Effects of Beta Glucan on IFNγ and IL-12 Production in Peripheral Blood Mononuclear Cells Induced by Whole Genome of M. Tuberculosis DNA
ABSTRACT
Background: In tuberculosis infections, the immune system is weakened and cannot produce enough cytokines to against the infection. Therefore, in order to increase the strength of immune cells to produce cytokines, the administration of external immunomodulators is very necessary. The primary cytokines in the process of Mycobacterium tuberculosis (M.
Tuberculosis) infection are interferon gamma (IFNγ) and interleukin 12 (IL-12). Beta glucan is known as one of the potential immunomodulatory polysaccharides. This study aimed to determine the effects of beta glucan on the production of IFNγ and IL-12 in peripheral blood mononuclear cells (PBMCs) induced by M. tuberculosis DNA.
Method: This study was a laboratory experimental study. In this study, PBMCs were isolated from 11 healthy respondents and were cultured in two kind of media culture. complete RPMI media containing beta-glucan (5 μg/ml) or without beta glucan. Those PBMCs were also induced with rpoB gene mutant or wild-type (WT) M. Tuberculosis DNA strain H37Rv (10 μg/ml) for six days in an incubator of 5% CO2. The production of cytokines IFNγ and IL-12 in the supernatant was performed using ELISA technique with an OD of 450 nm. To compare the mean cytokines in the group given beta glucan and without beta glucan using Kruskal Wallis statistical analysis
Results: The production of IFNγ in groups of PBMCs induced by rpoB mutant M.
tuberculosis DNA, wildtype M. tuberculosis DNA and control receiving beta glucan were
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higher (68.6+17.90 pg/ml; 66.4+10.50 pg/ml; and 55.4+10.90 pg/ml) than those without beta-glucan (36.4+4.87 pg/ml; 42.2+4.06 pg/ml; and 33.2+5.25 pg/ml) with p < 0.05. The same as IFNγ production, IL-12 production in groups of PBMCs induced by rpoB mutan M.
tuberculosis DNA, wildtype M. tuberculosis DNA and control receiving beta glucan were higher (122.9+11.16 pg/ml; 188.0+26.36 pg/ml; and 137.2+24.05 pg/ml) than those without beta glucan (82.3+26.23 pg/ml; 62.6+13.69 pg/ml; and 60.7+7.32 pg/ml) with p < 0.05.
Conclusion: These results indicated that the provision of beta-glucan could improve the performance of PBMCs in producing IFNγ and IL-12 as primary cytokines in tuberculosis infections, either in the cells induced by bacterial DNA or in the other cells without induction in vitro.
Keywords: Beta glucan, IFNγ, IL-12, M. Tuberculosis DNA
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INTRODUCTION
Tuberculosis (TB) is caused by bacteria (Mycobacterium tuberculosis) that most often affect the lungs (1). These bacteria belong to a group of pathogens that are difficult to kill because they have various abilities to attack and survive in host cells. Mycobacterium tuberculosis were able to penetrate the immune system which ultimately lead to infection in host cells (2).
Treatment of tuberculosis infections involves various combinations of anti-tuberculosis drugs. First-line anti-tuberculosis drugs consist of rifampicin, isoniazid, ethambutol and streptomycin (3). Rifampicin and isoniazid are the most potent drugs to kill M. tuberculosis bacteria (4). When this bacteria resistant to the first-line anti-tuberculosis drug, then the treatment given next is a second-line anti-tuberculosis drug that its side effects and price much higher than the first one (5-6).
The resistance of M. tuberculosis to anti-tuberculosis drugs is caused by mutations in the protein-coding gene which is the catch point of the anti-tuberculosis drugs (2). One of the mutations that occur in M. tuberculosis is the mutation of the rpoB gene that causes the bacteria become resistant to rifampicin (18). The target of rifampicin is the RNA polymerase protein. Rifampicin works by inhibiting RNA polymerase transcription. Rifampicin will specifically bound to the subunit β RNA polymerase encoded by the rpoB gene. This bond will inhibit the transcription process by stopping the extension of the RNA strands. Mutations in the rpoB gene will cause a confirmation changes in the binding site between rifampicin and the β subunit. Changes in the site of this bond cause rifampicin fail to bind the β subunit (8).
