TGF-β Isoforms and Receptors: A Gene Expression Analysis in Multiple Sclerosis Patients and Normal Individuals

Objective: Multiple sclerosis (MS) is a chronic inammatory disease of the central nervous system (CNS), depicted by lymphocytic inltration and demyelination. MS is associated with the up-regulation of pro-inammatory and down-regulation of anti-inammatory cytokines. The purpose of this experimental study was to evaluate the expression level of TGF-β1, TGF-β 2, TGF-β-R1 and TGF-β-R2 mRNAs in peripheral blood mononuclear cells (PBMCs) from MS patients and healthy controls using Real-Time PCR. Results: Our ndings indicated that the TGF-β-R1 expression level was 2.25 times higher in controls than MS patients. Also, a signicant correlation between normalized expression of TGF-β-R1 and TGF-β1, or TGF-β2 was observed. Therefore, these genes could likely play an important role in the etiology of MS.


Introduction
Multiple sclerosis (MS) is a chronic in ammatory disease, characterized by lymphocytic in ltration and demyelination in the central nervous system (CNS) [1][2][3]. Prognosis, diagnose and treatment of this disorder rely upon a greater determination of the mechanisms underlying MS onset and development [4]. In ammatory cascade and the role of different immune cells (including macrophages, natural killer cells and certain lymphocytes populations) are two considerable factors in the etiology of this disease [5].
This cytokine is a protein presented in three isoforms: TGF-β1, TGF-β2 and TGF-β3 [11]. All of the TGF-β isoforms could participate in the generation of chronic MS lesion [12]. Mechanistically, TGF-β signals are generally initiated through binding this factor to the heterodimer complex, composed of type I and II transmembrane receptor serine/threonine kinase, transforming growth factor-β receptor 1 (TGF-β-R1) and TGFβ-R2 [13]. Thus far, several functions have been determined for TGF-β in MS pathogenesis, including 1) suppressing the immune responses, 2) inhibiting T cells, B cells as well as many other cells, 3) inducing the production of regulatory T cells (Treg), 4) repressing leukocyte adhesion to endothelium, 5) downregulation of adhesion molecules, and 6) sustaining a state of immune tolerance [14,15].
With regards to the importance of TGF-β role in MS pathogenesis, in this experiment, we evaluated the gene expression level of two relevant isoforms (TGF-β1 and TGF-β2) as well as receptors (TGF-β-R1 and TGF-β-R2) in peripheral blood mononuclear cells (PBMCs) of MS patients, in comparison with normal individuals. In addition, we investigated correlation of the gene expressions with the expanded disability status scale (EDSS), the age of onset or the disease length.

Methods And Materials
Patient and control participants This experimental study was conducted in the genetics Laboratory of Tarbiat Modares University (Tehran, Iran) within 2013-2014. In this research, 61 MS patients and 36 age-, race-, and sex-matched controls were recruited from Multiple Sclerosis centers at Sina Hospital (Tehran, Iran) to investigate the mRNA expression level of TGF-β1, TGF-β2, TGF-β-R1 and TGF-β-R2 genes. The patients were diagnosed according to the McDonald criteria. informed consent was obtained from all human adult participants and the study was approved by the Ethics Committee of Tarbiat Modares University (ethical code: d52/6723).

RNA extraction and cDNA synthesis
After blood collection, PBMC separation was performed using density gradient Ficoll/Paque solution (lympholyte, Cedarlane, Netherlands) and total RNA was extracted from all patient and control samples, using RNXTM-plus reagent (Cinnagen, Iran) according to the manufacturer's protocol. The samples were subsequently reverse-transcribed into cDNA, using 3 µg total RNA and 250 μg oligo dT (MWG, Germany).
The reaction was incubated at 70° C for 10 minutes and cooled on ice for 3-5 minutes, followed by adding RNase inhibitor, 10 mM dNTPs and Reverse Transcriptase (all from Fermentas, Canada) to 20 ml total volume of reaction mixture. The mixture was ultimately incubated at 42° C and 80° C for 60-90 and 15 minutes, respectively.
Quantitative reverse transcriptase PCR cDNA for each sample was used to evaluate the mRNA expression level of TGF-β1, TGF-β2, TGF-β-R1 and TGF-β-R2 genes using relative quantitative reverse transcriptase PCR (qRT-PCR). In this experiment, glyceraldehyde3-phosphate dehydrogenase (GAPDH) was utilized as housekeeping gene.
Each qRT-PCR reaction was performed in a nal volume of 20 µl, using 10 ng cDNA, 2x SYBR Green I master mix (Takara, Shiga, Japan) and appropriate primer pair set (Table S1). Thermal condition, as onestep RT-PCR, was carried out by an initial step at 95° C for 15 minutes, followed by 40 cycles at 95° C, 60°C and 72° C for 15 seconds, 30 seconds and 30 seconds, respectively, in Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA). Termed cycle threshold (Ct) was determined for each sample, and the average Ct of duplicate samples was calculated.

Statistical analysis
Relative quanti cation data analysis was performed using arbitrary method (ΔCt). The signi cance of differences between control and test groups was determined by independent t-test using SPSS software (Version 20; SPSS Inc, Chicago, USA) and GraphPad Prism 5 (GraphPad Software, Inc., San Diego, USA). The correlation analysis was assessed by Pearson's correlation coe cient. A P-value of 0.05 was set as signi cant threshold.

