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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 1  |  Page : 11-15

Investigating the TANK-binding kinase expression in multiple sclerosis patients in comparison with control group in the Iranian population


1 Department of Immunology, Shahrekord University of Medical Sciences, Shahrekord, Iran
2 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
3 Department of Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
4 Lung Diseases and Allergy Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran

Date of Submission21-Apr-2019
Date of Decision13-Jun-2019
Date of Acceptance19-Jul-2019
Date of Web Publication03-Jan-2020

Correspondence Address:
Hedayatollah Shirzad
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AIHB.AIHB_45_19

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  Abstract 


Introduction: Multiple sclerosis (MS) is one of the most common inflammatory autoimmune diseases in the central nervous system, which affects individuals ranged between 20 and 40 years of age. There is still no gene or genes set that is unique to the disease. On the other hand, TANK-binding kinase 1 (TBK1) gene is one of the most important interferon signalling molecules and has been shown to affect many autoimmune diseases, and it is obvious that it is involved in CNS inflammation. In this study, we investigated the expression of TBK1 gene as a major factor in the production and activity of interferons in patients with MS. Materials and Methods: Ninety patients with MS and 30 healthy individuals referring to Kashani Hospital in Shahrekord which matched for age and sex were selected. According to the received treatments, MS patients were divided into three groups as follows: the newly diagnosed group not receiving treatment, interferon β1α (IFNβ1α) treatment group and IFNβ1β treatment group. After filling the questionnaire, blood was sampled, total RNA was extracted and finally, cDNA was synthesised from the individuals. The TBK1 kinase gene expression was investigated using the TaqMan real-time with ΔΔCt method. Obtained data were analysed using GRAPHPAD 5 and SPSS version 21 software. Results: In this study, the two studied groups did not show any significant differences in age and sex parameters. Our results showed that TBK1 expression in newly diagnosed patients was significantly higher than the control group (P < 0.006). However, no significant difference was observed between the two treated groups with the control group. Furthermore, there was a significant difference at the TBK1 gene expression level between the newly diagnosed group patients and the two groups receiving the treatments. There was also no significant correlation between age and sex with gene expression was demonstrated in this study (P = 0.130 and P = 0.310, respectively). Conclusion: Disruption of TBK1 gene expression can be one of the MS causes of onset at the molecular level. Besides, since the expression of this gene has been investigated in newly diagnosed individuals with MS, it may be possible to utilise this molecule in the early detection of the disease, especially in individuals at high risk of disease (with risk index). It can also be used as a new therapeutic approach by targeting this molecule to improve the progression of disease; furthermore, it can be used as a factor in responding to treatment in patients responding to IFN.

Keywords: Autoimmune disease, interferon, multiple sclerosis, TANK-binding kinase 1 gene


How to cite this article:
Helbi S, Bagheri N, Aminzadeh Z, Kooti W, Shirzad H. Investigating the TANK-binding kinase expression in multiple sclerosis patients in comparison with control group in the Iranian population. Adv Hum Biol 2020;10:11-5

How to cite this URL:
Helbi S, Bagheri N, Aminzadeh Z, Kooti W, Shirzad H. Investigating the TANK-binding kinase expression in multiple sclerosis patients in comparison with control group in the Iranian population. Adv Hum Biol [serial online] 2020 [cited 2020 Apr 4];10:11-5. Available from: http://www.aihbonline.com/text.asp?2020/10/1/11/275086