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The invasion of M. tuberculosis stimulates immune cells to produce various cytokines to kill and eliminate these bacteria, so that they do not cause infection. The main cytokines produced in the invasion of M. tuberculosis are IFNγ and IL-12. IFNγ is the main activator of macrophages and stimulates macrophages to inhibit the growth of M. tuberculosis. IFNγ also plays a role in the process of apoptosis and necrosis of macrophage cells infected by M.
tuberculosis (9). Tuberculosis infection stimulates macrophage cells to produce IL-12.
Expression of IL-12 during the course of the infection process regulates the natural immune response and separates the type of adaptive immune response. IL-12 induces IFNγ production (10).
In pathogenic M. tuberculosis infection, immune cells should be able to produce more amounts of IFNγ and IL-12. But these two cytokines are not produced in sufficient quantities because of weakening of immune cells. To increase the production of cytokines, immune cells need to get external immunomodulators. Beta glucan is known as a potent immunomodulator that can affect both natural and adaptive immunity. Beta glucan can increase the potential of the immune system through various communication mechanisms between the two immune systems. Dectin-1 is a type II transmembrane protein receptor that can bind beta glucan, that initiate and regulate the natural immune response (11-12), and needed in the process of eliminating bacterial infections and other pathogens.
The use of deoxyribonucleic acid (DNA) of bacteria as PBMCs inductor in this study was based on the potential of CpG oligodeoxynucleotide motifs in the evidenced DNA of the bacteria to stimulate the production of various cytokines and nitric oxide (NO) in immune cells, in which some are indicators of inflammation.(13-14) This work describes an in vitro
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study using mutant and WT M. tuberculosis bacterial DNA as the inductor of PBMCs. The selection of these two types of DNA was based on previous research which showed that the wildtype strain of M. tuberculosis bacteria found in Indonesia was Beijing strain H37Rv, whereas the most mutation of bacterial resistance to rifampicin first-line anti-tuberculosis drug was the mutation of the rpoB gene (7). The use of both types of DNA is expected to be able to describe the form of bacterial stimulation of host cells in vivo. The selection of M.
Tuberculosis bacteria in this study was mainly due to these bacteria being the pathogen causing tuberculosis infection. Treatment of these bacteria is still being developed to date, especially when the bacteria show resistance to anti-tuberculosis drugs. Thus, the clinical implications of this study are the potential of beta glucan as an immunomodulator in both types of tuberculosis infections caused by M. tuberculosis wildtype and mutants.
The purpose of this study was to investigate the effects of beta-glucan on the production of IFNγ and IL-12 in PBMCs that were induced by M. tuberculosis DNA in WT and mutant type.
METHODS
This study was a laboratory experimental study and conducted from October 2017 to March 2018. The culture of M. tuberculosis and bacterial resistance testing were carried out in the Health Laboratory of the Central Java Province, while for bacterial DNA isolation and determination of mutation were done at the Microbiology Laboratory, Institute of Tropical Disease, Airlangga University Surabaya. The measurement of cytokine production was carried out in the integrated laboratory of the Faculty of Medicine, Gadjah Mada University. This 81
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study received approval from the Health research ethics committee of the Faculty of Medicine Diponegoro University and Dr Kariadi Hospital Semarang with sertificate number 469/EC/FK- RSDK/VII/2017.
Respondent recruitment.
Respondent were healthy volunteers (six women and five men) who met the inclusion criteria, i.e., aged 18-25 years, not suffering from acute or chronic disease, negative HIV test results, having negative results on hepatitis B and hepatitis C tests, did not consume immunosuppressants and immunomodulators, and not undergoing surgery in the six months prior to the examination. All respondents involved in this study have volunteered to be involved in the research and signed an informed consent sheet.
Beta Glucan
The present study used beta glucan from baker’s yeast S. cerevisiae in the form of dry powder (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) with the purity of >90%. The dissolution process was performed by adding beta-glucan to ultrapure water at room temperature with vortex for 15 min until it was dissolved homogenly.
