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Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 58-61

Evaluation of Mycobacterium kansasii extracellular vesicles role in BALB/c mice immune modulatory

Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran

Date of Submission29-Dec-2019
Date of Acceptance01-Aug-2020
Date of Web Publication6-Mar-2020

Correspondence Address:
Seyed Davar Siadat
Pasteur Institute of Iran, No. 69, Pasteur Ave., Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_212_19

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Background:Mycobacterium kansasii as a nontuberculosis mycobacteria, naturally release extracellular vesicles (EVs) with widespread utilities. The aim of the present study was the extraction and biological evaluation of M. kansasii EV and its role in BALB/c mice immune modulatory by considering EVs medical usage specificities. Method: Density gradient ultracentrifugation method was used to EVs extraction from standard species of M. kansasii. Biologic validation of EVs has been performed by physicochemical experiments. Immunization has been done by subcutaneous injection to BALB/c mice, then spleen cell isolation and lymphocyte transformation test and eventually ELISA cytokine assays were made for interleukin-10 (IL-10) and interferon-gamma (IFN-γ). IBM SPSS version 22 software (SPSS. Inc., Chicago, IL, USA) was used for the data calculation. The evaluation of variables was conducted using one sample t-test. Results: Physicochemical experiment results contribute that extracted EVs have intransitive capability to use in immunization schedule. Finally, ELISA test results showed that EVs induced IL-10 production, but have no effect on IFN-γ. Conclusions: In this current study, EVs were prepared in high-quality composition. The results of cytokine assay revealed that the extracted EVs have anti-inflammatory property. Accordingly, this macromolecule can be used as immune modulatory agents to prevent severe immune reactions, especially in lungs disorders.

Keywords: Extracellular vesicles, immunomodulation, Mycobacterium kansasii

How to cite this article:
Tavassol ZH, Aziziraftar SK, Behrouzi A, Ghazanfari M, Masoumi M, Fateh A, Vaziri F, Siadat SD. Evaluation of Mycobacterium kansasii extracellular vesicles role in BALB/c mice immune modulatory. Int J Mycobacteriol 2020;9:58-61

How to cite this URL:
Tavassol ZH, Aziziraftar SK, Behrouzi A, Ghazanfari M, Masoumi M, Fateh A, Vaziri F, Siadat SD. Evaluation of Mycobacterium kansasii extracellular vesicles role in BALB/c mice immune modulatory. Int J Mycobacteriol [serial online] 2020 [cited 2021 Aug 3];9:58-61. Available from: https://www.ijmyco.org/text.asp?2020/9/1/58/280150

  Introduction Top

A group of pathogenic mycobacteria with similar bacilli to Mycobacterium tuberculosis that cause nontypical infections rather than infections caused by M. tuberculosis or Mycobacterium leprae, is known as nontuberculosis mycobacteria (NTM).[1] NTMs are also known as environmental mycobacteria, atypical mycobacteria, and mycobacteria other than tuberculosis.[2] Infection with NTM is increasingly reported around the world. The second most common cause of NTM infections is Mycobacterium kansasii (M. kansasii). M. kansasii infections frequently have been founded in immunodeficient individuals, such as HIV-positive patients.[3]M. kansasii is one of the closest mycobacterium species to M. tuberculosis due to phylogenetic analysis. However, the M. kansasii is an opportunistic pathogen.[4] As expected like all other cells, M. kansasii produces extracellular vesicles (EVs) in different biological situations.[5]

EVs are small membrane vesicles that are derived from the plasma membrane of all type of cells. These extracellular bacterial products could be considered as a bacterial nonclassic secretary system. It also have diverse features such as enzymes and toxins delivery, signaling, antigen presentation to innate and adaptive immune system, defense against other microorganisms, survival, genes transformation and biofilm formation that contribute to interaction of the microorganism with the environment, the modulation of microbe's physiology, and pathogenesis by immunologic molecules activation.[6]

One of the effects of EVs on adaptive immune responses is a tolerance induction. Proteomic analysis has demonstrated that EVs of the pathogenic strains have Toll-like receptor 2 (TLR2) agonist compounds. EVs interaction with macrophages isolated from mice stimulates cytokines and chemokines secretion in TLR2-dependent method.[7],[8]