  Introduction Top


Multiple sclerosis (MS) is one of the most common inflammatory autoimmune diseases in the central nervous system, which affects individuals ranged between 20 and 40 years of age. In this disease, myelin which plays a role in neuronal transmission along nerve fibres would be damaged.[1],[2] MS is known to be the second leading cause of impaired mobility in the world and a progressive trend has been reported in patients in recent years.[3] Unfortunately, in spite of extensive studies, there has not yet been found an effective way to achieve definitive treatment of the disease. Therefore, the need for fundamental and applied studies to identify the causes and involved mechanisms and new drug targets in the disease, and the subsequent design of modern therapies MS remains a research priority in most countries.[4],[5] Due to the unknown aetiology of MS disease and the common role of environmental and genetic factors in this disease, researchers are seeking to identify molecular pathways and genes involved in the disease. One of the proteins seem to have a role in MS pathogenesis is TANK-binding kinase 1 (TBK1). This protein is a serine/threonine kinase belongs to the IκB kinase (IKK) family expresses in all tissues.[6] TBK1 has the ability to phosphorylate interferon regulatory factors (IRF) 3 and 7. Each of these transcription factors plays a crucial role in the activation of Type 1 interferon (IFN-1) and interferon-induced genes (ISGs) in response to microbial infections.[7],[8],[9] By phosphorylation of IRF3 and IRF7 in the form of a dimer (homogeneous or heterogeneous) and transfer to the nucleus, IFN-1, ISGs and pro-inflammatory genes would be activated. On the other hand, many pattern recognition receptors, such as TLR3, 7, 9 and cytosolic sensors of DNA and RNA, when binding to the ligand, activate TBK1/IKKε and as a result, induce NF-κB and IRF3/7 signalling pathways. These innate immune signalling pathways are crucial for the immediate release of an antiviral state during an acute infection. In other words, signalling pathways should be restrictedly controlled, since the chronic activation of these pathways is harmful to the host and can lead to autoimmune diseases. In autoimmune diseases, mediated signals through TBK1 involved in the production of IFN-1, autoantibodies and chemokines such as CXCL10/IP-10. These molecules then initiate and strengthen the immune attack on the tissues, ultimately causing organ failure and disease. Another mechanism of this protein in MS is that TBK1 is associated with an important regulator of cellular metabolism called mTORC1 and suppresses its activity.[10] Metabolic disorders are very prevalent in autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières syndrome. Therefore, it seems logical to consider TBK1 as an interface between immune responses and metabolism.[11] Considering the above-mentioned evidence and the fact that gene expression level of this protein has not been studied up to now, the aim of this study was to investigate the TBK1 gene expression in newly diagnosed MS patients treated with IFNβ1α and IFNβ1β in comparison with control group.


  Materials and Methods Top


Studied population

In this study, ninety patients with MS and 30 healthy individuals referring to Kashani Hospital in Shahrekord which matched for age and sex were selected. According to the received treatments MS patients were divided into three groups as follows: the newly diagnosed group not receiving treatment, IFNβ1α treatment group and IFNβ1β treatment group. Newly diagnosed patients were selected after diagnosis and confirmation by a neurologist using clinical symptoms and positive magnetic resonance imaging before taking any medication. Sampling of patients was performed after obtaining the written consent form as well as filling the questionnaire form. Sample size was determined by the following formula: In this formula, α represents Type I error, β represents Type II error, d represents the desired error, Ơ represents the common standard deviation, and Z is the confidence intervals of 95%:[12]



Z1 − β = 0.59



d = 0.51

α = 0.05

β = 0.8

d = 0.51

Inclusion criteria

  • Affected by MS
  • Affected by newly diagnosed MS (new case) by a doctor who has not received any medication at the moment (in the newly diagnosed group)
  • Receiving IFNβ1α and IFNβ1β (in the treated group)
  • Age between 18 and 40
  • Ability to give informed consent
  • No individual or family history of autoimmune diseases such as lupus erythematosus, etc.
  • No history of malignancies
  • Having a normal Vitamin D level (both in the patient group and in the healthy group).


Exclusion criteria

  • Familial history of MS (in control group)
  • Affected by any underlying disease, metabolic disease, autoimmune or nervous system disease except MS
  • Recently affected by viral infections
  • Unwilling to continue the study
  • Receiving the medication during the study.