Selection of M. Tuberculosis isolates
This study used the culture of M. tuberculosis bacteria from Health laboratory of Central Java Province. Resistance testing started with bacterial culture on the medium of Lowenstein Jensen (LJ). The bacterial culture was carried out for 3-8 weeks and the growth of
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bacterial colonies was observed and recorded every week. When the bacterial colonies growed well, then the sensitivity test for anti-tuberculosis drugs was carried out using indirect
proportional modification methods. Before LJ medium was used to culture bacteria, medium quality testing was carried out first using Mycobacterium fortuitum bacteria with catalog number ATCC 6841. Each sample was cultured in one series of LJ medium containing first- line anti-tuberculosis drugs. Tubes that have been inoculated with bacteria are then incubated at 370C for 3 weeks. The bacteria usually grow confluently on the control medium and this growth was considered 100%. Determination of resistance was done by comparing the percentage of bacterial growth in drug media with controls. The control bacteria used in this study were BA 66 for comparison of streptomycin and rifampicin resistance, and BA 63 for comparison of resistance to isoniazid and ethambutol. If the number of bacterial colonies that grow on media contains more than 1% OAT, then the bacteria are considered resistant.
DNA isolation was carried out at the Microbiology Laboratory, Institute of Tropical Disease, Airlangga University, Surabaya. The isolation procedure was started by taking a bacterial culture of one ose and putting it in a tube containing PBS. Furthermore proteinase K and AL buffer were added to the tube and vortexed until the solution was homogeneous.
Ethanol was added to the tube and vortexed until evenly mixed. Then this solution was poured into the D-Neasy mini column and placed in a 2 ml collection tube. The tubes were centrifuged at 800 x g for 1 minute. The collection tube was removed and replaced with a new one. Then the AW1 buffer was added to the tube. The tubes were centrifuged at 800 x g for 1 minute and the collection tube was replaced with a new one. Furthermore, AW2 buffer was added to the tube and centrifuged at 1400 x g for 3 minutes. The collection tube was discarded
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and the tube was removed on a 1.5 ml microcentrifus. DNA was eluted with AE buffer and incubated at room temperature for 1 minute. Finally the tube was centrifuged at 800 x g for 1 minute. The whole process of DNA isolation was followed the procedure of the factory that produced Qiagen kits DNeasy Blood and Easy kit with catalog number 69504.
The final DNA amplification reaction was 50 μl, consisting of 20 μl mixture of PCR, 1μl forward primer, 1 μl reverse primer, 3 μl DNA, 20 μl Dw. Primer used as template: forward 5'- TAC GGT CGG CGA GCT GAT CC-3 '; reverse 5'-TAC GGC GTT AG C-3 TCG ATG 'with the target 411-bp fragment from the rpoB gene. PCR cycling process: 940C for 2 minutes; 35 cycles of denaturation at 940C for 20 seconds, annealing at 61.30C for 10 seconds, and
extension at 720C for 30 seconds; and the final extension at 720C for 5 minutes, and extension at 40C to completion. PCR products run on 1.5% agarose gel and stained with ethidium bromide. Then visualized with ultraviolet sinau. PCR products were ordered using automated DNA sequencing (Applied Biosystems, Inc., Foster City, Calif) and analyzed using BLAST bioinformatics tools available at the National Center for Biotechnology Information. compared to wild type M. tuberculosis (H37Rv). The process of PCR and sequencing was carried out in the Microbiology Laboratory, Institute of Tropical Disease at Airlangga University, Surabaya.
PBMC isolation and stimulation
Isolation of PBMCs based on the principle of the density gradient centrifugation method using Ficoll Histopaque by Panda and Ranvindran, 2013 with modification. This protocol started with pouring 4 ml peripheral blood in a tube with heparin and stored at room
temperature for an hour, and then centrifuged at 100 x g for 15 min. Buffy coat was taken and added to PBS. The blood was then isolated by Ficoll density gradient with centrifugation at
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100 x g for 30 min. In the next step, the buffy coat was taken and washed twice with PBS. In addition, a complete RPMI media of 1 ml was added to the buffy coat, and the cells were calculated using a haemocytometer (15). The composition of culture medium was FBS 10%, Penicillin-Streptomycin 2%, Fungizone 0.5% and RPMI 87.5%. PBMCs (5 x 105/ml) were co- cultured on 24-well plates of complete RPMI media with and without the addition of beta- glucan (5 μg/ml), and with M. tuberculosis DNA (10 μg/ml) WT and mutant with the
following distribution: group 1 was PBMCs induced with mutant DNA and given beta glucan, group 2 were PBMCs induced with mutant DNA without beta glucan, group 3 were PBMCs induced with wildtype DNA with beta glucan, group 4 were PBMCs which were induced by wildtype DNA without beta glucan, group 5 were PBMCs without inducers and were given beta glucan, and group 6 were PBMCs without induction and without beta glucan.
