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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 176-184

Analysis of Mycobacterium tuberculosis Uptake by Alveolar Macrophages after Ex vivo Expansion Indicates Processing Host Cells with Pathogen Actually from Lung Tissue of Patients with Pulmonary Tuberculosis


1 Laboratory of Medical Biotechnology, Research Institute of Biochemistry, Federal Research Center of Fundamental and Translational Medicine, Novosibirsk, Russia
2 Scientific Department, Ural Research Institute for Phthisiopulmonology, National Medical Research Center of Tuberculosis and Infectious Diseases of Ministry of Health of the Russian Federation, Yekaterinburg, Russia
3 Shared Center for Microscopic Analysis of Biological Objects, Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia

Date of Web Publication29-May-2020

Correspondence Address:
Elena Ufimtseva
Laboratory of Medical Biotechnology, Research Institute of Biochemistry, Federal Research Center of Fundamental and Translational Medicine, 2 Timakova Street, 630117 Novosibirsk
Russia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_39_20

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  Abstract 


Background: Previously, the ex vivo cultures of alveolar macrophages were developed from the surgical samples of the lungs in patients with pulmonary tuberculosis (TB) to establish the unique features of Mycobacterium tuberculosis (Mtb) lifestyle in host cells, but the question has remained whether Mtb-infected cells are isolated from the human lungs or they may be the result of Mtb phagocytosis in ex vivo culture. The study was aimed to investigate Mtb uptake by TB patients' cells after ex vivo expansion. Methods: Alveolar macrophages were infected with the Mtb clinical isolates in ex vivo culture, and the acid-fast Mtb loads in the cells were analyzed. Immunofluorescent staining and the examination of cytological and histological preparations by confocal microscopy were applied to detect Mtb ligands and macrophage surface markers. Results: The studies shown the lack of Mtb uptake by patients' alveolar macrophages during experimental infection with highly virulent Mtb clinical isolates containing pathogen-associated molecular patterns lipoarabinomannan and Ag38 at all used multiplicity of infection including a very high dose of infection. This fact was probably determined by the absence of pattern recognition receptors CD14, TLR2, and CD11b on the plasma membrane of human cells, likely, as a result of cellular processing from the resected lung tissues of patients. Conclusion: The findings indicate that alveolar macrophages with single Mtb or Mtb in colonies, including those with cord-morphology, found in the ex vivo cell cultures of all TB patients examined, were isolated from the lungs, and they characterize the Mtb infection in patients at the time of surgery.

Keywords: Alveolar macrophages, Mycobacterium tuberculosis clinical isolates, pathogen-associated molecular patterns, patients with pulmonary tuberculosis, pattern recognition receptors, phagocytosis


How to cite this article:
Ufimtseva E, Eremeeva N, Bayborodin S, Umpeleva T, Vakhrusheva D, Skornyakov S. Analysis of Mycobacterium tuberculosis Uptake by Alveolar Macrophages after Ex vivo Expansion Indicates Processing Host Cells with Pathogen Actually from Lung Tissue of Patients with Pulmonary Tuberculosis. Int J Mycobacteriol 2020;9:176-84

How to cite this URL:
Ufimtseva E, Eremeeva N, Bayborodin S, Umpeleva T, Vakhrusheva D, Skornyakov S. Analysis of Mycobacterium tuberculosis Uptake by Alveolar Macrophages after Ex vivo Expansion Indicates Processing Host Cells with Pathogen Actually from Lung Tissue of Patients with Pulmonary Tuberculosis. Int J Mycobacteriol [serial online] 2020 [cited 2020 Jul 11];9:176-84. Available from: http://www.ijmyco.org/text.asp?2020/9/2/176/285227




  Introduction Top


Tuberculosis (TB) remains to be the leading infectious cause of death worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, is a highly infectious and successful human pathogen, which is usually transmitted through aerosols.[1] In human lungs, Mtb is engulfed by alveolar macrophages, which serve as the major host cell niche for Mtb survival and propagation.[2] These phagocytic cells interact with microbes through the recognition of pathogen-associated molecular patterns (PAMPs) on Mtb, such as lipopolysaccharides, glycoproteins (for example, the 38-kDa phosphate-binding glycoprotein PstS-1 known as the 38-kDa antigen or Ag38), and glycolipids (for example, the largest and most complex lipoglycan lipoarabinomannan [LAM]), which are also essential for Mtb virulence.[3],[4] These PAMPs are recognized by a subset of cell surface pattern recognition receptors (PRRs), such as the phagocytic receptor CD14, complement receptors (CRs), toll-like receptors (TLRs), and others, leading to the generation of a cascade of signaling pathways, resulting in the initiation of the innate immune response against invading pathogens.[5],[6],[7],[8]

