Effects of β-D-mannuronic acid, as a novel non-steroidal anti-inflammatory medication within immunosuppressive properties, on IL17, RORγt, IL4 and GATA3 gene expressions in rheumatoid arthritis patients
Authors Barati A, Jamshidi AR, Ahmadi H, Aghazadeh Z, Mirshafiey A
Received 4 December 2016
Accepted for publication 15 February 2017
Published 30 March 2017 Volume 2017:11 Pages 1027—1033
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Rasika Samarasinghe
Peer reviewer comments 3
Editor who approved publication: Dr Anastasios Lymperopoulos
Anis Barati,1 Ahmad Reza Jamshidi,2,* Hossein Ahmadi,1 Zahra Aghazadeh,1 Abbas Mirshafiey1,*
1Department of Immunology, School of Public Health, 2Iranian Institute for Health Sciences Research, Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
*These authors contributed equally to this work
Abstract: Rheumatoid arthritis (RA) is the most common form of chronic inflammatory arthritis characterized by pain, swelling and destruction of joints, with a resultant disability. Disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs can interfere with the disease process. In this study, the effect of β-D-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive and anti-inflammatory effects together with antioxidant effects was evaluated on IL17, RORγt, IL4 and GATA3 gene expression in 12 RA patients. Previously, M2000 driven from sodium alginate (natural product; patented, DEU: 102016113018.4) has shown a notable efficacy in experimental models of multiple sclerosis, RA and nephrotic syndrome. This study was performed on 12 patients with RA who had an inadequate response to conventional treatments. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. M2000 was administered orally at a dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected before and after treatment to evaluate the expression levels of IL4, GATA3, IL17 and RORγt. The gene expression results showed that M2000 has a potent efficacy, so that it could not only significantly decrease IL17 and RORγt levels but also increase IL4 and GATA3 levels after 12 weeks of treatment. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments. In conclusion, M2000 as a natural novel agent has therapeutic and immunosuppressive properties on RA patients (identifier: IRCT2014011213739N2).
Keywords: M2000, IL17, RORγt, rheumatoid arthritis, inflammation, immunosuppressive
Alginic acid as a biomaterial has been successfully used in biomedical science, bioengineering and especially pharmaceutical application for over 40 years without any significant side effects.1 It has been applied in drug delivery systems such as controlled release, disintegrant, film former, thickening and stabilization.2 Moreover, the positive effect, safety and efficacy of sodium alginate on gastric reflux control and wound care have been completely proved.3 Accordingly, its comonomer β-D-mannuronic acid (M2000), patented (DEU: 102016113018.4), has been shown to have significant therapeutic and anti-inflammatory properties in several experimental models.4 Since sodium alginate comes from a natural polysaccharide (brown seaweeds), its comonomers have been shown to exhibit sufficient safety in pharmaceutical ingredients.1 In our previous animal experimental models, M2000 has been shown to have notable anti-inflammatory and immunosuppressive effects on multiple sclerosis, nephrotic syndrome, rheumatoid arthritis (RA) and immune complex glomerulonephritis.5–9 Prior studies have found some characterized molecular mechanisms of this novel drug, including inhibitory effects on the activity of matrix metalloproteinase-2, inhibition of infiltration of immune cells and reduction of IL-6 and antibody production.10 In the present clinical study, for the first time, we evaluated the effects of M2000 on RA patients since its administration has been reported to be completely safe in animal models.11
RA is the most common autoimmune and systemic inflammatory disease characterized by inflammation and deformity of synovial joints, leading to bone and cartilage erosion. There are some common symptoms in RA such as fatigue, low-grade fever, malaise, morning stiffness, headache, loss of appetite and loss of weight.12
Considering the molecular and pathological basis of immune-mediated chronic inflammatory disease, there is an interest in approaching to an effective therapy in RA. T-cell has a great role in RA disease; association of human leukocyte antigen (HLA)-DR known as an MHC class II cell surface receptor assigns a great proof that CD4+ T-cells (Th2) are involved in the development of the disease.13 Among the other types of T-cells, extensive evidence has shown that the subset of proinflammatory T helper cells such as TH17 play a notable role in the progression of RA.14 Th2 cytokines, especially IL4, may play an important role in anti-inflammatory effects. GATA3 as its transcription factor could strongly mediate the enhancement of IL4 promoter and regulate IL4 gene expression.15 Hereupon, an appropriate RA treatment should meet the increase in GATA3 and IL4 levels. Another transcription factor RORγt is required for the differentiation of Th17 cells and expression of IL17. Afterward, IL17-producing CD4+ Th17 cells, as a new subclass of cytokines with highly proinflammatory properties, could have a potential mechanism and role in the autoimmune diseases such as RA. Therefore, a treatment that is able to attenuate the levels of RORγt and IL17 could be able to control the progression of RA.16