Interferon-gamma (IFN-γ) is the most considered cytokine involved in the protective immune response in mycobacterial infection. The main function of IFN-γ is macrophage activation, in order to have microbicidal purposes. The studies show that IFN-γ is an essential cytokine for coping with mycobacteria in the intracellular environment. Moreover, in mycobacterial infection, interleukin-10 (IL-10) produced by macrophages and T lymphocytes that considered as an inhibitory cytokine. IL-10 is enrolled in balancing of the inflammatory and immune responses. Induction of IL-10 producing by EVs, indicating that EVs also contained components that could activate macrophages through a different ways from TLR2.[9]

EVs production has been observed in various pathogenic and nonpathogenic species of Mycobacterium,[6] indicating the release of EVs as a conserved feature among Mycobacterium species.

The aim of the present study was the evaluation of M. kansasii EVs immune modulatory role in BALB/c mice. The IL-10 and IFN-γ as anti-inflammatory and inflammatory cytokine have been considered.

  Methods Top

Bacteria strain and culture condition

Standard strain of M. kansasii (CRBIP 7. 42 standard bacterial collection of Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran). At first, the bacterial culture in Lowenstein–Jensen (Pasteur Institute of Iran, Tehran, Iran) in order to activate standard strain has been done. After early preparation, the bacteria adjusted to 1 MacFarland turbidity have been cultured in 150 ml of Middlebrook 7H9 Broth medium (Sigma, USA) for 4 weeks. After 4 weeks incubation at 37°C ± 1°C, the biomass was harvested by centrifuging at 1000 × g twice for 30 min (Eppendorf 5810 Centrifuge) and wet weight was evaluated.[10]

Extracellular vesicles extraction and validation

The EVs extraction and purification have been done by density gradient ultracentrifugation method (Claassen's method) using three extraction solutions described in previous publication. The physicochemical experiments used to confirm the spatial structure included protein assay (NanoDrop™ 1000 Spectrophotometer, Thermo Scientific Co. USA), sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) in 12% gel (using a Mini-PROTEAN® Tetra Cell-Bio-Rad), electron microscopy used FE-SEM (HITACHI S-4160 model with 5 nm image resolution, 20–30,000 times magnification and maximum accelerating voltage of 30 kV), and Limulus Amebocyte Lysate (LAL) test (Pierce® LAL Chromogenic Endotoxin Quantitation Kit, Thermo Fisher Scientific Co. USA).[10]

Immunization schedule

Subcutaneous injection of 50 μg/ml of extracted EVs to BALB/c mice (6 × BALB/c mice-Female-4 weeks old-on three occasions in 6 weeks) has been done. Furthermore, the control group considered with injection of distilled water in the same situation.

Spleen cell isolation and Lymphocyte Transformation Test

Lymphocyte transformation test is used to estimate the rate of proliferation of antigen-specific immune cells from exposure to the same antigen in vitro. After injection on days 0, 14, and 28, all mice were euthanized on day 42, and spleen isolation was performed in sterile conditions. In brief, spleens were removed from mice and placed in 5 ml Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma- Aldrich), designated isolation buffer. The spleen cells were homogenized by injection of the culture medium into the spleen tissue and squeezing it with the end of a syringe. Tris-buffered ammonium chloride as lysis buffer was used in order to red blood cells removing. After the cell count, dilute the cell suspension to 2 × 107 cell/ml. Three concentrations of purified antigen (EVs) 5, 10, and 20 μg/ml were prepared, and Con A was used as a positive control. One hundredmicroliters of culture medium containing spleen cells and 20 μl of stimulatory protein (EVs) were added to each well and incubate at 37°C for 72 h. After centrifugation at 1000 rpm for 5 min, supernatants are collected and maintained for the next step cytokines assay.

To investigate the role of EVs in stimulating the immune system, IL-10 and IFN-γ cytokines as an anti-inflammatory cytokine and a pro-inflammatory cytokine were selected, and eventually ELISA Cytokine assays were performed. The Mouse IL-10 ELISA development kit (3431-1H-6) and Mouse IFN-γ ELISA PRO kit (3321-1HP-2) (Mabtech Co. Sweden) have been used in this study.