Results
In this experiment, the age of MS Patients was between 18 and 52 years, with a female to male ratio of 51:10. The control individuals were between 22-45 years of age, with a female to male ratio of 25:11. The demographic characteristics of patients are given in the Table 1.

Gene expression analyses in MS patients compared to the controls
Findings showed that the TGF-β-R1 mRNA level mean was signi cantly increased in the PBMCs obtained from the controls compared to MS patients (2.25 times, p= 0.025). However, no signi cant difference was observed for TGF-β1, TGF-β2 and TGF-β-R2 mRNA expression levels (Fig. 1).

Analysis of the relationship between the expression levels of genes
We investigated the correlation between mean mRNA levels of the analyzed genes. There was a significant positive association between TGF-β-R1 mRNA level mean and TGF-β1, or TGF-β2 in MS patients, but no signi cant correlation was observed between the other genes mRNA expression level mean (Table 2).
Moreover, we observed no signi cant correlation between the normalized expression level of TGF-β1, TGF-β2 or TGF-β-R1 genes and disease length, EDSS scores, or age in MS patients. In contrast, data analysis showed a signi cant positive correlation between TGF-β-R2 mRNA expression level and disease length in the MS patients (r= 0.356, p= 0.006).

Discussion
TGF-β isoforms (TGF-β1, -β2, and -β3) are signaling ligands that promote expression of the extracellular matrix protein components, control growth and differentiation of the epithelial cells, and regulate immune cell function. Thus, the study of TGF-β isoforms and their receptors expression may be useful in determining different responses to the immunomodulatory as well as combinatory therapies. Curiously, TGF-β isoforms are important due to their conservation among vertebrates and their different roles in a variety of human diseases including tissue brosis, cancer and MS [15]. CNS degeneration and in ammation are important causes of demyelination in MS [16]. In 1998, Vincent et al. compared expression of TGF-β-R1 and TGF-β-R2 by immunohistochemistry in the brain tissue of MS patients and normal controls. They determined that TGF-β isoforms were expressed in microglia cells of the normal individuals' brain tissue. They also demonstrated that the TGF-β isoforms and relevant receptors are expressed in MS lesions [17].
Expression pro ling of the MS patients' peripheral blood showed misregulation of SMAD4, SMAD7 and TGF-β-R2. These nding revealed that TGF-β regulation is reduced in MS patients [18]. Here, we demonstrated no signi cant difference in TGF-β1, TGF-β2 and TGF-β-R2 mRNA expression levels in the MS patients compared to controls, while TGF-β-R1 expression level was 2.25 times higher in normal subjects compared to the MS patients.
Several investigations have so far represented difference of TGF-β1, TGF-β2, TGF-β-R1 and TGF-β-R2 mRNA levels in vitro and in vivo. TGF-β over-expression in MS patients, with little disability after thymectomy, disclosed that this protein could have a favorable effect on human diseases with autoimmune background [19]. Peress et al. have previously examined glial TGF-β expression of different isoforms in 14 MS patients. Active lesions illustrated TGF-β2 immuno-reactivity of lesion encircling rami ed microglia. In contrary, all three isoforms of this protein were expressed in the astrocytes of active white matter lesions. These results proposed that TGF-β cytokines could be locally expressed in demyelinated cells [20].
Further investigations showed that induction of rTGF-β1 suppressed IFN-γ, IL-4, IL-6, TNF-α and perforin up-regulation in MS, but it had no effects on IL-10 or TGF-β expression. The selective prohibitory effects of TGF-β1 on pro-in ammatory cytokines expression potentially turn it into an attractive treatment in MS disease [21]. MS patients with no or slight disability showed high level of TGF-β mRNA expression, while moderate or severe disability of MS patients was correlated with high level of IFN-σ-positive cells. Therefore, TGF-β and IFN-σ could have dissenting effects on MS pathology. Thus, administration of IFN-σ inhibitors and/or TGF-β activators might improve MS disease treatment [22].
These differences between our results and others may be due to environment factors including Vitamin D [23] or other drugs that patients use. We formerly studied TGF-β1 expression in 32 patients with MS and 32 healthy controls. Our ndings showed no signi cant change of TGF-β1 expression in the MS patients compared to control group [24], suggesting that large sample size could not affect gene regulation level.
Additionally, this study evaluated the correlation between these four gene expressions in MS patients. We detected a positive link only between the expression of TGF-β1 and TGF-β-R2 as well as the expression of TGF-β2 and TGF-β-R1 in MS patients; however, there was no signi cant association between the expressions of other genes. It is proposed that TGF-β-R1 might in uence on TGF-β1 and TGF-β2 expression by phosphorylating the receptor-speci c SMADs and promoting a feedback mechanism.
Altogether, expression level of TGF-β-R1 could be considered as a risk factor for MS disease which might be associated with in ammatory events in such patients.

Limitations
Investigations have previously implicated that heterogeneous nature of whole-blood samples may not show the optimal level of gene expressions [25,26], revealing some limitation in this study. Hence, assessment of gene expression at protein level MS patients' sorted PBMCs could help to better understand the role of TGF-β.

Availability of data and materials
All relevant data are included in the manuscript.

Competing interests
The authors declare that they have no competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx