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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 3  |  Page : 271-278

Sera from patients with tuberculosis increase the phagocytic-microbicidal activity of human neutrophils, and ESAT-6 is implicated in the phenomenon


1 Department of Immunology, National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico
2 Neumology Unit, General Hospital of Mexico “Eduardo Liceaga”, Mexico City, Mexico
3 Department of Immunology, Faculty of Medicine, Autonomous University of Nuevo Leon, Monterrey, Mexico

Date of Submission19-Jun-2021
Date of Acceptance03-Aug-2021
Date of Web Publication03-Sep-2021

Correspondence Address:
Oscar Rojas-Espinosa
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, Colonia Santo Tomás, 11340 Ciudad de México
Mexico
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_134_21

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  Abstract 


Background: It has been reported that sera from patients with active pulmonary tuberculosis (APT) induced nuclear changes in normal neutrophils that included pyknosis, swelling, apoptosis, and production of extracellular traps (NETs). Similar changes were observed with some sera from their household contacts but not with sera from healthy, unrelated individuals. It was suggested that those sera from household contacts that induced neutrophil nuclear changes might correspond to people with subclinical tuberculosis. Thus, our experimental approach might serve to identify individuals with early, ongoing disease. Methods: Nuclear changes in neutrophils were fully evident by 3 h of contact and beyond. Circulating mycobacterial antigens were the most likely candidates for this effect. We wanted to know whether the nuclear changes induced on neutrophils by the sera of APT patients would negatively affect the phagocytic/microbicidal ability of neutrophils exposed to APT sera for short periods. Results: We now provide evidence that short-term contact (30 min) with sera from patients with pulmonary tuberculosis increases several phagocytic parameters of normal neutrophils, including endocytosis, myeloperoxidase levels, production of free reactive oxygen species, phagolysosome fusion, and microbicidal activity on Staphylococcus aureus, with these effects not being observed with sera from healthy donors. We also give evidence that suggests that ESAT-6 and CFP-10 are involved in the phenomenon. Conclusion: We conclude that activation is a stage that precedes lethal nuclear changes in neutrophils and suggests that autologous neutrophils must circulate in an altered state in the APT patients, thus contributing to the pathology of the disease.

Keywords: Active pulmonary tuberculosis, ESAT-6, microbicidal activity, neutrophils


How to cite this article:
Rojas-Espinosa O, Rivero-Silva MA, Hernández-Solís A, Arce-Paredes P, Arce-Mendoza AY, Islas-Trujillo S. Sera from patients with tuberculosis increase the phagocytic-microbicidal activity of human neutrophils, and ESAT-6 is implicated in the phenomenon. Int J Mycobacteriol 2021;10:271-8

How to cite this URL:
Rojas-Espinosa O, Rivero-Silva MA, Hernández-Solís A, Arce-Paredes P, Arce-Mendoza AY, Islas-Trujillo S. Sera from patients with tuberculosis increase the phagocytic-microbicidal activity of human neutrophils, and ESAT-6 is implicated in the phenomenon. Int J Mycobacteriol [serial online] 2021 [cited 2021 Dec 3];10:271-8. Available from: https://www.ijmyco.org/text.asp?2021/10/3/271/325497




  Introduction Top


The granuloma is a hallmark of tuberculosis: The granuloma is not a static structure but a highly dynamic one where proinflammatory and anti-inflammatory cytokine-mediated events constantly occur. Many substances released from both cells and bacilli escape from the granuloma and are dispersed through the blood and lymph to distant sites of the body. Free and antibody-complexed mycobacterial antigens circulate in biological fluids of patients with pulmonary and meningeal tuberculosis.[1],[2],[3],[4] Mycobacterial antigens and immune complexes in the blood and other biological fluids, may probably affect the blood cells of tuberculous (TB) patients.[5] In vitro, isolated M. tuberculosis proteins, mycobacterial extracts, mycobacterial culture supernatants, PPD, and the whole bacteria, stimulate the proliferative response of lymphocytes of the diseased people and less markedly the response of normal cells.[6]