Currently, five lines of therapy are used in order to control the progression and symptoms of RA in patients, including disease-modifying anti-rheumatic drugs (DMARDs), biologic response modifiers (a type of DMARD), glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics (painkillers). All these medications help patients to attenuate symptoms and inflammation; however, in rare cases, the disease can go into the remission phase. On the other side, overuse of these medications can cause serious adverse effects, including damaging blood cells, liver, lungs, kidney and gastrointestinal tract, increasing the risk of some cancers, osteoporosis and muscle weakness.17–19 Therefore, not only is an effective treatment an essential target in medicine, but overcoming the side effects is also another substantial challenge in this area. Since RA itself is a painful and frustrating condition, it would indeed be difficult to struggle with the side effects of drugs. In this study, based on our previous approaches, M2000 as a natural medicine and novel designed NSAID with in immunosuppressive properties in T-cell-mediated autoimmune diseases was used for those RA patients who had failed response to the other therapies. Afterward, the effect of M2000 on these RA patients was figured out by the changes in the relative gene expression of IL4, GATA3, IL17 and RORγt since these genes have a great key role in autoimmune diseases such as RA.
Materials and methods
The study was approved by the ethics committee of Tehran University of Medical Sciences (TUMS) and was conducted under guidelines established by the American College of Rheumatology (ACR) and Helsinki manifest and its later amendments or comparable ethical standards. Written informed consent was obtained from all patients.
M2000 preparation and intake
M2000 as a small molecule (C6H10O7) with the molecular weight of 194.139 Da was prepared from sodium alginate (Sigma-Aldrich, St Louis, MO, USA) based on the method of Fattahi et al.11 Subsequently, the purity of M2000 was validated by characterizing the hydrolytic products using Fourier transform infrared (FTIR) spectroscopy and carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy.
Clinical characterization of patients and control
In this study, 12 patients with acute RA who had inadequate response to conventional drugs were selected for 12 weeks clinical trial. The mean age of the selected patients (females 10, males two) was 45±14.54 years, and the range of disease duration was 1–15 years. Treatment of these patients with M2000 based on the ACR criteria for RA was started on 16 May 2014 (identifier: IRCT2014011213739N2). At the baseline, although all patients were treated with DMARDs, steroids and TNF-α antagonists, the disease score (disease activity score in 28 joints [DAS28]) was quite high (Table 1). Prior to the enrollment of this clinical trial, the patients were informed of this study and asked to sign an informed consent. Afterwards, the patients received the follow-up clinic appointment at baseline, 4 weeks and 12 weeks at the Department of Rheumatology, Shariati University Hospital, Tehran, Iran, and the Division of Rheumatology Research, Rheumatism Center. Further follow-up was arranged by telephone for assessing the adverse effects of M2000 every week.
During this clinical trial, patients were allowed to use their routine medications, which included methotrexate (15–20 mg weekly), hydroxychloroquine (400 mg daily) and steroids (5–15 mg daily), and also seven of 12 patients received a subcutaneous injection of etanercept (25 mg twice weekly). However, patients were prohibited from using NSAIDs or other pharmacologic treatment during this 12-week follow-up. Based on the preclinical assessment, a minimum dosage (18 mg/kg/d) of M2000 was provided in a gelatinized capsule (500 mg of M2000) for oral administration. Finally, the M2000 capsule (500 mg) was prescribed twice daily for 12 weeks. Moreover, in this trial, we had 12 participants (nine females and three males) who had only used DMARDs without M2000 as the control (conventional) group, and also 12 healthy subjects (nine females and three males) without any background disease were selected as a normal group for immunological assessment.
Blood was obtained from normal donors and patients before and after treatment. Afterward, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and stored at −80°C. Total RNA was extracted from 2×106–5×106 cells using GeneAll® Hybrid-RTM kits (Qiagen, Valencia, CA, USA) based on manufacturer’s instructions and placed into 50 μL of RNase-free water. The concentration of total RNA was measured by NanoDrop 2000 UV–Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). It was then concentrated or diluted to the concentration <300 ng/μL for cDNA synthesis.
