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

TGF-β Isoforms and Receptors: A Gene Expression Analysis in Multiple Sclerosis Patients and Normal Individuals | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research note TGF-β Isoforms and Receptors: A Gene Expression Analysis in Multiple Sclerosis Patients and Normal Individuals Zeinab Shirvani-Farsani, Mehrdad Behmanesh, Mohammad Ali Sahraian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-145981/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Objective: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), depicted by lymphocytic infiltration and demyelination. MS is associated with the up-regulation of pro-inflammatory and down-regulation of anti-inflammatory 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 findings indicated that the TGF-β-R1 expression level was 2.25 times higher in controls than MS patients. Also, a significant 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. Molecular Biology TGF-β Pathway TGF- β receptors Multiple Sclerosis Expression Analysis Real-Time PCR Figures Figure 1 Introduction Multiple sclerosis (MS) is a chronic inflammatory disease, characterized by lymphocytic infiltration and demyelination in the central nervous system (CNS) [ 1-3 ]. Prognosis, diagnose and treatment of this disorder rely upon a greater determination of the mechanisms underlying MS onset and development [ 4 ]. Inflammatory 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 ]. In MS dependent inflammatory cascade, various cytokines are up-regulated or down-regulated, including pro-inflammatory ( e.g. IFN-γ, TNF-α and IL-12) and anti-inflammatory ( e.g. IL-10) cytokines [ 6 ]. TGF-β, as one of most critical anti-inflammatory factor, is secreted not only by the immune cells (mainly macrophages) but also the other non-hematopoietic cells [ 7 ]. The role of transforming growth factor- β (TGF-β) has been studied in several disorders, particularly in many autoimmune diseases like MS [ 8-10 ]. 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 trans-membrane receptor serine/threonine kinase, transforming growth factor-β receptor 1 (TGF-β-R1) and TGF-β-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) down-regulation 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 final 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 one-step 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 quantification data analysis was performed using arbitrary method (ΔCt). The significance 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 coefficient. A P-value of 0.05 was set as significant 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 significantly increased in the PBMCs obtained from the controls compared to MS patients (2.25 times, p= 0.025). However, no significant 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 significant correlation was observed between the other genes mRNA expression level mean (Table 2). Moreover, we observed no significant 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 significant 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 fibrosis, cancer and MS [ 15 ]. CNS degeneration and inflammation 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 profiling of the MS patients’ peripheral blood showed misregulation of SMAD4, SMAD7 and TGF-β-R2. These finding revealed that TGF-β regulation is reduced in MS patients [ 18 ]. Here, we demonstrated no significant 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 ramified 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-inflammatory 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 findings showed no significant 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-β 1 and TGF-β-R2 as well as the expression of TGF-β2 and TGF-β-R1 in MS patients; however, there was no significant association between the expressions of other genes. It is proposed that TGF-β-R1 might influence on TGF-β1 and TGF-β2 expression by phosphorylating the receptor-specific 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 inflammatory 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. Abbreviations MS: Multiple sclerosis CNS: central nervous system PBMCs: peripheral blood mononuclear cells EDSS: expanded disability status scale TGF-β: transforming growth factor-β TGF-β-R1: transforming growth factor-β receptor 1 Declarations Ethics approval and consent to participate This study was in accordance with the declaration of Helsinki. This study was approved by the Ethics Committee of Tarbiat Modares University. The informed consent was obtained from all the participants, and informed consent obtained was written. Consent for publication Not applicable. 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 specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ Contributions Z.SHF., M.B.; Performed all experiments, analyzed the data and wrote the manuscript. M.A.S.; Contributed to concept and design, manufactured the samples, and final approval of the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors gratefully acknowledge contribution of the patients and institutions in this study as well as Roya Amirinejad and Fahimeh Molaahmadi for their valuable laboratory supports. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors, other than Department of Research Affairs of Tarbiat Modares University. References Bhise V, Dhib-Jalbut S: Further understanding of the immunopathology of multiple sclerosis: impact on future treatments . Expert review of clinical immunology 2016, 12 (10):1069-1089. Touil H, Kobert A, Lebeurrier N, Rieger A, Saikali P, Lambert C, Fawaz L, Moore CS, Prat A, Gommerman J et al : Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis . Journal of neuroinflammation 2018, 15 (1):114. Tian Z, Song Y, Yao Y, Guo J, Gong Z, Wang Z: Genetic Etiology Shared by Multiple Sclerosis and Ischemic Stroke . Frontiers in genetics 2020, 11 :646. Baecher-Allan C, Kaskow BJ, Weiner HL: Multiple Sclerosis: Mechanisms and Immunotherapy . Neuron 2018, 97 (4):742-768. Pérez-Cerdá F, Sánchez-Gómez MV, Matute C: The link of inflammation and neurodegeneration in progressive multiple sclerosis . Multiple Sclerosis and Demyelinating Disorders 2016, 1 (1):9. Nali LH, Olival GS, Sousa FTG, de Oliveira ACS, Montenegro H, da Silva IT, Dias-Neto E, Naya H, Spangenberg L, Penalva-de-Oliveira AC et al : Whole transcriptome analysis of multiple Sclerosis patients reveals active inflammatory profile in relapsing patients and downregulation of neurological repair pathways in secondary progressive cases . Multiple Sclerosis and Related Disorders 2020, 44 :102243. Komai T, Inoue M, Okamura T, Morita K, Iwasaki Y, Sumitomo S, Shoda H, Yamamoto K, Fujio K: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals . Frontiers in Immunology 2018, 9 (1364). Sanjabi S, Oh SA, Li MO: Regulation of the Immune Response by TGF-β: From Conception to Autoimmunity and Infection . Cold Spring Harbor Perspectives in Biology 2017, 9 (6). Lee PW, Severin ME, Lovett-Racke AE: TGF-β regulation of encephalitogenic and regulatory T cells in multiple sclerosis . European Journal of Immunology 2017, 47 (3):446-453. Kashima R, Hata A: The role of TGF-β superfamily signaling in neurological disorders . Acta Biochimica et Biophysica Sinica 2017, 50 (1):106-120. Voisin A, Damon-Soubeyrand C, Bravard S, Saez F, Drevet JR, Guiton R: Differential expression and localisation of TGF-β isoforms and receptors in the murine epididymis . Scientific reports 2020, 10 (1):995. Compston A, Lassmann H, Smith K: Chapter 10 - The neurobiology of multiple sclerosis . In: McAlpine's Multiple Sclerosis (Fourth Edition). Edited by Wekerle ACCLMMNS. Edinburgh: Churchill Livingstone; 2006: 449-490. Hata A, Chen Y-G: TGF-β Signaling from Receptors to Smads . Cold Spring Harbor Perspectives in Biology 2016, 8 (9). Xu J, Wang Y, Jiang H, Sun M, Gao J, Xie A: TGF-β in Mice Ameliorates Experimental Autoimmune Encephalomyelitis in Regulating NK Cell Activity . Cell transplantation 2019, 28 (9-10):1155-1160. Nataf S, Guillen M, Pays L: TGFB1-Mediated Gliosis in Multiple Sclerosis Spinal Cords Is Favored by the Regionalized Expression of HOXA5 and the Age-Dependent Decline in Androgen Receptor Ligands . Int J Mol Sci 2019, 20 (23). Zhang X, Hamblin MH, Yin KJ: The long noncoding RNA Malat1: Its physiological and pathophysiological functions . RNA biology 2017, 14 (12):1705-1714. Vincent V, Tilders F, Van Dam A: Production, regulation and role of nitric oxide in glial cells . Mediators of inflammation 1998, 7 (4):239. Meoli EM, Oh U, Grant CW, Jacobson S: TGF-β signaling is altered in the peripheral blood of subjects with multiple sclerosis . Journal of neuroimmunology 2011, 230 (1):164-168. Ateke Mousavi N-k, Abbas M, Abdorreza Naser M, Mohammad Reza S-S, Mohammad Reza E, Maryam S, Mina A, Ali Akbar S-Y: Comparison of Cytokine Expression in Multiple Sclerosis Patients and Healthy Volunteers . Acta Medica Iranica 2018, 56 (2). Peress NS, Perillo E, Seidman RJ: Glial transforming growth factor (TGF)-β isotypes in multiple sclerosis: differential glial expression of TGF-β1, 2 and 3 isotypes in multiple sclerosis . Journal of neuroimmunology 1996, 71 (1):115-123. Araujo GR, Aglas L, Vaz ER, Machado Y, Huber S, Himly M, Duschl A, Goulart LR, Ferreira F: TGFβ1 mimetic peptide modulates immune response to grass pollen allergens in mice . Allergy 2020, 75 (4):882-891. Link J, Söderström M, Olsson T, Höjeberg B, Ljungdahl Å, Link H: Increased transforming growth factor‐β, interleukin‐4, and interferon‐γ in multiple sclerosis . Annals of neurology 1994, 36 (3):379-386. Shirvani-Farsani Z, Behmanesh M, Mohammadi SM, Naser Moghadasi A: Vitamin D levels in multiple sclerosis patients: Association with TGF-β2, TGF-βRI, and TGF-βRII expression . Life Sciences 2015, 134 :63-67. Shirvani-Farsani Z, Behmanesh M, Sahraian MA: Interleukin-10 but not transforming growth factor-β1 gene expression is up-regulated by vitamin D treatment in multiple sclerosis patients . Journal of the Neurological Sciences 2015, 350 (1):18-23. Pahlevan Kakhki M, Rakhshi N, Heidary M, Behmanesh M, Nikravesh A: Expression of suppressor of cytokine signaling 1 (SOCS1) gene dramatically increases in relapsing-remitting multiple sclerosis . J Neurol Sci 2015, 350 (1-2):40-45. Heidary M, Rakhshi N, Pahlevan Kakhki M, Behmanesh M, Sanati