As far as we know, except for our previous paper,[7] there is no information on the effect of sera from TB patients on the cell morphology or function of neutrophils. In this report, we first corroborated the effect of tuberculous sera (TBS) on the nuclear morphology of neutrophils, then we give evidence that TBS increase the phagocytic and microbicidal function of these cells when incubated for short (30 min) periods. We also provide data that indicate that ESAT-6, and CFP-10 in a lesser degree, are involved in the induction of nuclear changes in neutrophils, ESAT-6 and CFP-10 are two small proteins of the Mycobacterium tuberculosis complex coded within the RD1 genetic locus that participates in the pathology of the disease.


  Methods Top


Ethical issues

This project was presented to and approved by the Ethics Committee for Medical Research of the Hospital General de México “Dr. Eduardo Liceaga” on the Code DI/18/406/05/100, and the Ethics Committee for Research of ENCB, IPN, under the code SH-007-2020. The research was performed in adherence to the principles of the Helsinki Declaration (World Medical Association, 2013).

Reagents

Unless otherwise indicated, chemicals were purchased from SIGMA-ALDRICH, Toluca, México. ESAT-6 (Abcam 124574) and CFP-10 (Abcam 5303810) were from ABCAM, CB2 0AX, UK.

Blood samples

Blood samples were collected under informed consent from 26 selected TB patients of which 7 were female and 19 males, their age ranged from 18 to 70 (mean, 44) years old, 26 tested positive for tuberculosis by PCR, 14 had M. tuberculosis-positive cultures in Lowenstein-Jensen medium, none was positive for HIV, none had diabetes mellitus, 6 were hypertensive, 2 had neoplasia, one had rheumatoid arthritis, 20 had pulmonary tuberculosis, 3 had pulmonary-ganglionary tuberculosis, one had meningeal tuberculosis, one had miliary tuberculosis, one had pulmonary-renal tuberculosis, and 26 were under standard WHO treatment, according to the Norma Oficial Mexicana NOM-006-SSA2-2013. In addition, healthy blood was obtained from blood-bank donors who also signed informed consent.

Serum samples

Sera were separated from coagulated blood and centrifuged at 3000 rpm for 30 min to get rid of debris and platelets. They were then divided into 100 μl-aliquots and kept frozen at −20°C until used.

Neutrophil's separation

Five milliliters of heparinized blood from healthy donors were layered on 5.0 ml of Polymorphprep (Axis, Shield, PoC-AS, Oslo, Norway) in a 12-ml glass screw-capped tube, and the tube was centrifuged at 1500 rpm for 60 min at 25°C. This process produced two cell layers and the red blood package. The upper cell layer containing mononuclear cells and the lower cell layer containing polymorphonuclear (PMN) cells. After carefully removing the mononuclear cell layer, the PMN layer was collected, transferred to a 15 ml Nalgene conical tube, diluted to 12 ml with PBSG (0.01M phosphate, 0.15 M sodium chloride, 0.1% glucose, pH 7.4), mixed by inversion, and centrifuged at 1500 rpm for 5 min. Next, the cell sediment was gently loosened and suspended in 12 ml of PBSG for further centrifugation at 1300 rpm for 5 min. Finally, the washed cells were suspended in 2.0 ml of PBSG for counting in a Neubauer chamber with Turk liquid as the diluent. Over 95% were PMN neutrophil (NEU) cells. For most experiments, cells were adjusted to 1 × 106 cells per ml of PBSG.

Neutrophil monolayers

Forty-thousand NEU was deposited on circular 0.8 cm (Ө) areas on clean, degreased glass slides, and the slides were incubated for 30 min at 37°C, 5% CO2, to allow adherence of the cells. After carefully removing the supernatant fluid, the monolayers were covered with 40 μl of either normal or TB serum, and the slides were further incubated for 30 min (37°C, 5% CO2). Finally, sera were carefully removed and replaced with 40 μl of PBSG. These serum-treated monolayers were immediately used for the experiments detailed below.

Yeast preparation

A heavy 1.0% suspension of Baker's yeast in water was heat-inactivated and then centrifuged at 1000 rpm for 5 min. The supernatant suspension was collected and subjected to 5 similar centrifugation rounds to produce a fine suspension of yeast that was counted in a Neubauer chamber and adjusted to 100 × 106 cells per ml. One-ml aliquots of the suspension were prepared and stored frozen (−20°C) until used. To opsonize, one ml of yeast suspension was centrifuged (3000 rpm/5 min), and the sediment, suspended in 1.0 ml of fresh human serum, was incubated for 30 min at 37°C. At the end of the incubation time the suspension was centrifuged (3000 rpm/5 min) and the sediment suspended in 1.0 ml of 0.1% (nitro blue tetrazolium [NBT]) in PBSG.

Nuclear changes

NEU monolayers (40 × 103 cells) were incubated for 3 or 6 h with 40 μl of normal or TB serum at 37°C. At the end of the incubation time, the supernatant sera were carefully removed, and the cell monolayers were fixed with 4% paraformaldehyde in PBSG for 10 min. Then, the monolayer-containing slides were washed by immersion (3 dips) in distilled water, let to dry, and stained for 2 min with Hoechst solution 1:10,000 in water. After washing the monolayers were mounted under Vecta Shield mounting medium (Vector Laboratories, Inc., Burlingame, CA 94010) for microscope observation under ultraviolet light at 460–490 nm. Nuclear alterations were registered and quantified.

Ingestion (phagocytosis) of complement-opsonized yeast

NEU monolayers (40 × 103 cells), preincubated with normal or TBS and covered with 40 μl of PBSG were stimulated with 5 ul of the opsonized yeast suspension in 0.1% NBT and incubated for 30 min at 37°C, 5% CO2. At the end of the incubation time, the NEU monolayers were washed by immersion (3 dips) in water to get rid of non-phagocytosed yeast, stained with 0.5% fresh safranin for 10 min, washed by immersion in distilled water (3 dips), let to dry, and mounted under Entellan-new resin (107961 Sigma-Aldrich) for microscope observation. The percent of phagocytosing cells and the number of yeasts per cell were assessed, as was the number of cells showing reduction of NBT in the form of an insoluble blue formazan.

Contents of myeloperoxidase and phagolysosome fusion

NEU monolayers pretreated with normal or TBS were incubated with or without opsonized yeast for 60 min at 37°C, 5% CO2. At the end of the incubation time, monolayers were washed by immersion (3 dips) in PBSG and then covered with 40 μl of a solution containing 3.0 mg of orthodianisidine predissolved in 1.0 ml methanol, 100 μl of 4% hydrogen peroxide, and 9.0 ml of PBSG. After incubation for 20 min, the peroxidase reaction was stopped by immersing (3 dips) the monolayers in distilled water. Finally, the monolayers were let dry before being mounted under Entellan-new resin for microscope observation and counting. NEU monolayers incubated in the absence of yeast served to appreciate the presence of myeloperoxidase in the granular lysosomal granules while those incubated with yeast served to assess the grade of lysosome-phagosome fusion.

Superoxide production

Five hundred thousand NEU were placed into the wells of a 96-well microplate (44-2404-21 Nunc MaxiSorp™, Thermo Fisher Scientific) and let to adhere for 30 min (37°C, 5% CO2). No adherent cells in the supernatant fluid were carefully removed and replaced with 100 μl of normal or TB serum. After further incubation for 30 min (37°C, 5% CO2), the monolayers were washed once with PBSG, taking care not to disturb the monolayers, and the cells were stimulated by adding 100 μl of PBSG containing 500 ng of PMA (phorbol myristate acetate). After 30 min incubation (37°C, 5% CO2), the microplates were centrifuged (1500 rpm/5 min), the supernatant was removed and replaced with 200 μl of 10% SDS (sodium dodecyl sulfate) in 0.08N NaOH. Then the plates were left overnight at room temperature to promote dissolution of the formazan produced whose blue color was read at 600 nm in an ELISA reader (technic modified from Choi et al., 2006).[8]