Previously, based on the work on the isolation of cells from mouse granulomas,[9],[10] a technique was established to produce ex vivo cultures of cells, mainly of alveolar macrophages, from different parts of lung tissues surgically removed from patients with pulmonary TB to determine the level of Mtb infection, and the biological properties and functional status of the pathogen at the time of surgery.[11],[12],[13] At hours 16–18 of ex vivo culture, the number of Mtb-infected alveolar macrophages and the number of Mtb, referring mainly to the Beijing genotype family, in human cells strongly varied across TB patients: Only single Mtb were detected in alveolar macrophages of some patients, while in alveolar macrophages of other patients Mtb were actively replicating in colonies, including with the cord-morphology of Mtb growth, when replicating Mtb line up along their longitudinal axes, setting themselves into “braids,” and which was associated with increased virulence of Mtb in the guinea pig TB model.[11],[12],[13] The technique proposed for assessing the level of Mtb infection in alveolar macrophages has higher sensitivity than do traditional bacteriological or pathomorphological methods, but the question has remained whether Mtb-infected cells are actually isolated from patients' lung tissues, or they may be the result of Mtb uptake after ex vivo expansion. In this work, we studied the capabilities for the Mtb phagocytosis in TB patients' alveolar macrophages in ex vivo culture and analyzed the possible reasons for the lack of Mtb uptake by patients' cells after experimental infection with highly virulent Mtb clinical isolates at all multiplicity of infection (MOI) used.


  Methods Top


Patients

Lung tissue samples were obtained from four patients with pulmonary TB at the Department of Thoracic Surgery of the Ural Research Institute for Phthisiopulmonology (Yekaterinburg, Russia) affiliated with the National Medical Research Center of Tuberculosis and Infectious Diseases of the Ministry of Health of the Russian Federation (Moscow, Russia) in July 2018 (patients 27 ÷ 30) as described.[11] The patients' nomenclature used is explained previously.[11] All the patients (age, treatment, attendant diseases, surgery, and other parameters) were characterized in detail in [Table 1]. All the patients had fibrotic and caseotic TB lesions in the lungs and had been referred for the surgical management of treatment-refractory pulmonary TB. All procedures involving patients were fully reviewed and approved by the Ethical Committee of the Ural Research Institute for Phthisiopulmonology (27/2014/07/02) and conducted in accordance with the principles expressed in the Helsinki Declaration.
Table 1: The characteristics of patients with pulmonary tuberculosis and analysis of Mycobacterium tuberculosis and alveolar macrophages with Mycobacterium tuberculosis obtained from patients' resected lung tissues after surgery

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Ex vivo production of patients' alveolar macrophages

Alveolar macrophages from the samples of surgically resected lung tissue of the patients were produced as described.[11] Immediately after surgery, samples (~15 g) of lung tissues obtained from lung parts distant from macroscopic TB lesions (tuberculomas) were cut into small pieces and rubbed through a metal screen of a sieve with pores 0.5–2.0 mm in diameter in phosphate-buffered saline (PBS, pH 7.4) for separating cellular suspension containing alveolar macrophages from closed granulomatous-fibrotic tissue. Cellular pellets after centrifugation at 400 g for 5 min at room temperature were placed to 24-well plates (Orange Scientific, Belgium) with cover glasses (approximately 8 mm × 8 mm in size) in the bottom and cultured in 0.5 ml of RPMI 1640 complete growth medium containing 10% fetal bovine serum (FBS), 2 mM glutamine and 50 μg/ml gentamicin (BioloT, Russia) for 18 h at +37°C in an atmosphere containing 5% CO2. Simultaneously, the analysis of Mtb DNA and virulence in the lung tissue samples of patients 27 ÷ 30 was performed as described.[12],[13]

