot of variances is shown at the left bottom part of the map

Fig. 5

Factual proportions of infected population of cells among the 6 mycobacterial strains. The used cellular parameters for adjustments are included

Fig. 6

Photomicrographs of intracellular mycobacteria detected by double staining. a Macrophages (*), b Neutrophils (□), c Pneumocytes-II (∆).Slides stained by combination of auramine-rhodamine (for mycobacteria) and IHC of CD 68 (macrophages), NE (neutrophils) and SP-C (pneumocytes-II), respectively. The fluorescence signals of mycobacteria are red. Brightfield was used to capture immunohistochemical detections. Images were color-inversed to negative form which is why brown color of the color-substrate diaminobenzidine was shifted to blue. Overlays of the fluorescent and negative images were performed by using FixFoto. All images were acquired at × 400 magnification, scale bar =100 μm

Fig. 7

Cellular changes in infected cells. Intracellular bacilli were visualized by Ziel Neelsen staining and appear in pink color, scale-bars = 20 μm. a Cell schrinkage of infected macrophages (1) and normal macrophages (2), ×680 magnification. b Karyorrhexis of an infected macrophage (×1000). c Karyolysis of an infected macrophage (×1000)

Fig. 8

Morphologic changes in the infected phagocytes induced by mycobacteria. a Frequency of characteristic changes. The table reports absolute numbers of incidences and sums of morphological types. b Correspondence analysis map for mycobacterial strains and cellular changes. The map was derived from a two-cross tabulation: 1) cellular morphology types, which consisted of four elements (normal shaped macrophages, karyorrhectic, karyopyknotic, and karyolitic phagocytes), 2) six mycobacterial strains (AB1, AB2, AV1, AV2, TB1, TB2). Dispersions of variables produced an asymmetric map, its centroid (point of no correlation) was labeled by a star. All profile points were plotted around this weighted average. A bar plot of variances is shown at the right top part of the map

Fig. 9

Mycobacterial infections lead to changes in the cellular sizes and light densities. a Comparisons of nuclear and cytoplasmic sizes between infected and non-infected cells. Mann–Whitney U-tests were performed, the ranks are given and meanings of p values labeled as asterisks (*** = p ≤ 0.001, ns = not significant). b Comparisons of cytoplasmic (top) and nuclear (bottom) light densities (LDs) between infected and non-infected cells. Based on the test assumptions Mann–Whitney U tests were chosen and the graphs were created by transformed data of 2 groups, as particularly infected and non-infected cells of nuclear (N = 100) and cytoplasmic (N = 100) LDs, respectively