MH, Sanadgol N, Kamaladini H, Nikravesh A: The analysis of correlation between IL-1B gene expression and genotyping in multiple sclerosis patients . J Neurol Sci 2014, 343 (1-2):41-45. Tables Table 1 Demographic and disease characteristics of the MS patients and controls. MS Patient Control Number 61 31 Age (mean years ± SD) 32.54±8.45 29.03±6.81 RR/SP/PP 55/6/0 - EDSS (mean ± SD) 2.45±1.33 - Disease Duration 6.42±3.72 - Table 2 Correlation between normalized TGF-β1 , TGF-β2 , TGF-β-R1 or TGF-β-R2 expression level in MS patients. correlation r p-value TGF-β1-TGF-β2 0.077 0.551 TGF-β1-TGF-β RI 0.292 0.023 * TGF-β1-TGF-β RII 0.164 0.216 TGF-β2-TGF-β RI 0.477 0.0001 *** TGF-β2-TGF-β RII 0.010 0.9939 TGF-β RI-TGF-β RII 0.194 0.143 *significant p-value < 0.05 ***significant p-value < 0.001 Supplementary Files TableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Review # 2 received at journal 10 May, 2021 Review # 1 received at journal 10 May, 2021 Reviewer # 2 agreed at journal 14 Apr, 2021 Reviewers invited by journal 11 Apr, 2021 Reviewer # 1 agreed at journal 11 Apr, 2021 Submission checks completed at journal 12 Jan, 2021 Editor invited by journal 10 Jan, 2021 Editor assigned by journal 29 Nov, 2020 First submitted to journal 28 Nov, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-145981","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research note","associatedPublications":[],"authors":[{"id":8063485,"identity":"3f4230ac-b8c5-4792-87ca-ace410178015","order_by":0,"name":"Zeinab Shirvani-Farsani","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Shirvani-Farsani","suffix":""},{"id":8063486,"identity":"870608ed-6c8c-4e17-ab5f-80404868cd12","order_by":1,"name":"Mehrdad Behmanesh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACAwYeKIO9gQHGBosToYXnAMlaJBKQteAB5uy9Rzf83GFjby75OvHBm5p7cvINzA8/MBTcw6nFsudc2s3eM2mJO2fnbjacc6zY2OAAm7EEg0ExbofdyDG7wdt2OMHgdu42aR62hMQNDAxmQPEE3FruvzG7+bftv73BzbNALf8S6uc3sH/Dr+UGj9lt3rYDjBtu8G6T5m1LSGA4wIPfFsueHLPbsm3JiRvOAP0yty/BcMNhnmKJBDxazNnPmN1822Znb3D87MYHb74lyMu3t2/88OEPbi1YADMQk6RhFIyCUTAKRgEGAAC7UlUhP58oDwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mehrdad","middleName":"","lastName":"Behmanesh","suffix":""},{"id":8063487,"identity":"0d523554-91d4-4d0f-91d6-d9b473308b74","order_by":2,"name":"Mohammad Ali Sahraian","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Sahraian","suffix":""}],"badges":[],"createdAt":"2021-01-12 16:08:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-145981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-145981/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5105788,"identity":"54d36020-4b31-47aa-a8af-155b7f96d838","added_by":"auto","created_at":"2021-01-19 23:46:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26185,"visible":true,"origin":"","legend":"TGF-β-R1 gene expression in PBMCs. A) The mRNA expression of this gene shows significant increase in the controls compared to MS patients (2.25 times). B) ΔCt values in the patient and control samples. The results were normalized relative to the level of GAPDH mRNA expression.","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145981/v1/c9ea50b4223f47724c8b6ac2.jpg"},{"id":13650619,"identity":"33bc6247-e215-406a-93b1-5095ef3a57aa","added_by":"auto","created_at":"2021-09-17 09:41:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":861932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-145981/v1/250da19c-40f9-41e3-ac63-4afa506c00aa.pdf"},{"id":5105663,"identity":"78d62337-8db1-4ffc-85f9-32856a2090b8","added_by":"auto","created_at":"2021-01-19 23:43:48","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12714,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-145981/v1/2ba54307760376218755e109.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTGF-β Isoforms and Receptors: A Gene Expression Analysis in Multiple Sclerosis Patients and Normal Individuals\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) is a chronic inflammatory disease, characterized by lymphocytic infiltration and demyelination in the central nervous system (CNS) [\u003ca href=\"#_ENREF_1\"\u003e1-3\u003c/a\u003e]. Prognosis, diagnose and treatment of this disorder rely upon a greater determination of the mechanisms underlying MS onset and development [\u003ca href=\"#_ENREF_4\"\u003e4\u003c/a\u003e]. Inflammatory 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 [\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIn MS dependent inflammatory cascade, various cytokines are up-regulated or down-regulated, including pro-inflammatory (\u003cem\u003ee.g.\u003c/em\u003e IFN-\u0026gamma;, TNF-\u0026alpha; and IL-12) and anti-inflammatory (\u003cem\u003ee.g.\u003c/em\u003e IL-10) cytokines [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. TGF-\u0026beta;, as one of most critical anti-inflammatory factor, is secreted not only by the immune cells (mainly macrophages) but also the other non-hematopoietic cells [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. The role of transforming growth factor- \u0026beta; (TGF-\u0026beta;) has been studied in several disorders, particularly in many autoimmune diseases like MS [\u003ca href=\"#_ENREF_8\"\u003e8-10\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThis cytokine is a protein presented in three isoforms: TGF-\u0026beta;1, TGF-\u0026beta;2 and TGF-\u0026beta;3 [\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e]. All of the TGF-\u0026beta; isoforms could participate in the generation of chronic MS lesion [\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e]. Mechanistically, TGF-\u0026beta; signals are generally initiated through binding this factor to the heterodimer complex, composed of type I and II trans-membrane receptor serine/threonine kinase, transforming growth factor-\u0026beta; receptor 1 (TGF-\u0026beta;-R1) and TGF-\u0026beta;-R2 [\u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. Thus far, several functions have been determined for TGF-\u0026beta; 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) down-regulation of adhesion molecules, and 6) sustaining a state of immune tolerance [\u003ca href=\"#_ENREF_14\"\u003e14\u003c/a\u003e, \u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eWith regards to the importance of TGF-\u0026beta; role in MS pathogenesis, in this experiment, we evaluated the gene expression level of two relevant isoforms (\u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e) as well as receptors (\u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e) 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.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cp\u003e\u003cstrong\u003ePatient and control participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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 \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and cDNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 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\u0026rsquo;s protocol. The samples were subsequently reverse-transcribed into cDNA, using 3 \u0026micro;g total RNA and 250 \u0026mu;g oligo dT (MWG, Germany). The reaction was incubated at 70\u0026deg; 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\u0026deg; C and 80\u0026deg; C for 60-90 and 15 minutes, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative reverse transcriptase PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA for each sample was used to evaluate the mRNA expression level of \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e genes using relative quantitative reverse transcriptase PCR (qRT-PCR). In this experiment, glyceraldehyde3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) was utilized as housekeeping gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEach qRT-PCR reaction was performed in a final volume of 20 \u0026micro;l, using 10 ng cDNA, 2x SYBR Green I master mix (Takara, Shiga, Japan) and appropriate primer pair set (Table S1). Thermal condition, as one-step RT-PCR, was carried out by an initial step at 95\u0026deg; C for 15 minutes, followed by 40 cycles at 95\u0026deg; C, 60\u0026deg; C and 72\u0026deg; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative quantification data analysis was performed using arbitrary method (\u0026Delta;Ct). The significance 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\u0026rsquo;s correlation coefficient. A P-value of 0.05 was set as significant threshold.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn 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\u0026ndash;45 years of age, with a female to male ratio of 25:11. The demographic characteristics of patients are given in the Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analyses in MS patients compared to the controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFindings showed that the \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e mRNA level mean was significantly increased in the PBMCs obtained from the controls compared to MS patients (2.25 times, p= 0.025). However, no significant difference was observed for \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e mRNA expression levels (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the relationship between the expression levels of genes \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the correlation between mean mRNA levels of the analyzed genes. There was a significant positive association between \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e mRNA level mean and \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, or \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e in MS patients, but no significant correlation was observed between the other genes mRNA expression level mean (Table 2).\u003c/p\u003e\n\u003cp\u003eMoreover, we observed no significant correlation between the normalized expression level of \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e or \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e genes and disease length, EDSS scores, or age in MS patients. In contrast, data analysis showed a significant positive correlation between \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e mRNA expression level and disease length in the MS patients (r= 0.356, p= 0.006).