Bacterial strains and cultures

The highly virulent Mtb clinical isolates 14–329 and 14–319 obtained from the lung tissue homogenates of TB patients 6 and 8, respectively, during our earlier work[11],[12] and here named as Mtb 6 and Mtb 8, respectively, and the virulent Mtb laboratory strain H37Rv (TMC#102, FSBI Tarasevich State Control Institute, Moscow, Russia) were used in this study. The aliquots of each Mtb strain were stored in 50% glycerol at −70°C. The aliquots of the Mtb strains used were thawed at room temperature and inoculated on Lowenstein–Jensen solid medium (BD, USA). Colonies of Mtb cultivated for 3 weeks were recovered from solid medium, collected in PBS, and vigorously shaken in tubs with glass beads on a Vortex-type device. The number of bacteria/ml was determined using optical density at 600 nm as a function of colony-forming units (CFU)/ml. The stock concentration of Mtb clinical isolates and laboratory strain H37Rv was prepared in PBS according to the 0.5 McFarland standard, which contained 1.5 × 108 CFU of Mtb.

Experimental infection

At hour 18 of ex vivo culture, after the removal of growth medium with dead cell debris and nonadherent cells, monolayer cultures of patients' cells on glass coverslips were washed twice with PBS to prepare them for experimental infection. Mtb 6 and Mtb 8 were added to cells of patient 27 at a MOI (mycobacteria to alveolar macrophage) of 1 and 10, and to cells of patients 28 and 29 at an MOI of 50. The cells of patient 30 were exposed to both Mtb clinical isolates and Mtb laboratory strain H37Rv at an MOI of 500. For all the experiments, the Mtb-infected samples were prepared to achieve the MOI required for each experiment, from the bacterial solutions with the stock concentration of Mtb. The nomenclature of Mtb-infected samples with different MOI is Mtb 6-1, Mtb 6–10, Mtb 6–50, Mtb 6–500, Mtb 8-1, Mtb 8–10, Mtb 8–50, Mtb 8–500, and Mtb H37Rv-500. Patients' cells on cover glasses were incubated with 200 μl of each Mtb-infected sample in the complete growth medium without antibiotics in 24-well tissue culture plates for 1 h at + 37°C in an atmosphere containing 5% CO2. The cells were further extensively washed twice with PBS to remove extracellular bacteria and were cultured for various time periods under the same conditions as cells without experimental infection (the control cell cultures). Before experimental infection of patients' cells, a 50 μl aliquot of Mtb from each Mtb-infected sample was added to an uncoated glass microscope slide, dried, fixed, and stained by the Ziehl–Neelsen (ZN) method and at an MOI of 500, additionally by antibodies to Mycobacteria LAM and Ag 38 to control the presence of acid-fast Mtb and Mtb ligands in the cell wall of bacteria, respectively. The size of each dried spot with Mtb on the microscope slide approximately corresponded to the size of the cover glasses with anchored patients' cells used in these experiments.

Cell staining

At hour 18 of ex vivo culture, some cell cultures of patients on cover glasses were washed with PBS and fixed with 4% formaldehyde solution in PBS for 10 min at room temperature. The same procedure was applied to human cells at hours 4, 24, 48, 72, 96, 120 (for patient 27) and at hours 4, 72, and 144 (for patients 28 ÷ 30) following experimental infection with Mtb and to control cell cultures without experimental infection. To visualize acid-fast Mtb within host cells, the preparations were washed with PBS and stained by the ZN method. After the ZN staining, the cells were further counterstained with Mayer's hematoxylin. Some cell preparations fixed at hour 18 of ex vivo culture were washed with PBS, blocked in PBS solution containing 2% BSA, and incubated with rabbit monoclonal primary antibodies to human CD14 (Spring Bioscience, USA, M492) diluted 1:100 and PE-conjugated mouse monoclonal primary antibodies to human TLR2 (BioLegend, USA, 309708) diluted 1:200 or mouse monoclonal primary antibodies to the Mtb 38-kDa protein (Abcam, England, ab183165) diluted 1:1000. Fluorescent visualization of CD14 and Mtb Ag38 was enabled using secondary goat polyclonal DyLight 488-conjugated secondary antibodies to rabbit IgG (ThermoFisher Scientific, USA, A11034) diluted 1:400 and Alexa 555-conjugated secondary antibodies to mouse IgG (Invitrogen, USA, A21422) diluted 1:500. Other fixed cell preparations were washed with PBS, blocked and incubated with rat monoclonal APC-conjugated primary antibodies to human CD11b (eBioscience, USA, 17-0112) diluted 1:200. The Mtb spots fixed on microscope slides were washed with PBS, blocked and incubated first with rabbit polyclonal primary antibodies to Mycobacteria LAM (Abcam, England, ab20832) diluted 1:200 and mouse monoclonal primary antibodies to the Mtb 38-kDa protein diluted 1:1000, then with Alexa 488-conjugated goat anti-rabbit IgG secondary antibodies diluted 1:400 and Alexa 555-conjugated secondary antibodies to mouse IgG diluted 1:500. The cell preparations were incubated with the appropriate antibodies for 60 min at room temperature. Fluorescent staining was analyzed using the VECTASHIELD Mounting Medium with DAPI (4´,6-diamidino-2-phenylindole) (Vector Laboratories, USA, H-1200).