Quantitative real-time polymerase chain reaction (PCR)
Reverse-transcribed random hexamer primers were used for conventional PCR based on cDNA Synthesis Kit protocol (Takara Co., Ltd., Dalian, China). Quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ II (Takara Co., Ltd.) with specific primer (Sigma-Aldrich; Table 2) based on the provided guideline. The analysis of IL4, GATA3, IL17, RORγt and β-actin transcripts was carried out in StepOne™ and StepOnePlus™ Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). The relative quantities of target gene mRNA compared against one internal control, β-actin mRNA, were measured by following a ΔCT method using an amplification plot (fluorescence signal vs cycle number). The difference (ΔCT) between the mean values in the replicate samples of target genes and β-actin mRNA was calculated. Subsequently, the difference (ΔΔCT) between changes in the expression of target genes and the normal group was calculated and expressed as 2−ΔΔCT.
Table 2 IL4, GATA3, IL17, RORγt and β-actin real-time PCR primers
Data were representative of three independent experiments. The data were expressed as mean ± standard deviation, and the analysis was performed by SPSS software (19.0; IBM Corporation, Armonk, NY, USA). Moreover, the parametric data were subjected to analysis of variance (ANOVA) and the Newman–Keuls test to determine significant differences in the gene expression level of before and after treatment. The statistical significance was classified as *P<0.05, **P<0.01 or ***P<0.001. A P-value of <0.05 was considered as statistically significant.
The improvement in the patient status was observed after 2 weeks and also continued during the treatment course. The mean of disease activity (DAS28), morning stiffness, rheumatoid factor (RF) and C-reactive protein (CRP) had met a significant reduction after 12 weeks of treatment (Table 1). In addition, improvement was observed in the other clinical laboratory tests, including anti-CCP, anti-dsDNA and ESR levels, which returned to the normal range.
Effects of M2000 on IL4, GATA3, IL17 and RORγt gene expressions
The results represented that after 12 weeks therapy with M2000, IL17 and RORγt gene expressions in patient’s, PBMCs were decreased by 22.39- and 2.36-fold, respectively, in comparison to the gene expressions of the patients before therapy. As shown in Figures 1 and 2, after treatment, IL17 and RORγt fold changes were 1.7 and 1.03, respectively, which were approximately near to the normal range. Furthermore, the statistical analyses revealed that the differences between the control group and other groups were significant (P=0.0001 and P=0.034, respectively).
On the other side, the IL4 and GATA3 gene expressions in patients’ PBMCs were increased by 0.92- and 0.48-fold, respectively, in comparison to the gene expressions of the patients before therapy. Furthermore, in the normal group, the fold change was 1.9 and 1 for IL4 and GATA3, respectively. However, the statistical analyses revealed that the differences between control group and other groups for IL4 and GATA3 (Figures 3 and 4) were not significant (P=0.165 and P=0.084, respectively). The results also demonstrated that although they had a reduction in the expression level, it was not sufficient.
In RA, the immune system is misdirected and attacks the joints. This misdirecting could promote inflammation not only in joints but also in other various organs and tissues of body, occasionally.20 Generally, the conventional DMARDs and TNF-α blockers were used to reduce both disease activity and progression in patients.21,22 However, some patients do not respond to these treatments and present a diminished response to them overtime. Furthermore, RA patients are always suffering from the adverse effects of these chemicals and biological drugs.23 Therefore, M2000 as a novel and natural anti-inflammatory agent was used during 3 months clinical trial and showed a suitable response in the proposed gene expression and clinical and paraclinical results.
In RA, autoimmune response is involved by immune cells, including various white blood cells, such as B- and T-cells, neutrophils, dendritic cells, macrophages and mast cells, which focus on the joint cavity. These cells release a wide range of chemical agents such as inflammatory mediators, cytokines, leukotrienes and prostaglandins.24–26 These chemicals develop the growth of blood vessels, which supply nutrients for the growth of leukocytes and fibrous tissues in synovial cartilage.27 Among these chemical mediators, IL4 and IL17 as T-cell cytokines and their transcription factors GATA3 and RORγt have a critical role in RA.15,16 Our presented data indicate that IL4 and its transcription factor (GATA3) are expressed at a low level in the peripheral blood cells of RA patients before treatment. IL4 has anti-inflammatory property and disease-restricting role. The potential function of IL4 level in RA patients using antibody-based delivery of IL4 has been reported. On the other hand, it has been illustrated that the lack of IL4 does not appear to affect the activation of autoreactive T-cells and also could display various courses of disease, including no arthritis, transient mild arthritis and severe arthritis.15 There are two possible explanations for this variability; first, IL4 could decrease the collaboration of T-cells and B-cells, and the other could refer to the other possible cytokines such as IL13 that may lead IL4 function to a level that appears to change the disease condition.15,28 Therefore, the reason why in this experiment induction of IL4 and GATA3 gene expressions was not significant might be due to the abovementioned reason.