Statistical analysis

IBM SPSS version 22 software (SPSS. Inc., Chicago, IL, USA) was used for the data calculation. The evaluation of variables was conducted using one-sample t-test.

  Results Top

In outcome of extraction and validation of EVs, the total protein concentration was determined about 1.4 mg/ml, and the Bradford protein assay confirms this result. The SDS-PAGE result showed more than five strong protein bands between 60 and 180 kDa molecular weight approved protein profiles of EVs. The intactness of EVs permitted with spherical shape in diameter of 100–250 nm, using scanning electron microscope that performed as described previously [Figure 1]. According to the performed LAL test, the amount of lipopolysaccharide contamination existing in EVs was 1.5 EU/ml that is in the specified application range of biological products.
Figure 1: Scanning electron micrograph of Mycobacterium kansasii extracellular vesicles

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Cytokines assay

After culture of the immunized BALB/c mice spleen cells as previously mentioned, ELISA for IL-10 and IFN-γ has been performed. The IL-10 titer was raised by spleen cell stimulating with EVs (P ≤ 0.01) compared to negative control. The IL-10 concentration in 10 μg/ml dilution of EVs has no significant different with positive control, so it has the most amount of induction of IL-10 [Figure 2]. No significant effect on the titer of IFN-γ had been detected.
Figure 2: Interleukin-10 ELISA Cytokine assay result from mouse spleen cell culture stimulate with Mycobacterium kansasii extracellular vesicles (*** =0.002, ** =0.02)

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  Discussion Top

The release of EVs is a conserved feature in microorganisms that have role in parts of interaction with the environment and other physiological and pathological processes.

The properties of EVs released by acid-fast bacteria such as mycobacteria, are less well known.[5],[11] Hence, studies of mycobacterial EVs can declare its potential in treatment and detection of NTM infections.

Nowadays, the incidence of pulmonary diseases caused by NTMs due to the prevalence of immunodeficiency diseases and antibiotics resistance is increasing. In addition, the prevalence of M. kansasii in polluted areas and its clinical and antigenic similarity to M. tuberculosis,[12] this NTM species should be considered more seriously.

Sato et al. in an immunological study on M. kansasii according to existing of specific antigens of M. tuberculosis in this bacteria, indicated that M. kansasii antigens are not as capable as M. tuberculosis antigens to induce IFN-γ.[13] Moreover, in Prados-Rosales et al. study on producing EVs in two important species, M. tuberculosis and Mycobacterium bovis (Bacillus Calmette Guerin) and some other nonpathogenic species of bacteria, pointed out that only the rich lipoprotein EVs of pathogenic strains not opportunist Mycobacteria, act as TLR2 agonist.[7]

Therefore, the lack of IFN-γ induction by the EVs derived from M. kansasii is verifiable. Furthermore, as stated in the opportunistic feature of M. kansasii and physiopathology and clinically its similarity to M. tuberculosis, the difference in EVs can be noted to differentiate these two species of mycobacteria.

The study that was conducted in 2014 determined the ways of dealing with an outbreak of tuberculosis and showed the role of B cells and humoral immunity in the face of mycobacteria. According to the afore said study, B cells can regulate the level of cytokines and T cell activity to manage intracellular bacteria such as M. tuberculosis.[14]M. kansasii EVs by induction of IL-10 guided the immune system to humoral immunity. Therefore, induction of these cytokines can partially be effective against M. kansasii infection.

The significant increase observed in the level of IL-10 indicates anti-inflammatory property of extracted EV. According to Abdalla et al. study, IL-10 transgenic mice show more severe lung injury in M. tuberculosis infection in comparison with untransformed mice. M. kansasii EVs inhibit activation macrophages and dendritic cells to produce IL-12, the main IFN-γ motivate, through IL-10 induction; consequently, cause inflammation decline.[15]

  Conclusions Top

In conclusion, M. kansasii EVs can be used as an immunosuppressive agent to prevent traumatic reactions. For instance according to fusion capability of the EVs with eukaryotic cells,[16] it can used as a nasal aerosol in M. tuberculosis-infected patients endangered respiratory disorder. Considering biological origin, it can be corticosteroid medications replacement. Important questions still to be answered include the composition variability of EVs, the variability parameters, and the introduction of immunogenicity-enhancing molecules in EVs.