Histology

The histological sections of the resected lung tissues of the TB patients were prepared as described.[13] Briefly, the resected lungs of patients were cut into pieces. One portion of lung pieces was collected for producing alveolar macrophages, as described above. The other portion of lung pieces was fixed with 4% formaldehyde solution in PBS for 20 h at +4°C. After fixation, the lung tissues were washed with PBS, incubated with 30% sucrose in PBS for 20 h at +4°C, frozen in Tissue-Tek O. C. T. Compound at −25°C, and sectioned at 16-μm slides on the Microtome Cryostat HM550 (Microm, Germany) at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics (Novosibirsk, Russia). Sections were air-dried on SuperFrost Plus slides (ThermoFisher Scientific, USA) and immunofluorescent stained as described above. The histological sections were incubated with the appropriate primary antibodies for 20 h at +4°C. To visualize acid-fast Mtb within host cells, sections were stained by the ZN method and further counterstained with Mayer's hematoxylin.

Microscopy

The histological sections, cytological and other preparations were examined at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, SB RAS (Novosibirsk, Russia), using an Axioscop 2 plus microscope (Zeiss) and an LSM 780 laser scanning confocal microscope (Zeiss) as described.[11],[12],[13] All human cells and Mtb in human cells were counted separately on each cover glass for each patient and phagocytosis assay. More than 1000 alveolar macrophages were analyzed at each preparation for each patient. Statistical data processing was performed using MS Excel 2007 (Microsoft, Redmond, Washington, USA).


  Results Top


The ex vivo cultures of cells were isolated from the specimens of lung tissue resected during elective surgery from TB patients 27 ÷ 30 and characterized in [Table 1]. The ex vivo cell cultures studied were largely composed of alveolar macrophages and some dendritic and multinucleated Langhans giant cells that were viable and had neither apoptotic nor necrotic morphology after ex vivo culture for 18 h. Some of the alveolar macrophages had a large number of denser dark inclusions in the cytoplasm and were what is called smokers' macrophages. Acid-fast Mtb was detected only in single alveolar macrophages from patients 27 ÷ 29. Alveolar macrophages each had only a single low virulent Mtb, referred mainly to the Beijing genotype family. No Mtb in colonies or with cord-morphology were identified in the ex vivo cell cultures. No Mtb-infected alveolar macrophages were observed in the ex vivo cell culture for patient 30. Alveolar macrophages with Mtb were also rare on the histological sections from the resected lung tissues distant from macroscopic TB lesions for patients 28 and 29 and were absent for patients 27 and 30. Thus, the ex vivo cell cultures of patients 27 ÷ 30 contained very small numbers of Mtb-infected alveolar macrophages and might be used for experimental infection.

At hour 18 of ex vivo culture, alveolar macrophages of patients 27 ÷ 30 were infected with two highly virulent Mtb clinical isolates obtained earlier in our work[11],[12] and named here as Mtb 6 and Mtb 8. In the infectious samples under microscope examination, Mtb 6 and Mtb 8 were acid-fast [Figure 1]a and c], contained Mtb PAMPs such as LAM and Ag 38 on the bacterial cell wall [Figure 1]b and d], and were located as independent bacteria or in some Mtb aggregations, including cords [Figure 1]a, [Figure 1]b, [Figure 1]c.
Figure 1: Acid-fast clinical isolates (a and b) Mtb 6 and (c and d) Mtb 8 contain different PAMPs on the bacterial cell wall. (a and c) Acid-fast Mtb from infectious samples are stained by the ZN method. (c and d) Representative confocal fluorescent images of Mtb stained by Mtb LAM-specific (left panels, green signal) and Ag38-specific antibodies (central panels, red signal) show the colocalization of Mtb ligands (right panels with phase-contrasted images, yellow signal). The scale bars are 10 μm each. PAMPs: Pathogen-associated molecular patterns, Mtb: Mycobacterium tuberculosis, LAM: Lipoarabinomannan, ZN: Ziehl–Neelsen