Our results demonstrated that IL17 and its transcription factor (RORγt) showed a significant reduction in PBMCs. Previous evidence has indicated the critical role of IL17 in RA and that suitable therapies are able to reduce its level. Since the increase of IL17 production and upregulation of Th17 cells which characterized by expression of the RORγt are the most common features of RA.26 Regarding the previous studies, IL17-producing CD4+ T (Th17) cells as a unique T-helper, which is known as T-cell-mediated tissue injury, could promote inflammation by proinflammatory cytokines and chemokines in autoimmune disease.29–31 In the present study, the IL17 level after treatment with M2000 significantly decreased 22.39-fold the level of these cytokines compared to the before treatment group. The results of this trial are in agreement with the other studies that reported the potential role of IL17 in mediating joint damage and the ability of IL17 to induce collagen release from cartilage.31,32 In this study, there were significant correlations between the gene expression results and clinical and paraclinical assessments. The levels of these cytokines were in accordance with the disease activity (DAS28), tender joint and swelling, all of which showed a reduction in mean after treatment. Moreover, the range of ESR and CRP, which was quite high before M2000, faced a significant reduction and back to the normal range.
This clinical trial revealed potent anti-inflammatory and immunosuppressive properties of M2000 in 12 RA patients. During this trial, due to the good response of patients to this natural agent, the rheumatologist started to reduce the corticosteroid dosage, and at the end of 3 months, the intake dosage of corticosteroid was decreased to the half. In addition, etanercept injection was eliminated in patients.
Finally, based on the clinical assessment and genes expression results, it can be concluded that oral administration of M2000 as a novel NSAID with immunosuppressive property and a natural agent could help in the process of RA treatment. Moreover, this finding brings a perspective view for further trials with a higher number of patients and also other areas in autoimmune diseases.
The authors would like to thank nurses and staff of Rheumatology Department of Shariati University Hospital of TUMS. This study was not funded by any organization or person.
The authors report no conflicts of interest in this work.
Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012;37(1):106–126.
Tønnesen HH, Karlsen J. Alginate in drug delivery systems. Drug Dev Ind Pharm. 2002;28(6):621–630.
Savarino E, Pohl D, Zentilin P, et al. Functional heartburn has more in common with functional dyspepsia than with non-erosive reflux disease. Gut. 2009;58(9):1185–1191.
Mirshafiey A, Rehm BH. Alginate and its comonomer mannuronic acid: medical relevance as drugs. In: Rehm BH, editor. Alginates: Biology and Applications. Berlin Heidelberg: Springer; 2009:229–260.
Mirshafiey A, Rehm B, Abhari RS, Borzooy Z, Sotoude M, Razavi A. Production of M2000 (β-d-mannuronic acid) and its therapeutic effect on experimental nephritis. Environ Toxicol Pharmacol. 2007;24(1):60–66.
Mirshafiey A, Rehm B, Sotoude M, Razavi A, Abhari RS, Borzooy Z. Therapeutic approach by a novel designed anti-inflammatory drug, M2000, in experimental immune complex glomerulonephritis. Immunopharmacol Immunotoxicol. 2007;29(1):49–61.
Mirshafiey A, Cuzzocrea S, Rehm B, Matsuo H. M2000: a revolution in pharmacology. Med Sci Monit. 2005;11(8):I53–I63.
Mirshafiey A, Matsuo H, Nakane S, Rehm BH, Koh CS, Miyoshi S. Novel immunosuppressive therapy by M2000 in experimental multiple sclerosis. Immunopharmacol Immunotoxicol. 2005;27(2):255–265.
Mirshafiey A, Cuzzocrea S, Rehm B, Mazzon E, Saadat F, Sotoude M. Treatment of experimental arthritis with M2000, a novel designed non-steroidal anti-inflammatory drug. Scand J Immunol. 2005;61(5):435–441.