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTGF-\u0026beta; isoforms (TGF-\u0026beta;1, -\u0026beta;2, and -\u0026beta;3) 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-\u0026beta; isoforms and their receptors expression may be useful in determining different responses to the immunomodulatory as well as combinatory therapies. Curiously, TGF-\u0026beta; isoforms are important due to their conservation among vertebrates and their different roles in a variety of human diseases including tissue fibrosis, cancer and MS [\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e]. CNS degeneration and inflammation are important causes of demyelination in MS [\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e]. In 1998, Vincent et al. compared expression of TGF-\u0026beta;-R1 and TGF-\u0026beta;-R2 by immunohistochemistry in the brain tissue of MS patients and normal controls. They determined that TGF-\u0026beta; isoforms were expressed in microglia cells of the normal individuals\u0026rsquo; brain tissue. They also demonstrated that the TGF-\u0026beta; isoforms and relevant receptors are expressed in MS lesions [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eExpression profiling of the MS patients\u0026rsquo; peripheral blood showed misregulation of SMAD4, SMAD7 and TGF-\u0026beta;-R2. These finding revealed that TGF-\u0026beta; regulation is reduced in MS patients [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. Here, we demonstrated no significant difference in \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2 \u003c/em\u003emRNA expression levels in the MS patients compared to controls, while \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e expression level was 2.25 times higher in normal subjects compared to the MS patients.\u003c/p\u003e\n\u003cp\u003eSeveral investigations have so far represented difference of \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e, TGF-\u0026beta;-R1 and TGF-\u0026beta;-R2 mRNA levels in vitro and in vivo. TGF-\u0026beta; 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 [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e]. Peress et al. have previously examined glial TGF-\u0026beta; expression of different isoforms in 14 MS patients. Active lesions illustrated TGF-\u0026beta;2 immuno-reactivity of lesion encircling ramified microglia. In contrary, all three isoforms of this protein were expressed in the astrocytes of active white matter lesions. These results proposed that TGF-\u0026beta; cytokines could be locally expressed in demyelinated cells [\u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eFurther investigations showed that induction of rTGF-\u0026beta;1 suppressed IFN-\u0026gamma;, IL-4, IL-6, TNF-\u0026alpha; and perforin up-regulation in MS, but it had no effects on IL-10 or TGF-\u0026beta; expression. The selective prohibitory effects of TGF-\u0026beta;1 on pro-inflammatory cytokines expression potentially turn it into an attractive treatment in MS disease [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eMS patients with no or slight disability showed high level of \u003cem\u003eTGF-\u0026beta;\u003c/em\u003e mRNA expression, while moderate or severe disability of MS patients was correlated with high level of IFN-\u0026sigma;\u0026ndash;positive cells. Therefore, TGF-\u0026beta; and IFN-\u0026sigma; could have dissenting effects on MS pathology. Thus, administration of IFN-\u0026sigma; inhibitors and/or TGF-\u0026beta; activators might improve MS disease treatment [\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThese differences between our results and others may be due to environment factors including Vitamin D [\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e] or other drugs that patients use. We formerly studied \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e expression in 32 patients with MS and 32 healthy controls. Our findings showed no significant change of \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e expression in the MS patients compared to control group [\u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e], suggesting that large sample size could not affect gene regulation level.\u003c/p\u003e\n\u003cp\u003eAdditionally, this study evaluated the correlation between these four gene expressions in MS patients. We detected a positive link only between the expression of \u003cem\u003eTGF-\u0026beta;\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e as well as the expression of \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e in MS patients; however, there was no significant association between the expressions of other genes. It is proposed that TGF-\u0026beta;-R1 might influence on \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e and \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e expression by phosphorylating the receptor-specific SMADs and promoting a feedback mechanism. Altogether, expression level of TGF-\u0026beta;-R1 could be considered as a risk factor for MS disease which might be associated with inflammatory events in such patients.