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First, alveolar macrophages of patient 27 (the cell density of them was close to the monolayer on cover glasses) were infected with Mtb clinical isolates at low MOI of 1 or 10. The number of alveolar macrophages with Mtb in the Mtb-infected cell cultures was largely consistent with the data obtained for the control cell cultures without experimental infection [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d and [Figure 3]a, [Figure S1]a. We detected only single regions of patient' cells (no more than 200 × 200 μm) after experimental infection where almost half of the alveolar macrophages contained individual Mtb or their colonies, including those with cord-morphology [Figure 2]c and [Figure 2]d. Two such cell regions were found in the cell cultures at hour 48 following infection with Mtb 6-1 and Mtb 6–10, and represented the small pieces of lung tissue where many alveolar macrophages contained a large number of Mtb. Of note, adjacent cells not associated with these lung tissue pieces were not infected with Mtb. In parallel, only a single alveolar macrophage with one Mtb was defined in the piece of lung tissue saved in the control cell culture at hour 48 without experimental infection. Other four such regions with the increased number of Mtb-infected cells were located on the edges of the cover glasses. Only few dendritic cells with Mtb were observed in all cell cultures analyzed [Figure 2]d. Further, we infected alveolar macrophages of patients 28 and 29 with the same Mtb clinical isolates at medium MOI (50 Mtb per cell) and again identified only single alveolar macrophages with Mtb, whose numbers were mainly comparable to the data obtained for the control cell cultures without experimental infection [Figure 3]b and [Figure S1]b and [Figure S2]a, [Figure S2]b, [Figure S2]c, [Figure S2]e. The main difference between experimental infection at different MOI was that, at low MOI, alveolar macrophages of patient 27 mainly contained 1–2 Mtb, whereas at medium MOI, an increased number of Mtb, predominantly in colonies, including those with cord-morphology, was revealed in some patients' cells. This situation prompted us to infect alveolar macrophages of patient 30 with the same Mtb clinical isolates and for the first time, a laboratory strain H37Rv at high MOI of 500 [Figure 3]c and [Figure S1]c and [Figure S4]a, [Figure S4]b, [Figure S4]e. Note that, the number of Mtb used for experimental infection of alveolar macrophages had to be about three times as high as the number of Mtb that was observed in the images of microbes in the infectious samples on the same surface area of the cover glasses with human cells [Figure S4]a. At hour 4 following experimental infection, we did not find human cells with Mtb in the cell cultures both without experimental infection (control) and after experimental infection with Mtb 6. Only single alveolar macrophages with two Mtb 8 were identified. Alveolar macrophages were better infected with the Mtb laboratory strain H37Rv than with the Mtb clinical isolates, but the number of infected cells, each contained from one to more than twenty Mtb H37Rv, including in colonies with cord-morphology, remained generally low. After prolonged culture following experimental infection at medium and high MOI, we found a large number of Mtb-infected alveolar macrophages with morphological signs of cell death (nuclei with intense chromatin condensation, nuclear fragmentation, compromised cytoplasmic membranes, leakage of cell components, the occurrence of nucleus-free cells and chromatolysis), as a rule with increased acid-fast Mtb loads in them, and only live alveolar macrophages without Mtb in the cell cultures of patients [Figure S2]b, [Figure S2]d and [Figure S2]e, [Figure S3]a and [Figure S3]b and [Figure S4]e. Notably, viable Mtb-infected alveolar macrophages were revealed near patients' dead cells at hours 72 and 144 [Figure S2]b, [Figure S2]d and [Figure S2]e. Probably, the infection of neighboring alveolar macrophages by Mtb from the dead human cells was responsible for some increase in the number of Mtb-infected alveolar macrophages in these cell cultures [Figure 3]a, [Figure 3]b, [Figure 3]c. In parallel, we observed a decrease in the number of patients' cells containing a small number of Mtb in each and a gradual increase in the number of alveolar macrophages with increased Mtb loads in them during prolonged culture after experimental infection at all MOI used [Figure S1]a, [Figure S1]b, [Figure S1]c. No alveolar macrophages with ingested nonacid-fast Mtb were detected both in viable and in dead cells of patients 27 ÷ 30. Thus, lack of Mtb uptake by patients' alveolar macrophages after experimental infection at all MOI used in ex vivo culture was observed, although in the infectious samples acid-fast Mtb clinical isolates expressed LAM and Ag 38 ligands, which belong to Mtb PAMPs and must be recognized by alveolar macrophages with subsequent initiation of Mtb phagocytosis.
Figure 2: Single alveolar macrophages with Mtb are mostly found in the cell cultures of patient 27 exposed to Mtb clinical isolates at low MOI. (a-d) Alveolar macrophages with acid-fast Mtb, stained by the ZN method, are indicated by black and ([c], left panel) white arrows. The scale bars are 10 μm each. (a) Some alveolar macrophages with (left panel) single Mtb in the ex vivo cell culture without experimental infection and (other panels) Mtb cords in the cell cultures at hour 4 following infection with (central-left panel) Mtb 8-1 and (right and central-right panels) Mtb 8–10. (b) Alveolar macrophages with engulfed Mtb cords are viable and have no morphological sings of cell death in the cell cultures at (left and central-right panels) hour 48 following infection with Mtb 6–10 and Mtb 8-1, respectively, and (right and central-left panels) hour 120 following infection with Mtb 6–10 and Mtb 8–10, respectively. ([c], left panel) Single alveolar macrophage with one Mtb and ([c], right panel) many alveolar macrophages with Mtb cords are detected in the pieces of lung tissue saved in the cell cultures at hour 48 without experimental infection and following infection with Mtb 6–10, respectively. (d) The regions of cells with a large number of Mtb-infected alveolar macrophages in the cell cultures at hour 96 following infection with (left panel) Mtb 6-1 and (right panel) Mtb 8–10. The dendritic cell with a single acid-fast Mtb is indicated by the green arrow. Mtb: Mycobacterium tuberculosis, MOI: Multiplicity of infection