Hydrogen peroxide production

Five hundred thousand NEU were placed into the wells of a 96-well microplate and let to adhere for 30 min (37°C, 5%CO2). Then, after a single careful washing with warm PBSG, the cells were incubated with 100 μl of normal or TB serum for 30 min. At the end of the incubation time, sera were removed and replaced with 200 μl of a solution containing 0.01% horse-radish peroxidase and 0.02% phenol red in PBSG. PMA (500 ng/10 μl) was used as the stimulus. Thirty min after incubation (37°C, 5% CO2) the reaction was stopped by adding 20 μl of 1.0 N NaOH, and the resulting fuchsia color was read at 600 nm in an ELISA reader. Optical density readings were transformed into nanograms in reference to a calibration curve with authentic hydrogen peroxide (Technic modified from Pick and Keisari, 1980).[9]

Microbicidal activity

One-million NEU in 0.5 ml of PBSG were incubated for 30 min at 37°C in the presence of 100 μl of normal or TB serum and 1 × 107 Staphylococcus aureus in 5.0 ml-round bottom polystyrene tubes (Falcon). After the incubation time the tubes were centrifuged (1500 rpm, 5 min) and the supernatant was removed and replaced with 0.5 ml of Triton X-100 to dissolve, by gentile shaking, the cellular sediment. The lysate was then serially diluted 1:10, 1:100, and 1:1000 in PBSG. Ten microliters of each dilution were then dropped in triplicate on nutritious agar plates (the micro drop plaquing technic) and the plates were incubated for 16–18 h at 37°C. Surviving bacteria were counted and referred to as colony-forming units (CFU); drops with colonies in the range of 30–100 CFU were used to calculate the number of surviving bacteria in reference to the number of colonies in drops seeded with bacteria alone. Volume seeded (10 μl) and dilution (1:10, 1:100 or 1:1000) were used to calculate the number of surviving bacteria per ml (modified from Malcom, 2018).[10]


  Results Top


Nuclear changes induced by tuberculous sera

Sera from patients with pulmonary tuberculosis but not sera from healthy (“normal”) people induced clear changes in the nuclear morphology of normal neutrophils. These changes included pyknosis, nuclear swelling, apoptosis, and DNA extrusion, all of them suggestive of apoptosis and netosis. This result corroborates the ones reported in a previous article.[7] [Figure 1] depicts the changes induced by 6 normal (NL) and 6 TBS after 6 h incubation. It can be seen the homogeneous nuclear shape in NEU treated with normal (NL) sera, and the marked heterogeneous changes induced by TBS.
Figure 1: Sera from tuberculous individuals induce nuclear changes in neutrophils. Sera from healthy individuals (normal, NL) do not induce nuclear changes in neutrophils. In contrast, sera from tuberculous patients produce a variety of changes that include pyknosis (p), nuclear swelling (s), apoptosis (a), and netosis (n). Sera from 6 NL or TB individuals were incubated with neutrophils of the same healthy donor for 6 h at 37°C (5% CO2). Fluorescent stain with Hoechst's reagent, ×40

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Yeast ingestion is increased when neutrophils are preincubated with tuberculous sera

Sera from TB patients increase the phagocytic ability of neutrophils over this ability of neutrophils incubated in the presence of normal (NL) serum. As illustrated in [Figure 2], the ability of individual TBS to induce ingestion of opsonized yeast varied widely. Still, the mean ability was higher than the one with normal sera, and the difference was statistically significant (P = 0.0014). Furthermore, the average number of yeasts ingested per cell was higher in the group preincubated with TB (4.7) sera than in the group treated with normal sera (3.8) (P = 0.0029). Twenty TBS and 20 sera from healthy individuals (normal, NL) were tested in this assay.
Figure 2: Tuberculous sera increase neutrophils' phagocytic activity. The number of neutrophils (%) that ingest yeasts is higher in the group of cells preincubated with tuberculous sera than in the group preincubated with normal sera (NLS) (P = 0.0014). Also, the average number of yeasts ingested per cell is higher in the group pretreated with tuberculous sera than in the group pretreated with normal sera (P = 0029) (n = 3). (Mann–Whitney test)