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eInvestigations have previously implicated that heterogeneous nature of whole-blood samples may not show the optimal level of gene expressions [\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e, \u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e], revealing some limitation in this study. Hence, assessment of gene expression at protein level MS patients\u0026rsquo; sorted PBMCs could help to better understand the role of TGF-\u0026beta;.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eMS:\u003c/strong\u003e Multiple sclerosis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCNS:\u003c/strong\u003e central nervous system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBMCs:\u003c/strong\u003e peripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDSS:\u003c/strong\u003e expanded disability status scale\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF-\u0026beta;:\u003c/strong\u003e transforming growth factor-\u0026beta;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF-\u0026beta;-R1:\u003c/strong\u003e transforming growth factor-\u0026beta; receptor 1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was in accordance with the declaration of Helsinki. This study was approved by the Ethics Committee of Tarbiat Modares University. The informed consent was obtained from all the participants, and informed consent obtained was written.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.SHF., M.B.; Performed all experiments, analyzed the data and wrote the manuscript. M.A.S.; Contributed to concept and design, manufactured the samples, and final approval of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge contribution of the patients and institutions in this study as well as Roya Amirinejad and Fahimeh Molaahmadi for their valuable laboratory supports. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors, other than Department of Research Affairs of Tarbiat Modares University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBhise V, Dhib-Jalbut S: \u003cstrong\u003eFurther understanding of the immunopathology of multiple sclerosis: impact on future treatments\u003c/strong\u003e. \u003cem\u003eExpert review of clinical immunology \u003c/em\u003e2016, \u003cstrong\u003e12\u003c/strong\u003e(10):1069-1089.\u003c/li\u003e\n\u003cli\u003eTouil H, Kobert A, Lebeurrier N, Rieger A, Saikali P, Lambert C, Fawaz L, Moore CS, Prat A, Gommerman J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eHuman central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis\u003c/strong\u003e. \u003cem\u003eJournal of neuroinflammation \u003c/em\u003e2018, \u003cstrong\u003e15\u003c/strong\u003e(1):114.\u003c/li\u003e\n\u003cli\u003eTian Z, Song Y, Yao Y, Guo J, Gong Z, Wang Z: \u003cstrong\u003eGenetic Etiology Shared by Multiple Sclerosis and Ischemic Stroke\u003c/strong\u003e. \u003cem\u003eFrontiers in genetics \u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e:646.\u003c/li\u003e\n\u003cli\u003eBaecher-Allan C, Kaskow BJ, Weiner HL: \u003cstrong\u003eMultiple Sclerosis: Mechanisms and Immunotherapy\u003c/strong\u003e. \u003cem\u003eNeuron \u003c/em\u003e2018, \u003cstrong\u003e97\u003c/strong\u003e(4):742-768.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-Cerd\u0026aacute; F, S\u0026aacute;nchez-G\u0026oacute;mez MV, Matute C: \u003cstrong\u003eThe link of inflammation and neurodegeneration in progressive multiple sclerosis\u003c/strong\u003e. \u003cem\u003eMultiple Sclerosis and Demyelinating Disorders \u003c/em\u003e2016, \u003cstrong\u003e1\u003c/strong\u003e(1):9.\u003c/li\u003e\n\u003cli\u003eNali LH, Olival GS, Sousa FTG, de Oliveira ACS, Montenegro H, da Silva IT, Dias-Neto E, Naya H, Spangenberg L, Penalva-de-Oliveira AC\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eWhole transcriptome analysis of multiple Sclerosis patients reveals active inflammatory profile in relapsing patients and downregulation of neurological repair pathways in secondary progressive cases\u003c/strong\u003e. \u003cem\u003eMultiple Sclerosis and Related Disorders \u003c/em\u003e2020, \u003cstrong\u003e44\u003c/strong\u003e:102243.\u003c/li\u003e\n\u003cli\u003eKomai T, Inoue M, Okamura T, Morita K, Iwasaki Y, Sumitomo S, Shoda H, Yamamoto K, Fujio K: \u003cstrong\u003eTransforming Growth Factor-\u0026beta; and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals\u003c/strong\u003e. \u003cem\u003eFrontiers in Immunology \u003c/em\u003e2018, \u003cstrong\u003e9\u003c/strong\u003e(1364).\u003c/li\u003e\n\u003cli\u003eSanjabi S, Oh SA, Li MO: \u003cstrong\u003eRegulation of the Immune Response by TGF-\u0026beta;: From Conception to Autoimmunity and Infection\u003c/strong\u003e. \u003cem\u003eCold Spring Harbor Perspectives in Biology \u003c/em\u003e2017, \u003cstrong\u003e9\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eLee PW, Severin ME, Lovett-Racke AE: \u003cstrong\u003eTGF-\u0026beta; regulation of encephalitogenic and regulatory T cells in multiple sclerosis\u003c/strong\u003e. \u003cem\u003eEuropean Journal of Immunology \u003c/em\u003e2017, \u003cstrong\u003e47\u003c/strong\u003e(3):446-453.\u003c/li\u003e\n\u003cli\u003eKashima R, Hata A: \u003cstrong\u003eThe role of TGF-\u0026beta; superfamily signaling in neurological disorders\u003c/strong\u003e. \u003cem\u003eActa Biochimica et Biophysica Sinica \u003c/em\u003e2017, \u003cstrong\u003e50\u003c/strong\u003e(1):106-120.