Mirshafiey A, Khorramizadeh MR, Saadat F, Rehm BHA. Chemopreventive effect of M2000, a new anti-inflammatory agent. Med Sci Monit. 2004;10(10):I105–I109.
Fattahi MJ, Abdollahi M, Agha Mohammadi A, et al. Preclinical assessment of β-d-mannuronic acid (M2000) as a non-steroidal anti-inflammatory drug. Immunopharmacol Immunotoxicol. 2015;37(6):535–540.
Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31(3):315–324.
Cope A, Schulze-Koops H, Aringer M. The central role of T cells in rheumatoid arthritis. Clin Exp Rheumatol. 2007;25(5):S4.
Sarkar S, Cooney L, Fox DA. The role of T helper type 17 cells in inflammatory arthritis. Clin Exp Rheumatol. 2010;159(3):225–237.
Ohmura K, Nguyen LT, Locksley RM, Mathis D, Benoist C. Interleukin-4 can be a key positive regulator of inflammatory arthritis. Arthritis Rheum. 2005;52(6):1866–1875.
Gaffen SL. The role of interleukin-17 in the pathogenesis of rheumatoid arthritis. Curr Rheumatol Rep. 2009;11(5):365–370.
Singh JA, Furst DE, Bharat A, et al. 2012 Update of the 2008 American College of Rheumatology recommendations for the use of disease-modifying antirheumatic drugs and biologic agents in the treatment of rheumatoid arthritis. Arthritis Care Res (Hoboken). 2012;64(5):625–639.
Gøtzsche PC, Johansen HK. Short-term low-dose corticosteroids vs placebo and nonsteroidal anti-inflammatory drugs in rheumatoid arthritis. Cochrane Library. 2005;1.
Laine L, Bombardier C, Hawkey CJ, et al. Stratifying the risk of NSAID-related upper gastrointestinal clinical events: results of a double-blind outcomes study in patients with rheumatoid arthritis. Gastroenterology. 2002;123(4):1006–1012.
Bingham CO. The pathogenesis of rheumatoid arthritis: pivotal cytokines involved in bone degradation and inflammation. J Rheumatol. 2002;65:3–9.
Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986–1000.
McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7(6):429–442.
McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365(23):2205–2219.
McKenzie GJ, Fallon PG, Emson CL, Grencis RK, McKenzie AN. Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2–mediated responses. J Exp Med. 1999;189(10):1565–1572.
Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev. 2008;223(1):87–113.
Miossec P. IL-17 and Th17 cells in human inflammatory diseases. Microbes Infect. 2009;11(5):625–630.
Al-Saadany HM, Hussein MS, Gaber RA, Zaytoun HA. Th-17 cells and serum IL-17 in rheumatoid arthritis patients: correlation with disease activity and severity. Egypt Rheumatol. 2016;38(1):1–7.
Koshy P, Henderson N, Logan C, Life PF, Cawston TE, Rowan AD. Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines. Ann Rheum Dis. 2002;61(8):704–713.
Peters TL, McClain KL, Allen CE. Neither IL-17A mRNA nor IL-17A protein are detectable in Langerhans cell histiocytosis lesions. Mol Ther. 2011;19(8):1433–1439.
Ratajewski M, Walczak-Drzewiecka A, Sałkowska A, Dastych J. Upstream stimulating factors regulate the expression of RORγT in human lymphocytes. J Immunol. 2012;189(6):3034–3042.
Kehrmann J, Tatura R, Zeschnigk M, et al. Impact of 5-aza-2′-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells. J Immunol. 2014;142(3):384–395.
Chiu YH, Chen H. GATA3 inhibits GCM1 activity and trophoblast cell invasion. Sci Rep. 2016;6:21630.
Nathan C, Ding A. Nonresolving inflammation. Cell. 2010;140(6):871–882.
Singh JA, Christensen R, Wells GA, et al. Biologics for rheumatoid arthritis: an overview of Cochrane reviews. Sao Paulo Med J. 2010;128(5):309–310.
Lee YH, Rho YH, Choi SJ, Ji JD, Song GG. Association of TNF-alpha–308 G/A polymorphism with responsiveness to TNF-α-blockers in rheumatoid arthritis: a meta-analysis. Rheumatol Int. 2006;27(2):157–161.
Day R. Adverse reactions to TNF-α inhibitors in rheumatoid arthritis. Lancet. 2002;359(9306):540–541.
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