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Phagolysosome fusion in the presence of tuberculous and normal sera

Phago-lysosomal fusion is a key part of the phagocytic process and is related to the microbicidal activity of neutrophils. Phagolysosome fusion allows the direct contact of the phagosome-confined microorganism with the toxic lysosomal contents including acid- and neutral hydrolases, myeloperoxidase, and antibiotic peptides.[11] In the present study, we found that neutrophils preincubated with TBS showed a higher frequency of phagolysosome fusion (83 ± 3.014) than neutrophils preincubated with normal sera (NLS) (65 ± 4.56), and the difference was statistically significant (P = 0.006) [Figure 3].
Figure 3: Phagolysosome fusion in neutrophils preincubated with tuberculous sera or normal (NLS) sera. Panel A illustrates the presence of fine granular peroxidase in the cytoplasm of resting neutrophils (upper image) and the phagolysosome fusion (lower image) in neutrophils that have ingested yeasts (dark ingested yeasts). MPO stain (×100). Panel B shows the percent of phagolysosome fusion in neutrophils preincubated with 10 tuberculous or NL sera. The difference was statistically significant (P = 0.030) (n = 3) (Mann–Whitney test)

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Superoxide production is increased when neutrophils are preincubated with tuberculous sera

The ingestion of microorganisms is associated with the ability of neutrophils to produce superoxide anion, a process dependent on the NADPH-oxidase activity. This system captures molecular oxygen and transforms it into superoxide anion, which gives origin to hydrogen peroxide, hydroxyl radicals, oxygen singlet, and other secondary derivatives (reactive oxygen species [ROS]). ROS serve several physiological functions and participate in the killing of microorganisms[12] but at supraphysiological concentrations, ROS may promote damage in several tissues.[13] The ex-vivo production of superoxide anion is frequently measured by the reduction of the NBT dye which produces a blue insoluble formazan[8] [Figure 4]a that can be extracted and read in a photo colorimeter. By using the NBT reduction test, it was found that the production of superoxide anion was higher in NEU preincubated with TBS than in NEU preincubated with sera from normal people (NLS) [[Figure 4]b, graph at the right], and the difference was statistically significant (P = 0.006).
Figure 4: The NBT test to detect the production of superoxide anion. Panel A shows a monolayer of resting neutrophils (upper panel) and a monolayer of cells after ingestion of yeast in the presence of NBT (lower panel). Panel B illustrates the ability of neutrophils preincubated with tuberculous sera or normal (NLS) sera to produce anion superoxide (biochemical assay). Panel C depicts the ability of NEU preincubated with tuberculous sera or NLS to produced hydrogen peroxide (biochemical assay). In both cases, NEU preincubated with tuberculous sera showed greater activity than NEU preincubated with NLS, and the differences were statistically significant (P = 0.006, and P = 0.033, respectively) (n = 3) (t-test in both cases)

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Production of hydrogen peroxide is increased when neutrophils are preincubated with tuberculous sera

Correspondingly, neutrophils preincubated with sera from TB patients (TBS) produced higher amounts of hydrogen peroxide than neutrophils preincubated with normal sera (NLS), and the difference was statistically significant (P = 0.033) [Figure 4]c.