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Figure 3: A small quantity of Mtb-infected alveolar macrophages of patients (a) 27, (b) 28, 29, and (c) 30 is detected both in the ex vivo cell cultures without experimental infection (control) and following experimental infection with Mtb clinical isolates at all MOI used. The number of alveolar macrophages with Mtb expressed as the percentage of the total number of examined alveolar macrophages stained by the ZN method after ex vivo culture for several hours. Mtb: Mycobacterium tuberculosis, ZN: Ziehl–Neelsen, MOI: Multiplicity of infection

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As is known, LAM- and Ag38-regulated phagocytosis of Mtb is dependent mainly on the plasma membrane PRRs CD14, TLR2, and CR3 with the CD11b subunit.[5],[14],[15] At hour 18 of ex vivo culture, PRRs CD14, TLR2, and CD11b were found only in the intracellular vesicles, but not on the plasma membrane of patients' alveolar macrophages [Figures 4a and b and S5a], studied both here and previously.[11],[13] At the same time, the markers were identified both in large numbers on the cell surface, including in microdomains, and in the vesicles in the cytoplasm of all alveolar macrophages on the histological sections from the resected lungs of the same TB patients [Figures 4c and d and S5b-e]. On the histological sections, CD14 often colocalized with TLR2 [Figure S5c and e], which is an important coreceptor for CD14 for Mtb internalization. Taken together, our findings suggest that Mtb phagocytosis in TB patients' alveolar macrophages during experimental infection was strongly inhibited, likely, due to the lack of PRRs on the cell surface after ex vivo expansion.