The cells were incubated on a humidified incubator of 5% CO2 at 37 °C for six days.
Next, the supernatant was harvested and analyzed for its cytokine production with enzyme- linked immune-sorbent assay (ELISA). The supernatant, which was not immediately analyzed after harvesting, was stored in the freezer at a temperature of -80 °C for further study.
Cytokine measurements
The cytokines measured in this study were interferon-gamma (IFNγ) and interleukin 12 (IL-12) using ELISA technique. The analysis of cytokine was performed using the Human IFNγ ELISA kit and the Human IL-12 ELISA kit (Fine Test, Hubei, China). The ELISA procedure was done by following the instruction manual issued by the manufacturer. In bief, the supernatant was centrifuged at 1,000 ×g for 20 min. The plate was rinsed twice before use.
The sample of 100 μl was put into each well and incubated at 37 °C for 90 min. In each well,
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as much as 100 μl of Biotin-detection antibody working solution was added and it was then incubated at 37 °C for 60 min. After being aspirated, the supernatant was rinsed three times.
Next, 100 μl of HRP-Streptavidin Conjugate (SABC) working solution was added to each well and incubated again at 37 °C for 30 min. It was then aspirated and washed five times. As much as 90 μl of tetramethylbenzidine (TMB) substrate was added and the mixture was incubated for 15 min at 37 °C. In the final step, 50 μl of stop solution was added. The result was read using a Biorad microplate reader on O.D. with the absorbance of 450 nm.
Statistical Analysis
Kruskal-Wallis analysis was used for all paired comparisons between beta glucan and non beta glucan stimulated cells, and control. to compare the mean differences in IFN gamma and IL-12 production with a confidence level of 95% and p < 0.05. Post hoc analysis was used to compare the mean values of cytokines between the groups induced by mutant and wild-type (WT) DNA, and the control. The software used for statistical analysis was IBM SPSS statistics 20.
RESULTS
DNA of M. tuberculosis
The PCR product of DNA M. tuberculosis used in this study were described in Figure 1.
rpoB gene of M. tuberculosis indicated by PCR product 411 bp.
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Figure 1. The PCR products of rpoB gene of M. tuberculosis. M: marker ladder of 100 bp., A- H: isolates of DNA M. tuberculosis., K: control. The size of PCR product of rpoB gene of is 411 bp.
After the rpoB gene in M. tuberculosis was identified through PCR, then sequencing was carried out to determine the base composition of the gene. Sequencing results showed a variation of the rpoB gene on codon 351.
Figure 2. Variation of the rpoB gene in M. tuberculosis. The bases on codon 513 which initially TCG changed to TTG
Cytokines Production
100 bp 411 bp 500 bp
T T G T C G
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The production of cytokines was made between cells receiving beta-glucan and without beta-glucan, and the cells induced by mutant and WT M. tuberculosis DNA (strain H37Rv), as well as the cells which were not induced by any DNA.
Table 1. Mean distribution of IFNγ and IL-12 PBMC levels based on the provision of beta glucan
Type of Induction Provision of Beta Glucan
Mean+SD (pg/ml)
P- value Production of IFNγ
- PBMCs induced with mutant M. tuberculosis DNA
With beta glucan
68.5+17.90 < 0.05
Without beta glucan
36.3+4.87 - PBMCs induced with
wild-type M.
tuberculosis DNA
With beta glucan
66.3+10.50 < 0.05
Without beta
glucan 42.2+4.06
- Non-induced PBMC (control)
With beta
glucan 55.3+10.90 < 0.05 Without beta
glucan
33.1+5.25
Production of IL-12 - PBMCs induced with
mutant M. tuberculosis DNA
With beta glucan
122.8+11.16 < 0.05
Without beta
glucan 82.3+26.23
- PBMCs induced with wild-type M.
tuberculosis DNA
With beta
glucan 188.0+26.36 < 0.05 With beta 62.5+13.69
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glucan - Non-induced PBMC
(control)
With beta
glucan 137.2+24.05 < 0.05 Without beta
glucan
60.6+7.32
In general, the administration of beta glucan in PBMC with various treatments, increases the production of IL-12 and IFNγ, both those induced by mutant M. tuberculosis DNA, wildtype M. tuberculosis DNA, or without induction. IL-12 is produced in greater amounts than IFNγ. The presence of IFNγ and IL-12 shows the occurrence of immune cell activity, specifically T lymphocytes and macrophages known as the secretory cells of both cytokines.
In this study, administration of beta glucan was shown to significantly increase the activity of immune cell function (T cells and macrophages) in producing the main cytokines in tuberculosis infections, namely IFNγ and IL-12.
PBMCs induced by mutant M. tuberculosis bacteria and beta glanans produced an average IFNγ of 68.6 + 17.90 pg / ml, and were significantly different compared to IFNγ produced by PBMCs induced by mutant bacterial DNA without beta glucan administration (p
<0.01) . Similar conditions also occur in the production of IL-12, where PBMCs induced with mutant M. tuberculosis bacterial DNA and given beta glucan produce IL-12 averaging 122.9 + 11.66 pg / ml, and differ significantly compared to IL-12 produced by PBMC induced mutant bacterial DNA without beta glucan administration (p <0.05).
PBMCs induced by wildtype M. tuberculosis DNA and beta glucan produced an average IFNγ of 66.4 + 10.50 pg / ml, and were significantly different compared to IFNγ produced by PBMCs induced by wildtype DNA without beta glucan administration (p <0.05) . PBMCs
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induced with wildtype M. tuberculosis DNA and beta glucan produced an average IL-12 of 188.0 + 26.36 pg / ml, and were significantly different compared to IL-12 produced by wild- type DNA-induced PBMC without beta glucan administration ( p <0.05).
PBMCs that were not induced and only given beta glucan produced an average IFNγ of 55.4 +10.90 pg / ml, and were significantly different compared to IFNγ produced by PBMC without induction and without beta glucan administration (p <0.05). PBMC without induction and only beta glucan produced an average IL-12 of 137.2 + 24.05 pg / ml, and was significantly different compared to IL-12 produced by PBMC without induction and without beta glucan administration (p <0.05).
DISCUSSION
IFNγ and IL-12 are the main cytokines produced by T-lymphocytes and macrophages during tuberculosis infection, which function as one form of body defense to avoid infection.(16). To counter and prevent tuberculosis infection, IFNγ and IL-12 must be produced in sufficient quantities. The results of this study showed that PBMCs cultured with beta glucan produced IFNγ more than PBMCs that were cultured without beta glucan, both in the induction of mutant M. tuberculosis DNA, wildtype M. tuberculosis DNA and without induction. The results of this study are in line with the results of the 2015 Javmen study which showed that orally-administered β-glucan from S. cerevisiae enhanced IFN-γ production in BALB/c mice (17). Increased IFNγ production modulated by beta glucan is not only observed in PBMCs, but also in serum (16). At the molecular level, oral mushroom beta-glucan treatment significantly increased IFN-γ and IL- 12 mRNA expression (18). IFN-γ was induced by a branched β-
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glucan from S. crispa (SCG) in adherent splenocytes, but IFN-γ production was most significantly increased by SCG in instances involving coexistence of adherent and
nonadherent splenocytes. In fact, inhibition of cell-cell contact reduced IFN-γ induction by SCG. In addition, interleukin-12 p70 (IL-12p70) was induced by SCG in DBA/2 mice in vitro (19). In addition to whole blood and serum, the potential of beta glucan as a modulator of IFNγ production can also be observed in Peyer's patches. The level of induction of IFN-γ by SCG was significantly increased in SCHWE-treated mice. This activity was more clearly observed when chlorpromazine was administered as a pretreatment in SCHWE-treated mice.
The production of IFN-γ by immune cells in Peyer's patches was higher in SCHWE-treated mice than in control mice. These results suggest that orally administered β-glucan may modulate cytokine induction by SCG in the spleen through the activation of Peyer's patches (20).
In addition to IFNγ production, the results of this study also showed that PBMCs cultured with beta glukan produced more IL-12 than PBMCs cultured without beta glucan, both in the induction of mutant M. tuberculosis DNA, wildtype M. tuberculosis and without induction. The research conducted by Pelizon in 2015 found results that were in line with this study, where in vivo beta glucan administration primed spleen cells for a higher production of IL-12, ant another cytokines (21). Beta glucan does not only affect PBMCs in producing IFNγ, but also signişcantly upregulated IFN-γ production. In contrast, neutralization of IL-12 activity by anti-IL-12 decreased IFN-γ synthesis. These data suggest that beta glucan may support anti-tumour and anti-infective immune responses by increasing IL-12-induced IFNγ production in T cells (10). Beta-glucan from S. cerevisiae is one of the polysaccharides that
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has been proven to increase the production of IFNγ and IL- 12 in vitro (16). In previous study, orally administered beta glucans reduced the progression of decreased white blood cell (WBC) count and increased the production of IL-4 and IL-12 in breast cancer patients when compared with the placebo control group (22).