Neutrophils preincubated with tuberculous sera exhibit higher microbicidal activity than neutrophils preincubated with normal sera

Staphylococcus aureus is a convenient target microorganism to assess the bactericidal activity of neutrophils.[10] In this study, neutrophils preincubated with sera from TB patients (TBS) showed increased microbicidal activity than neutrophils preincubated with normal sera and the difference was statistically significant [Figure 5]. For this experiment, sera from TB patients that induced significant changes on the nuclear morphology of neutrophils were selected and tested.
Figure 5: Tuberculous serum-activated neutrophils efficiently kill S. aureus. (a) shows the number of CFU resulting when 10 μl of dilutions 1:10, 1:100 and 1:1000 of Staphylococcus aureus were dropped on nutritious agar plates. (b) shows the number of CFU recovered from neutrophils preincubated for 60 min with a normal serum, and (c) shows the number of CFU recovered from neutrophils preincubated with a tuberculous serum. There were differences at any dilution tested but the counting of CFU was made at the dilution 1:1000. (d) Illustrates the bactericidal of neutrophils preincubated for 60 min with 24 sera from healthy (NLS) or tuberculous sera individuals. P <0.0001 (Shapiro–Wilk, t-test)

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Mycobacterial antigens induce nuclear changes in neutrophils

Soluble mycobacterial antigens in the culture medium (PBY synthetic medium) of M. tuberculosis induced changes in the nuclear morphology of neutrophils similar to the changes induced by TBS, and the changes were directly proportional to the antigen concentration. In the experiment depicted in [Figure 6], 40 ng of mycobacterial protein in the test were enough to induce the changes, but lesser amount of protein has been change-inducers in other experiments.
Figure 6: Neutrophil nuclear changes induced by mycobacterial antigens. Forty nanograms of soluble protein from Mycobacterium tuberculosis are enough to induce visible changes in the nuclear morphology of neutrophils (40 × 103 cells). In this 3-h experiment, the most noticeable changes were pyknosis, nuclear swelling, and images suggestive of apoptosis, no NETs were observed. Hoechst's stain, ×40

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Mycobacterial antigens in QuantiFERON TB gold induce changes in the nuclear morphology of neutrophils

QuantiFERON TB gold's components induced the expected changes in the nuclear morphology of neutrophils. Tubes 1 (negative control), 2 (unspecified mitogen), and 3 (ESAT-6, CFP-10, and TB7.7) were rehydrated with 1.0 ml of PBSG and 10 μl of the resulting solutions were used to stimulate neutrophil monolayers (40 × 103 cells) for 3 h. [Figure 7] shows that the contents of tubes 2 (mitogen) and 3 (ESAT-6, CFP10, TB7.7) induced changes in the neutrophils' nuclei similar to the changes induced by the soluble M. tuberculosis antigens and tuberculous sera.
Figure 7: ESAT-6, CFP-10, and TB7.7 of the QuantiFERON kit induce nuclear changes in neutrophils. Two different lots (upper and lower rows) of QuantiFERON TB Gold were tested on two different neutrophil monolayers with similar results. ESAT-6, CFP-10, and TB7.7 in tube 3 were able to induce nuclear changes in NEU analogous to the ones induced by tuberculous sera. Hoechst's stain, ×40

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Recombinant ESAT-6 and CFP-10 induce changes in the nuclear morphology of neutrophils

ESAT-6 and CFP-10 are two proteins of the tuberculosis complex that participate in the inhibition of the phago-lysosome formation in macrophages[14] and have deleterious effects on neutrophils. In the present study, we found that both proteins were able to induce nuclear changes in neutrophils similar to the ones produced by soluble proteins of M. tuberculosis, and similar to the changes induced by the sera of patients with active pulmonary tuberculosis. [Figure 8] illustrates the results of 3 independent experiments. At the same concentrations used (1 μg/40 × 103 cells), ESAT-6 resulted in a more potent change inducer than CFP-10 and the changes were comparable to the changes generated by PMA.
Figure 8: Recombinant CFP-10 and ESAT-6 induce changes in the nuclear morphology of neutrophils similar to the ones induced by phorbol-myristate acetate. Three independent experiments (a-c) showing the nuclear morphology of neutrophils when incubated alone (NEU), with PMA, recombinant CFP-10, or recombinant ESAT-6. One microgram of each compound was added to NEU monolayers (40 × 103 cells) and the monolayers were incubated for 3 h at 37°C (5% CO2). Fluorescent Hoechst stain, ×40

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Soluble mycobacterial antigens, CFP-10, and ESAT-6 are inducer of nuclear changes in neutrophils

Summarizing, to corroborate that mycobacterial antigens were possibly involved in the capacity of tuberculous sera to induce nuclear changes in neutrophils, sera from 20 patients with pulmonary tuberculosis and 20 sera from healthy individuals were tested on monolayers of neutrophils from different healthy blood donors. Sera were aseptically collected and centrifugated (10,000 RPM for 10 min) to remove any protein aggregates and tested directly on the neutrophil monolayers. The whole process was described in the previous paper of this series.[7] Antigens of three lots of QuantiFERON gold were prepared by adding 1.0 ml of PBSG to each one of the 3 tubes of the kit. Tube 1 is a no reagent control, tube 2 contains a proprietary mitogen, and tube 3 is a cocktail of ESAT-6, CFP-10, and TB7.7 antigens. Ten microliters of each solution were added to neutrophil monolayers (40 × 103 cells protected with 40 μl of PBSG) and the monolayers were incubated for 3 or 6 h at 37°C/5% CO2. At the end of the incubation time, the monolayers were fixed for 10 min with 4% paraformaldehyde, then they were washed by 3-dips in PBSG, stained for 2 min with Hoechst' stain, protected with anti-fade Vecta Shield, and microscopically examined under ultraviolet light. In separate experiments, neutrophil monolayers were stimulated with 1.0 μg of recombinant ESAT-6 or FP-10 for 3 and 6 h and processed as before [Figure 6],[Figure 7],[Figure 8].

Statistical analysis

The GraphPad Prism software was used to analyze the results. The Shapiro–Wilk normality test with α = 0.05 was used to decide whether a t-test (for a normal distribution) or a Mann–Whitney test (for a no normal distribution) should be applied in each experiment.


  Discussion Top


The granuloma in tuberculosis is not a static, but a highly dynamic structure where many simultaneous biological processes occur. It is not a confining structure developed to limit the spreading and proliferation of M. tuberculosis; on the contrary, the granuloma is a propitious niche for surviving and replication of the bacillus. Many cells participate in the granuloma structure and function, namely alveolar macrophages (AM), dendritic cells (DC), adaptive and innate T-lymphocytes, and PMN neutrophils. The TB granuloma is indeed a battlefield with many cells being recruited and killed, with M. tuberculosis in the core structure.[15] AM and DC are indeed the first cells that contact M. tuberculosis. This early contact results in the immediate defensive response of AM and DC with the release of a series of cytokines including the neutrophil attracting chemokines CXCL-3 and CXCL-5.[16],[17] Neutrophils are the first cell population to immigrate in large numbers from the blood circulation to the incipient granuloma; once in there, they become activated by macrophage-derived cytokines and bacillary components and respond phagocytosing macrophage-debris and bacilli and releasing free ROS,[18] and type-1 and type-2 cytokines, thus contributing to the inflammatory environment of the granuloma.[19] By this time, DC have carried out variably injured bacilli to regional lymph nodes to present bacilli-derived peptides to reactive T-cells in an immunological-synapsis context. Activated T cells proliferate and pass to the blood circulation to eventually accumulate into the developing TB granuloma to help macrophages fighting the TB bacilli. In the majority of the infected people, the disease will stay latent, but it would take over under disturbing situations affecting the individuals' cell-mediated immunity.[20] Many substances originated in the granuloma, including cell-and bacilli-debris, free bacillary antigens, pro-inflammatory and anti-inflammatory cytokines, as well as cell-and bacillary-metabolites will reach the circulation to be distributed throughout the body. All these granuloma-derived products might also have effects on the blood cells themselves, including neutrophils, and the effect of tuberculous sera on the nuclear morphology and the phagocytic-microbicidal activity of neutrophils here reported, support this possibility. Anti-mycobacterial antibodies, and cytokine (tumor necrosis factor-alpha [TNFα], interferon-gamma [IFNγ], interleukin [IL]-6, IL-8) levels, although appeared elevated in tuberculous sera did not correlate with their capacity to induce nuclear changes in neutrophils.[7] Preliminary experiments reported in that paper suggested that soluble mycobacterial antigens might be responsible for the changes. In the present article, we confirm that mycobacterial antigens, ESAT-6, and CFP-10 among them, are involved in the phenomenon, and this finding reinforces the observations of Goyal et al.[1] who found significant levels of circulating immune complexes containing ESAT-6, CFP-10, and CFP-21 in the serum of patients with tuberculosis, and Francis et al.,[5] who found a deleterious effect of ESAT-6 on neutrophils, including necrosis and NETosis. An additional finding related to our observations was reported by Poulakis et al.[21] who found, by flow cytometry, that neutrophils from TB patients contain intracellular ESAT-6 in 90% of the cases whereas 46% of close contacts, and none of the unrelated healthy people, were positive for that protein. All these possibly negative effects on circulating neutrophils are compensated by the moderate neutrophilia observed in TB patients as a bone marrow response to the neutrophils' harm in the patients; neutrophilia, however, was associated with increased risk of mortality.[22]