  Discussion Top


In the present study, we established that highly virulent rod-shaped Mtb clinical isolates, obtained earlier in our work and containing PAMPs, such as LAM and Ag38, on the bacterial cell wall, were not internalized by patients' alveolar macrophages during experimental infection at all MOI used, including a very high dose of infection, probably, due to the lack of PRRs, such as CD14, TLR2, and CD11b, on the surface of human cells after ex vivo expansion. Furthermore, it was demonstrated the absence of phagocytosis of BCG-mycobacteria by human THP-1 and hamster CHO cells without functional PRRs, whereas the cells stably transfected with CD14, TLR2, and CR3 markedly enhanced bacterial internalization.[5] In our work, Mtb was incubated with patients' alveolar macrophages in the presence of 10% FBS with complement proteins in the infectious samples during experimental infection. Consequently, the CR3-pathway could be activated for Mtb uptake by human cells if CD11b receptors were expressed on the alveolar macrophage after ex vivo expansion. It is likely that the ability to phagocytosis is restored in alveolar macrophages after prolonged ex vivo culture since the infection of neighboring viable alveolar macrophages by Mtb from dead cells was detected in the cell cultures of patients at hours 72 and 144 following experimental infection. The removal of PRRs from the surface of patients' cells was likely the result of cellular processing from the surgically resected lung tissues and characterized the specific features of patients' alveolar macrophage cultivation on cover glasses at early times in ex vivo culture. The influence of the above processes is indirectly confirmed by the observation that a large number of Mtb was phagocytized by most alveolar macrophages during experimental infection even at low MOI, which were preserved in the small pieces of lung tissue in some cell cultures of patient 27 and presumably, retained the localization of PRRs on the plasma membrane. Interestingly, in the case of Mtb internalization by patients' alveolar macrophages in ex vivo culture, we have identified in general the same dynamics of Mtb behavior in human cells during prolonged culture after experimental infection with a gradual increase in Mtb loads in them and increased death rates for alveolar macrophages, each containing >20 Mtb, as in our previous work with acute BCG infection of mouse cells in in vitro culture.[16]

A significantly larger number of alveolar macrophages were infected with the Mtb laboratory strain H37Rv with a larger number of the engulfed microbes than with Mtb clinical isolates in the cell cultures of patient 30. In addition, the number of Mtb-infected alveolar macrophages with morphological sings of death was lower in the cell cultures following experimental infection with the Mtb laboratory strain H37Rv than Mtb clinical isolates for few days of cultivation. It can be assumed that the Mtb laboratory strain H37Rv with a century-long history of its use in various research laboratories of the world has acquired such changes in the bacterial cell wall that allow it to better infect mammalian cells and survive in them. Of note, nonacid-fast (and therefore destroyed) Mtb, such as clinical isolates and the laboratory strain H37Rv, were not detected both in viable and dead patients' alveolar macrophages after experimental infection at all MOI used, whereas in acute BCG infection of mouse cells in in vitro culture, nonacid-fast BCG-mycobacteria were found in some bone marrow and peritoneal macrophages.[16] Differences both in infectious agents (virulent Mtb vs. attenuated BCG) and in cells (those from intact mice vs. those from the lung tissues of TB patients) probably influenced the results of the experiments. Accordingly, the BCG-infected cells of intact mice were able to kill some bacteria, but alveolar macrophages of patients could not do as much in all ex vivo cell cultures, although they could control the reproduction of their “own” low virulent Mtb both during TB disease before surgery and in ex vivo culture. It is likely that a similar situation can be observed in the human population in the case of the secondary infection of TB patients with Mtb, which are characterized by other signs, for example, a higher virulence. The identified events with significant local rearrangements of the plasma membrane components, including the surface receptors, after ex vivo expansion are likely common to all alveolar macrophages of all TB patients studied, whose cells were obtained from the resected lung tissues according to the method proposed by us and also shown the intracellular localization of the receptor CD14 as demonstrated previously.[11],[13] This hypothesis is confirmed by the fact that approximately the same small number of human cells with Mtb was identified in the ex vivo cell cultures of the patients after experimental infection with Mtb clinical isolates at all MOI used.


  Conclusions Top


The findings indicate that the alveolar macrophages with single Mtb or Mtb in colonies, including those with cord-morphology, a significant number of which were obtained in the ex vivo cultures of cells in previously performed studies, were isolated actually from the resected lung tissues of the patients, and consequently, they characterize the TB infection in patients' lungs at the time of surgery. In clinical practice, rapid quantification of Mtb infection level and functional status of the pathogen in alveolar macrophages is very important for a personalized revision of treatment regimens in postoperative TB patients.