Ethical consideration

This study was approved at the Ethical Committee of Shahrekord University of Medical Sciences (with code of IR.SKUMS.1395.164). Required explanations were given to the volunteers whom participated in the study, and after receiving written consent sampling were performed. All of the patients' information were kept in privacy and instead of the patients' names, each individual was given a code and all information were recorded private, without a name and with a unique code.

Sampling method, RNA extraction and cDNA synthesis

A volume of 5 ml of blood sample was obtained from the patient using a syringe and collected into a tube containing EDTA. Simple non-randomised sampling was done after receiving written consent from the all patients. Obtained samples were transferred to the laboratory on dry ice. RNA extraction was done using Qiagen kit according to the company's standard protocol. The concentration and quality of the extracted RNA was determined using a nanodrop device. In the cDNA synthesis, the extracted RNA was converted to cDNA using the Genall cDNA synthesis kit.

Performing real-time polymerase chain reaction

Initially, a master mix containing deionised water, primers, SYBR ® Green was prepared and 9 μl added to each of the real-time polymerase chain reaction tubes then cDNA was added to each tube and placed on the device. The tubes were placed in the device and the locking ring was mounted on the samples. Then, the required temperature and time schedule were given to the device software and eventually the programme initiated. The real-time temperature and time conditions were as follows: Taq polymerase enzyme activation (45 s in 94°C), denaturation (15 s in 94°C), annealing (20 s in 60°C), extension (30 s in 72°C) and finally, a melting cycle was performed to scan final products from the reaction at a temperature range of 65°C–95°C. In this study, Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control gene. The sequences of used primers are listed in [Table 1].
Table 1: The sequences of used primers in this study

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Statistical analysis

Obtained data were analysed using GraphPad Prism Version 5 and SPSS version 22 computer software. One way analysis of variance method and Tukey's test were used as a complement test for comparison between groups. In addition, logistic regression analysis and Pearson correlation coefficient were used to assess the age and sex with expression of the gene.


  Results Top


In this study, the expression of TBK1 gene in MS patients was compared with the control group in the Iranian population. There were no significant differences between the studied groups in terms of age and sex. According to [Figure 1], the expression of TBK1 gene was significantly higher in the new diagnosed patient group than in the control group (P < 0.001). While, there was no significant difference between the medication receiving group and the control group. Furthermore, there was a significant difference between the newly diagnosed patients with those receiving the medication (P < 0.01) [Figure 1]. Furthermore, there was no significant correlation between age and sex with gene expression in the study (P = 0.130 and P = 0.310, respectively) [Figure 2] and [Figure 3].
Figure 1: Comparison of TANK-binding kinase 1 gene expression in the studied groups. The data are shown as (2 ^ − ΔΔCT ± standard deviation) ** Significant difference at level of P<0.01.

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Figure 2: The correlation between age and gene expression.

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Figure 3: The correlation between sex and gene expression (1: Male; 2: Female).