\u003c/li\u003e\n\u003cli\u003eVoisin A, Damon-Soubeyrand C, Bravard S, Saez F, Drevet JR, Guiton R: \u003cstrong\u003eDifferential expression and localisation of TGF-\u0026beta; isoforms and receptors in the murine epididymis\u003c/strong\u003e. \u003cem\u003eScientific reports \u003c/em\u003e2020, \u003cstrong\u003e10\u003c/strong\u003e(1):995.\u003c/li\u003e\n\u003cli\u003eCompston A, Lassmann H, Smith K: \u003cstrong\u003eChapter 10 - The neurobiology of multiple sclerosis\u003c/strong\u003e. 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Edinburgh: Churchill Livingstone; 2006: 449-490.\u003c/li\u003e\n\u003cli\u003eHata A, Chen Y-G: \u003cstrong\u003eTGF-\u0026beta; Signaling from Receptors to Smads\u003c/strong\u003e. \u003cem\u003eCold Spring Harbor Perspectives in Biology \u003c/em\u003e2016, \u003cstrong\u003e8\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eXu J, Wang Y, Jiang H, Sun M, Gao J, Xie A: \u003cstrong\u003eTGF-\u0026beta; in Mice Ameliorates Experimental Autoimmune Encephalomyelitis in Regulating NK Cell Activity\u003c/strong\u003e. \u003cem\u003eCell transplantation \u003c/em\u003e2019, \u003cstrong\u003e28\u003c/strong\u003e(9-10):1155-1160.\u003c/li\u003e\n\u003cli\u003eNataf S, Guillen M, Pays L: \u003cstrong\u003eTGFB1-Mediated Gliosis in Multiple Sclerosis Spinal Cords Is Favored by the Regionalized Expression of HOXA5 and the Age-Dependent Decline in Androgen Receptor Ligands\u003c/strong\u003e. \u003cem\u003eInt J Mol Sci \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e(23).\u003c/li\u003e\n\u003cli\u003eZhang X, Hamblin MH, Yin KJ: \u003cstrong\u003eThe long noncoding RNA Malat1: Its physiological and pathophysiological functions\u003c/strong\u003e. \u003cem\u003eRNA biology \u003c/em\u003e2017, \u003cstrong\u003e14\u003c/strong\u003e(12):1705-1714.\u003c/li\u003e\n\u003cli\u003eVincent V, Tilders F, Van Dam A: \u003cstrong\u003eProduction, regulation and role of nitric oxide in glial cells\u003c/strong\u003e. \u003cem\u003eMediators of inflammation \u003c/em\u003e1998, \u003cstrong\u003e7\u003c/strong\u003e(4):239.\u003c/li\u003e\n\u003cli\u003eMeoli EM, Oh U, Grant CW, Jacobson S: \u003cstrong\u003eTGF-\u0026beta; signaling is altered in the peripheral blood of subjects with multiple sclerosis\u003c/strong\u003e. \u003cem\u003eJournal of neuroimmunology \u003c/em\u003e2011, \u003cstrong\u003e230\u003c/strong\u003e(1):164-168.\u003c/li\u003e\n\u003cli\u003eAteke Mousavi N-k, Abbas M, Abdorreza Naser M, Mohammad Reza S-S, Mohammad Reza E, Maryam S, Mina A, Ali Akbar S-Y: \u003cstrong\u003eComparison of Cytokine Expression in Multiple Sclerosis Patients and Healthy Volunteers\u003c/strong\u003e. \u003cem\u003eActa Medica Iranica \u003c/em\u003e2018, \u003cstrong\u003e56\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003ePeress NS, Perillo E, Seidman RJ: \u003cstrong\u003eGlial transforming growth factor (TGF)-\u0026beta; isotypes in multiple sclerosis: differential glial expression of TGF-\u0026beta;1, 2 and 3 isotypes in multiple sclerosis\u003c/strong\u003e. \u003cem\u003eJournal of neuroimmunology \u003c/em\u003e1996, \u003cstrong\u003e71\u003c/strong\u003e(1):115-123.\u003c/li\u003e\n\u003cli\u003eAraujo GR, Aglas L, Vaz ER, Machado Y, Huber S, Himly M, Duschl A, Goulart LR, Ferreira F: \u003cstrong\u003eTGF\u0026beta;1 mimetic peptide modulates immune response to grass pollen allergens in mice\u003c/strong\u003e. \u003cem\u003eAllergy \u003c/em\u003e2020, \u003cstrong\u003e75\u003c/strong\u003e(4):882-891.\u003c/li\u003e\n\u003cli\u003eLink J, S\u0026ouml;derstr\u0026ouml;m M, Olsson T, H\u0026ouml;jeberg B, Ljungdahl \u0026Aring;, Link H: \u003cstrong\u003eIncreased transforming growth factor‐\u0026beta;, interleukin‐4, and interferon‐\u0026gamma; in multiple sclerosis\u003c/strong\u003e. \u003cem\u003eAnnals of neurology \u003c/em\u003e1994, \u003cstrong\u003e36\u003c/strong\u003e(3):379-386.\u003c/li\u003e\n\u003cli\u003eShirvani-Farsani Z, Behmanesh M, Mohammadi SM, Naser Moghadasi A: \u003cstrong\u003eVitamin D levels in multiple sclerosis patients: Association with TGF-\u0026beta;2, TGF-\u0026beta;RI, and TGF-\u0026beta;RII expression\u003c/strong\u003e. \u003cem\u003eLife Sciences \u003c/em\u003e2015, \u003cstrong\u003e134\u003c/strong\u003e:63-67.\u003c/li\u003e\n\u003cli\u003eShirvani-Farsani Z, Behmanesh M, Sahraian MA: \u003cstrong\u003eInterleukin-10 but not transforming growth factor-\u0026beta;1 gene expression is up-regulated by vitamin D treatment in multiple sclerosis patients\u003c/strong\u003e. \u003cem\u003eJournal of the Neurological Sciences \u003c/em\u003e2015, \u003cstrong\u003e350\u003c/strong\u003e(1):18-23.\u003c/li\u003e\n\u003cli\u003ePahlevan Kakhki M, Rakhshi N, Heidary M, Behmanesh M, Nikravesh A: \u003cstrong\u003eExpression of suppressor of cytokine signaling 1 (SOCS1) gene dramatically increases in relapsing-remitting multiple sclerosis\u003c/strong\u003e. \u003cem\u003eJ Neurol Sci \u003c/em\u003e2015, \u003cstrong\u003e350\u003c/strong\u003e(1-2):40-45.