Acknowledgments

The authors are thankful to T. Aleshina (Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, Novosibirsk), E. Petrunina and L. Lavrenchuk (Ural Research Institute for Phthisiopulmonology, Yekaterinburg) for technical support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Pieters J, McKinney JD, editors. Pathogenesis of Mycobacterium tuberculosis and its Interaction with the Host Organism. Curr. Topics Microbiol. Immunol. Vol. 374. Berlin: Springer; 2013.  Back to cited text no. 1
    
2.
Guirado E, Schlesinger LS, Kaplan G. Macrophages in tuberculosis: Friend or foe. Semin Immunopathol 2013;35:563-83.  Back to cited text no. 2
    
3.
Källenius G, Correia-Neves M, Buteme H, Hamasur B, Svenson SB. Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations. Tuberculosis 2016; 96:120-30.  Back to cited text no. 3
    
4.
Forrellad MA, Klepp LI, Gioffré A, Sabio y García J, Morbidoni HR, de la Paz Santangelo M, et al. Virulence factors of the Mycobacterium tuberculosis complex. Virulence 2013;4:3-66.  Back to cited text no. 4
    
5.
Sendide K, Reiner NE, Lee JS, Bourgoin S, Talal A, Hmama Z. Cross-talk between CD14 and complement receptor 3 promotes phagocytosis of mycobacteria: Regulation by phosphatidylinositol 3-kinase and cytohesin-1. J Immunol 2005;174:4210-9.  Back to cited text no. 5
    
6.
Ferguson JS, Weis JJ, Martin JL, Shlesinger LS. Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid. Infect. Immun. 2004; 72: 2564-73.  Back to cited text no. 6
    
7.
Gopalakrishnan A, Salgame P. Toll-like receptor 2 in host defense against Mycobacterium tuberculosis: To be or not to be-that is the question. Curr Opin Immunol 2016;42:76-82.  Back to cited text no. 7
    
8.
Dey B, Bishai WR. Crosstalk between Mycobacterium tuberculosis and the host cell. Semin Immunol 2014;26:486-96.  Back to cited text no. 8
    
9.
Ufimtseva E. Investigation of functional activity of cells in granulomatous inflammatory lesions from mice with latent tuberculous infection in the new ex vivo model. Clin Dev Immunol 2013;2013:14.  Back to cited text no. 9
    
10.
Ufimtseva E. Mycobacterium-host cell relationships in granulomatous lesions in a mouse model of latent tuberculous infection. Biomed Res Int 2015;2015:16.  Back to cited text no. 10
    
11.
Ufimtseva E, Eremeeva N, Petrunina E, Umpeleva T, Karskanova S, Baiborodin S, et al. Ex vivo expansion of alveolar macrophages with Mycobacterium tuberculosis from the resected lungs of patients with pulmonary tuberculosis. PLoS One 2018;13:e0191918.  Back to cited text no. 11
    
12.
Ufimtseva EG, Eremeeva NI, Petrunina EM, Umpeleva TV, Baiborodin SI, Vakhrusheva DV, et al. Mycobacterium tuberculosis cording in alveolar macrophages of patients with pulmonary tuberculosis is likely associated with increased mycobacterial virulence. Tuberculosis 2018;112:1-10.  Back to cited text no. 12
    
13.
Ufimtseva E, Eremeeva N, Baiborodin S, Umpeleva T, Vakhrusheva D, Skornyakov S. Mycobacterium tuberculosis with different virulence reside within intact phagosomes and inhibit phagolysosomal biogenesis in alveolar macrophages of patients with pulmonary tuberculosis. Tuberculosis 2019;114:77-90.  Back to cited text no. 13
    
14.
Jung SB, Yang CS, Lee JS, Shin AR, Jung SS, Son JW, et al. The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes. Infect Immun 2006;74:2686-96.  Back to cited text no. 14
    
15.
Turner J, Torrelles JB. Mannose-capped lipoarabinomannan in Mycobacterium tuberculosis pathogenesis. Pathog Dis 2018;76:fty026: 16.  Back to cited text no. 15
    
16.
Ufimtseva E. Differences between mycobacterium-host cell relationships in latent tuberculous infection of mice ex vivo and mycobacterial infection of mouse cells In vitro. J Immunol Res 2016;2016:21.  Back to cited text no. 16
    


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