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  Discussion Top


In this study, the expression of TBK1 gene in MS patients in the Iranian population was evaluated and the expression of this gene was compared with healthy controls in different patients (newly diagnosed and receiving medication). In the present study, TBK1 expression in newly diagnosed patients was significantly higher than that in the control group. However, there was a significant difference in the expression of this gene in the patients receiving medication in comparison with the newly diagnosed individuals, but there was no significant difference in the gene expression level between the control subjects and those receiving the medication. Even though, the aetiology of the disease is unknown, MS seems to be a disease that initiates in a genetically susceptible patient as a result of exposure to a biological agent, more likely a pathogen. Although there are many genes associated in MS disease, there is still no gene or gene set specifically identified for MS.[13],[14] The unique function of TBK1 is the ability of this molecule to phosphorylate IRF 3 and 7.[9] Each of these transcription factors plays a crucial role in the activation of IFN-1 and ISGs in response to microbial infections. As a result, TBK1 is one of the most important components of the interferon signalling pathway and induces interferons, particularly type-1 interferon, in various ways.[7],[15] As indicated, the activation of the signalling pathway in response to DNA detection in the cytoplasm, which leads to interferon beta induction, acts as a stimulant for autoimmune diseases such as SLE.[16],[17] Fitzgerald et al. in order to demonstrate the role of TBK1 in innate immune responses transferred TBK1-expressing vector into their desired cells and revealed that the expression of INF-ß and RANTES increased significantly. It was also shown that this action is via the IRF3 pathway.[7] Takeshita and Ishii studied the role of DNA intracellular sensors in immunity. In their study, they found that several specific immune molecules and their regulatory elements involved in detecting foreign and ectopic DNA within the cell. These molecules include TBK1, IRF3, TLR9 and type I interferons that are interconnected through different pathways.[18] Increasing the expression of this gene and as a result increased interferon production seems to be the response of the body to autoimmune diseases and intracellular infectious agents. Increasing the production of this gene and the final product of this pathway, interferon, by reducing inflammatory factors such as interleukin-1, reduces the extent of damage induced into different parts of the body during autoimmune diseases.[19] Considering the role of TBK in activating the expression of the interferons, targeting TBK1 as a kinase can be effective in blocking the interferon expression. In other autoimmune diseases, such as SLE, the increased expression of TBK1 gene in patients is shown compared to the control group.[20] These results were in concordance with our results on MS disease. In our study, considering the higher expression of TBK1 in patients in comparison to the control group, it is possible that by reducing the expression of this gene and its effects the inflammatory factors would reduce and the clinical manifestations of the MS disease in patients would suppress. The results of our study showed that the expression of this gene decreased significantly in the medication receiving groups. It can be expected that the medications used for these patients impose their effect by targeting the expression of the TBK1 gene. Smith et al. suggested that TBK1 plays an important role in the expression of pro-inflammatory cytokines, adhesion molecules, growth factors and anti-apoptotic proteins in systemic lupus erytromatosis and given the elevated level of TBK1 in these patients, can contribute to the pathogenesis of this disease.[21] The results of their study were in accordance to ours and demonstrated the increased expression of TBK1 gene in autoimmune diseases. Smith et al. also found that medications used in patients reduced the expression of this gene. In our study, the reduction in TBK1 gene expression was observed in comparison with newly diagnosed patients. Therefore, TBK1 gene can be considered as one of the genes involved in the pathogenesis of autoimmune diseases, especially MS, whose therapeutic agents can facilitate the improvement process of the disease by reducing the expression of this gene. As Lee et al. found, inflammatory inhibitors in the nervous system reduced TBK1 expression in patients receiving the inhibitors compared to the control group.[22] It can be said that the therapeutic agents targeting the TBK1 gene have a better efficacy in treatment, and reduced expression of this gene may be used as a prognostic factor in the treatment process.


  Conclusion Top


From the present study, it can be concluded that increased TBK1 expression in newly diagnosed patients is a process for reducing inflammation and symptoms of MS. Therefore, it seems reasonable that TBK1 expression in patients differs from that of the control group and significantly increases expression, which can be due to the important role of TBK1 gene in patients. On the other hand, due to the reduction in the expression of TBK1 in patients undergoing treatment, TBK1 can be said to be an interesting target for treatment, susceptibility, diagnosis, prognosis and treatment follow-up.

Acknowledgements

This study was approved by Shahrekord University of Medical Sciences, Iran.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Kamińska J, Koper OM, Piechal K, Kemona H. Multiple sclerosis – Etiology and diagnostic potential. Postepy Hig Med Dosw (Online) 2017;71:551-63.  Back to cited text no. 1
    
2.
Foroutan P, Zarezadeh Y, Choobdarian H, Faridi A, Azadi NA, Boshagh MA. Comparison of epstein-barr virus antibodies in the serum of patients with multiple sclerosis and normal people in Sanandaj, Iran. Int J Biomed Public Health 2018;1:127-31.  Back to cited text no. 2
    
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Joseph D, Kumar S. Identifying clues to molecular etiology of multiple sclerosis in South Indian patients. Mult Scler Relat Disord 2016;5:7-11.  Back to cited text no. 4
    
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Li S, Lu LF, Wang ZX, Lu XB, Chen DD, Nie P, et al. The P protein of spring viremia of carp virus negatively regulates the fish interferon response by inhibiting the kinase activity of TANK-binding kinase 1. J Virol 2016;90:10728-37.  Back to cited text no. 6
    
7.
Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 2003;4:491-6.  Back to cited text no. 7
    
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Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway. Science 2003;300:1148-51.  Back to cited text no. 8
    
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Tamura T, Yanai H, Savitsky D, Taniguchi T. The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol 2008;26:535-84.  Back to cited text no. 9
    
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Kim JK, Jung Y, Wang J, Joseph J, Mishra A, Hill EE, et al. TBK1 regulates prostate cancer dormancy through mTOR inhibition. Neoplasia 2013;15:1064-74.  Back to cited text no. 10
    
11.
Wu T, Xie C, Han J, Ye Y, Weiel J, Li Q, et al. Metabolic disturbances associated with systemic lupus erythematosus. PLoS One 2012;7:e37210.  Back to cited text no. 11
    
12.
Pokatayev V, Hasin N, Chon H, Cerritelli SM, Sakhuja K, Ward JM, et al. RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS-STING innate immune-sensing pathway in mice. J Exp Med 2016;213:329-36.  Back to cited text no. 12
    
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Shi JH, Xie X, Sun SC. TBK1 as a regulator of autoimmunity and antitumor immunity. Cell Mol Immunol 2018;15:743-5.  Back to cited text no. 13
    
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Jiao B, Sun Q, Yuan Z, Wang J, Zhou L, Yan X, et al. Rare TBK1 variants in patients with frontotemporal dementia and amyotrophic lateral sclerosis in a Chinese cohort. Transl Neurodegener 2018;7:31.  Back to cited text no. 14
    
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Tohnai G, Nakamura R, Sone J, Nakatochi M, Yokoi D, Katsuno M, et al. Frequency and characteristics of the TBK1 gene variants in Japanese patients with sporadic amyotrophic lateral sclerosis. Neurobiol Aging 2018;64:158.e15-21.  Back to cited text no. 15
    
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Choubey D. DNA-responsive inflammasomes and their regulators in autoimmunity. Clin Immunol 2012;142:223-31.  Back to cited text no. 16
    
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Crisafulli SG, Brajkovic S, Cipolat Mis MS, Parente V, Corti S. Therapeutic strategies under development targeting inflammatory mechanisms in amyotrophic lateral sclerosis. Mol Neurobiol 2018;55:2789-813.  Back to cited text no. 17
    
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Takeshita F, Ishii KJ. Intracellular DNA sensors in immunity. Curr Opin Immunol 2008;20:383-8.  Back to cited text no. 18
    
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Hasan M, Dobbs N, Khan S, White MA, Wakeland EK, Li QZ, et al. Cutting Edge: Inhibiting TBK1 by compound II ameliorates autoimmune disease in mice. J Immunol 2015;195:4573-7.  Back to cited text no. 20
    
21.
Smith S, Gabhann JN, Higgs R, Stacey K, Wahren-Herlenius M, Espinosa A, et al. Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus. Arthritis Rheum 2012;64:1601-9.  Back to cited text no. 21
    
22.
Lee SH, Kim KW, Min KM, Kim KW, Chang SI, Kim JC. Angiogenin reduces immune inflammation via inhibition of TANK-binding kinase 1 expression in human corneal fibroblast cells. Mediators Inflamm 2014;2014:861435.  Back to cited text no. 22
    


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