\u003c/li\u003e\n\u003cli\u003eHeidary M, Rakhshi N, Pahlevan Kakhki M, Behmanesh M, Sanati MH, Sanadgol N, Kamaladini H, Nikravesh A: \u003cstrong\u003eThe analysis of correlation between IL-1B gene expression and genotyping in multiple sclerosis patients\u003c/strong\u003e. \u003cem\u003eJ Neurol Sci \u003c/em\u003e2014, \u003cstrong\u003e343\u003c/strong\u003e(1-2):41-45.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Demographic and disease characteristics of the MS patients and controls.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003eMS Patient\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003eNumber\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003e31\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003eAge (mean years \u0026plusmn; SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003e32.54\u0026plusmn;8.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003e29.03\u0026plusmn;6.81\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003eRR/SP/PP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003e55/6/0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003eEDSS (mean \u0026plusmn; SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003e2.45\u0026plusmn;1.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"206\"\u003e\n\u003cp\u003eDisease Duration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"161\"\u003e\n\u003cp\u003e6.42\u0026plusmn;3.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Correlation between normalized \u003cem\u003eTGF-\u0026beta;1\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;2\u003c/em\u003e, \u003cem\u003eTGF-\u0026beta;-R1\u003c/em\u003e or \u003cem\u003eTGF-\u0026beta;-R2\u003c/em\u003e expression level in MS patients.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003ecorrelation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003er\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta;1-TGF-\u0026beta;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.077\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e0.551\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta;1-TGF-\u0026beta; RI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.292\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.023\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta;1-TGF-\u0026beta; RII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.164\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e0.216\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta;2-TGF-\u0026beta; RI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.477\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.0001\u003csup\u003e***\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta;2-TGF-\u0026beta; RII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.010\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e0.9939\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTGF-\u0026beta; RI-TGF-\u0026beta; RII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.194\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e0.143\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*significant p-value \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003e***significant p-value \u0026lt; 0.001\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TGF-β Pathway, TGF- β receptors, Multiple Sclerosis, Expression Analysis, Real-Time PCR","lastPublishedDoi":"10.21203/rs.3.rs-145981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-145981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), depicted by lymphocytic infiltration and demyelination. MS is associated with the up-regulation of pro-inflammatory and down-regulation of anti-inflammatory 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Our findings indicated that the TGF-β-R1 expression level was 2.25 times higher in controls than MS patients. Also, a significant 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.\u003c/p\u003e","manuscriptTitle":"TGF-β Isoforms and Receptors: A Gene Expression Analysis in Multiple Sclerosis Patients and Normal Individuals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-19 23:43:46","doi":"10.21203/rs.3.rs-145981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-05-11T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-05-11T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-04-15T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-04-12T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-04-12T00:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-12T16:08:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-11T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-11-30T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-11-29T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3daa9779-40fc-4d80-91f4-90929a73d153","owner":[],"postedDate":"January 19th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1914161,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-01-19T23:43:46+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-19 23:43:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-145981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-145981","identity":"rs-145981","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}