Research carried out by Hetland in 2002 obtained results indicate that β-glucans inhibit growth of M. tuberculosis in host cells in vitro, probably due to cellular stimulation and/or competitive inhibition of uptake of bacteria via CR3 (CD11b/18) (23), where the study showed that beta glucan has the potential to treat tuberculosis infections. There was a dose- dependent effect of beta-glucan injected before BCG challenge on the number of BCG bacilli found in spleen and liver homogenates. In addition, antibody cross-reactivity was
demonstrated between M. tuberculosis cell wall and beta-glucan. The results suggest that beta- glucan has a protective effect against M. bovis, BCG infection in susceptible mice (24).
Beta glucan can stimulate immune cells because they are recognized by immune cells themselves. Special emphasis is placed on Dectin‐1, as we know the most about how this key β‐glucan receptor translates recognition into intracellular signaling, stimulates cellular
responses, and participates in orchestrating the adaptive immune response. Dectin-1 was almost exclusively responsible for the beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. these results identify Dectin-1 as a new target for
examining the immunomodulatory properties of beta-glucans for therapeutic drug design (4).
Increased production of cytokines in this study has occurred because beta glucan has been evidenced to increase the ability of differentiation of monocyte cells into macrophages,
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(25) with the help of other cytokines during differentiation process through nonapoptotic, and caspase-3-dependent mechanisms(26) .
The ability of beta-glucan to induce macrophage cells is due to the presence of Dectin-1 receptor on the cells. Through this receptor, the cells can recognize and bind the surrounding beta glucan. The dectin-1 receptor is often referred to as beta-glucan receptor since it more predominantly binds beta glucan than any other receptors present in the cells (27).
The limitation of the study: this study used two types of inducers, mutant DNA M.
tuberculosis and DNA M. tuberculosis wildtype in one study, so that the strength of both inducers were not clearly identified.
CONCLUSIONS
The beta-glucan improves the performance of PBMCs in producing IFNγ and IL-12, in mutant DNA M. tuberculosis induction, wildtype DNA M. tuberculosis or without induction, and for the future studies it is better to use only one type of inducer.
ACKNOWLEDGEMENTS
The authors would like to express acknowledgment to Suprihatin for her technical assistance with laboratory work and all participants. Furthermore, the authors also express gratitude to Ministry of Research, Technology and Higher Education of the Republic of Indonesia for the research funding of this study. No. of the Contract. 275-22/UN7.5.1/PG/2017
CONFLICT OF INTEREST
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None of the authors of this paper have a conflict of interest.
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References
1. World Health Organization; 2018. Global tuberculosis report 2018. Geneva.
2. Smith I. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence. Clinical Microbiology Reviews. 2003;16(3):463-96.
3. Sotgiu G, Centis R, D’ambrosio L, and Migliori G. Tuberculosis Treatment and Drug Regimens. Cold Spring Harb Perspect Med 2015;5:a017822..
4. Genestet C, Ader F, Pichat C, Lina G, Dumitrescu O, Goutelle S, the Lyon TB Study Group. 2018. Assessing the combined antibacterial effect of isoniazid and rifampin on four Mycobacterium tuberculosis strains using in vitro experiments and response- surface modeling. Antimicrob Agents Chemother. 2018:1-12.
5. Jnawali H and Ryoo S. First and Second Line Drugs and Drug Resistance.
Tuberculosis - Current Issues in Diagnosis and Management. 2013:163-180.
6. Laing R, McGoldrick K. Tuberculosis drug issues: prices, fixed-dose combination products and second-line drugs. Int J Tuberc lung dis 2000;4(12):S194–S207.
7. Erawati M, Andriany M, Kusumaningrum NSD. Mutation in the rpoB gene of multidrugs-resistant Mycobacterium tuberculosis isolates from Semarang. International journal of Molecular and Clinical Microbiology. 2017:7(2):818-823
8. Campbell E, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst S.
Structural Mechanism for Rifampicin Inhibition of Bacterial RNA Polymerase. Cell.