The authors gratefully acknowledge all the personnel and researchers from the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran and Pasteur Institute of Iran for supporting this study.

Financial support and sponsorship

This study was financially supported by Pasteur Institute of Iran.

Conflicts of interest

There are no conflicts of interest.

  References Top

Porvaznik I, Solovič I, Mokrý J. Nontuberculous mycobacteria: Classification, diagnostics, and therapy. Adv Exp Med Biol 2017;944:19-25.  Back to cited text no. 1
Johnson MM, Odell JA. Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 2014;6:210-20.  Back to cited text no. 2
deStefano MS, Shoen CM, Cynamon MH. Therapy for Mycobacterium kansasii infection: Beyond 2018. Front Microbiol 2018;9:2271.  Back to cited text no. 3
Wang J, McIntosh F, Radomski N, Dewar K, Simeone R, Enninga J, et al. Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii. Genome Biol Evol 2015;7:856-70.  Back to cited text no. 4
Rodriguez GM, Prados-Rosales R. Functions and importance of mycobacterial extracellular vesicles. Appl Microbiol Biotechnol 2016;100:3887-92.  Back to cited text no. 5
Wang J, Wang Y, Tang L, Garcia RC. Extracellular vesicles in mycobacterial infections: their potential as molecule transfer vectors. Front Immunol 2019;10:1929.  Back to cited text no. 6
Prados-Rosales R, Baena A, Martinez LR, Luque-Garcia J, Kalscheuer R, Veeraraghavan U, et al. Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. J Clin Invest 2011;121:1471-83.  Back to cited text no. 7
Prados-Rosales R, Carreño LJ, Batista-Gonzalez A, Baena A, Venkataswamy MM, Xu J, et al. Mycobacterial membrane vesicles administered systemically in mice induce a protective immune response to surface compartments of Mycobacterium tuberculosis. mBio 2014;5:e01921-14.  Back to cited text no. 8
Cavalcanti YV, Brelaz MC, Neves JK, Ferraz JC, Pereira VR. Role of TNF-alpha, IFN-gamma, and IL-10 in the development of pulmonary tuberculosis. Pulm Med 2012;2012:745483.  Back to cited text no. 9
Tavassol HZ, Vaziri F, Siadat S. Extraction and biological evaluation of Mycobacterium kansasii extracellular vesicles as a vaccine candidate against mycobacterial pulmonary infections. Vaccine Research 2017;4:19-22.  Back to cited text no. 10
Gardiner C, di Vizio D, Sahoo S, Théry C, Witwer KW, Wauben M, et al. Techniques used for the isolation and characterization of extracellular vesicles: Results of a worldwide survey. J Extracell Vesicles 2016;5:32945.  Back to cited text no. 11
Mortazavi Z, Bahrmand A, Sakhaee F, Doust RH, Vaziri F, Siadat SD, et al. Evaluating the clinical significance of nontuberculous mycobacteria isolated from respiratory samples in Iran: An often overlooked disease. Infect Drug Resist 2019;12:1917-27.  Back to cited text no. 12
Sato R, Nagai H, Matsui H, Kawabe Y, Takeda K, Kawashima M, et al. Interferon-gamma release assays in patients with Mycobacterium kansasii pulmonary infection: A retrospective survey. J Infect 2016;72:706-12.  Back to cited text no. 13
Chan J, Mehta S, Bharrhan S, Chen Y, Achkar JM, Casadevall A, et al. The role of B cells and humoral immunity in Mycobacterium tuberculosis infection. Semin Immunol 2014;26:588-600.  Back to cited text no. 14
Abdalla AE, Lambert N, Duan X, Xie J. Interleukin-10 Family and Tuberculosis: An old story renewed. Int J Biol Sci 2016;12:710-7.  Back to cited text no. 15
Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles 2014;3:24641.  Back to cited text no. 16


  [Figure 1], [Figure 2]


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