It is worth noting that ESAT-6 not only impacts on the neutrophils' activity, but it also exerts modulatory effects on T cells as shown by Mvungi et al.,[23] who found that ESAT-6 in the QuantiFERON plus system activates peripheral blood cells to release significant amounts of IFNγ, and IL-4 but not TNF, IL-6, or IL-10. On the contrary, Sharma et al.[24] observed that ESAT 6 interfered with the activation of T cells by blocking the MHC-II/CD28 pathway in Jurkat T cells, a phenomenon that in vivo might ease tuberculosis progression.[24] A full review on ESAT-6 on innate and acquired immunity is being prepared by the authors of the present article.

The presence of granuloma-derived toxic substances made us assume a deleterious effect of tuberculous sera on neutrophils from healthy donors, but we found instead a stimulatory effect reflected on their increased phagocytic and microbicidal activity; yeast-ingestion, phagolysosome fusion, production of superoxide anion, hydrogen peroxide, and microbicidal activity on S. aureus, were all increased. However, this stimulatory effect was only observed when neutrophils were in contact with tuberculous sera for short periods (30 min) because it shifted to a deleterious effect at longer incubation times (3 h, and beyond) which means that preactivation is a step that precedes the induction of morphologic changes in neutrophils. A critical point that must be considered is the physical and physiologic states of neutrophils in the blood and tissue (granuloma) of the patients themselves; unraveling this issue will contribute to better understand the pathology of tuberculosis. Activated neutrophils in the granuloma are surely responsible for the extensive inflammatory damage seen in active tuberculosis.[25] Preliminary results from an ongoing study of our group indicate that compared to neutrophils from healthy donors, neutrophils from TB patients show a higher frequency of spontaneous (nonstimulated) chromatin decompaction and other nuclear changes.

We conclude that the ability of sera from patients with pulmonary tuberculosis to induce changes in the morphology of neutrophils obeys to the presence of circulating soluble mycobacterial antigens among which ESAT-6 and CFP-10 are key participants. These proteins might also be the serum factors responsible for the biochemical activation on neutrophils related to phagocytic and microbicidal activity. From these results, we propose that measuring the levels of these antigens in the blood might be a rational approach to detect those cases of subclinical, not latent, disease, among the patients' household contacts, a hypothesis currently investigated in our laboratory.

Acknowledgment

To COFAA, SIP, EDI, and SNI (Conacyt) for the scholarships provided.

Ethical clearance

This project was presented to and approved by the Ethics Committee for Medical Research of the Hospital General de Mexico Dr. Eduardo Liceaga on the Code DI/18/406/05/100, and the Ethics Committee for Research of Escuela Nacional de Ciencias Biológicas del IPN, under the code SH0072020. The research was performed in adherence to the principles of the Helsinki Declaration (World Medical Association, 2013).

Financial support and sponsorship

Secretaría de Investigación y Posgrado (SIP) del Instituto Politécnico Nacional, COFAA, EDI and SNI (CONACYT), México.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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