2001;104: 901–12.
9. Lee J, Hartman M, Kornfeld H. Macrophage apoptosis in tuberculosis. Yonsei Med J.
2009;50(1):1–11
337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358
10. Budak F, Goral G, Oral HB. Saccharomyces Cerevisiae Beta-Glucan Induces Interferon-Gamma Production in Human T Cells Via IL-12. Turk J Immunol. 2008;
13: 21-6.
11. Brown G, Herre J, Williams D, Willment J, Marshall A, Gordon S. Dectin-1 mediates the biological effects of β-glucans. J Exp Med. 2003;197:1119–24.
12. Herre J, Marshall A, Caron E, Edwards A, Williams D, Schweighoffer E, et al. Dectin- 1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood. 2004.
104:4038–45
13. Wattrang E, Palmb A, Wagnerc B. Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells Vet Immunol Immunopathol. 2012;146:113– 24
14. Tavakoli Z, Ardestani SK, Lashkarbolouki T, Kariminia A, Salehi TZ, Tavassoli N.
DNAs from Brucella strains activate efficiently murine immune system with production of cytokines, reactive oxygen and nitrogen species. Iran J Allergy Asthma Immunol. 2009;8(3):127–34.
15. Panda, S. K., S. Kumar, N. C. Tupperwar, T. Vaidya, A. George, S. Rath, V. Bal and B. Ravindran (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.
16. Zhang B., Guo, Y. M., & Wang, Z. The modulating effect of beta-1,3/1,6-glucan supplementation in the diet on performance and immunological responses of broiler chickens. Asian-Australas J Anim Sci, (2008). 21(2), 237–244.
359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
377 378 379
17.Javmen A, Nemeikaitė-čėnienė A, Bratchikov M, Grigiškis S, Grigas F, Jonauskienė I, et al. β-Glucan from Saccharomyces cerevisiae Induces IFN-γ Production In Vivo in BALB/c Mice. In vivo. 2015; 29: 359-364.
18. Wang W, Wu Y, Chen S, Liu C, Chen S. Mushroom �-Glucan May Immunomodulate the Tumor-Associated Macrophages in the Lewis Lung Carcinoma. BioMed Research International. 2014:1-16.
19. Harada T, Miura N, Adachi, Nakajima M, Yadomae T, and Naohito Ohno.Journal of Interferon & Cytokine Research. 2004;22(12).
20. Hida T, Kawaminami H, Ishibashi K, Miura N, Adachi, Ohno N. Oral Administration of Soluble β-Glucan Preparation from the Cauliflower Mushroom, Sparassis crispa (Higher Basidiomycetes) Modulated Cytokine Production in Mice. International Journal of Medicinal Mushrooms. 2013;15(6)
21. Pelizon AC, Kaneno R, Soares AMVC, Meira DA, Sartori A. Immunomodulatory Activities Associated with β-Glucan Derived from Saccharomyces cerevisiae. Physiol.
Res. 2005; 54: 557-64.
22.Lee SB, Kim YH, Hyun MC, Kim YH, Kim HS, Lee YH. T-Helper Cytokine Profiles in Patients with Kawasaki Disease. Korean Circ J. 2015; 45(6): 516–21.
23. Hetland G, Sandven P. 1,3-glucan reduces growth of Mycobacterium tuberculosis in macrophage cultures. FEMS Immunol Med Microbiol. 2002;33(1):41–5
380 381 382
383 384 385
386 387
388 389 390 391 392 393 394 395 396 397 398
24. Saito M, Nagasawa M, Takada H, Hara T, Tsuchiya S, Agematsu K, et al. Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells. J Exp Med. 2011;208(2):235-49.
25.Salem SAM, Diab HM, Fathy G, Obia LM, El Sayed SB. Value of Interleukins 4, 10, 12, 18 and Interferon Gamma in Acute Versus Chronic Atopic Dermatitis. J Egypt Women Dermatol Soc. 2010;7(1):56-60.
26. Miura M. Apoptotic and nonapoptotic Caspase function in animal development. Cold Spring Harb Perspect Biol. 2016;4(10):a008664.
, 399 400 401 402 403 404 405 406 407
408 409 410 411
