Methotrexate Mechanism of Action in Plaque Psoriasis: Something New in the Old View

J Clin Aesthet Dermatol. 2022;15(8):42–46.

by Fatma Mohamed El-Esawy, MD; Inas A. Ahmed, MD;
Asmaa Adel El-Fallah, MD; and Rehab Mohammed Salem, MD

Drs. Salem and El-Esawy are with the Dermatology Department and Faculty of Medicine at Benha University, in Banha, Egypt. Dr. Ahmed is with Medical Biochemistry and Molecular Biology, Faculty of Medicine at Benha University in Banha, Egypt. Dr. El-Fallah is Assistant Professor of Clinical and Chemical Pathology with the Faculty of Medicine at Benha University in Banha, Egypt.

FUNDING: No funding was provided for this article.

DISCLOSURES: The authors report no conflicts of interest relevant to the content of this article.

ABSTRACT: Background. Despite the era of biologic therapy in the management of psoriasis, methotrexate, the traditional backbone of psoriasis treatment, does not stop surprising us with what it can offer.

Objective. In this study, we aimed to evaluate the peripheral expression and the serum levels of TWEAK in patients with psoriasis vulgaris before and after receiving methotrexate treatment.

Methods. The study included 58 patients with moderate to severe psoriasis vulgaris, and 90 apparently healthy individuals as a control group. Before starting the treatment course, all patients were evaluated clinically using Psoriasis Area Severity Index (PASI) score and were subjected to TWEAK serum levels and peripheral expression measurement using ELISA and qRT-PCR techniques, respectively. After 12 weeks of treatment with methotrexate (intramuscular methotrexate; up to 30mg per week) the patients were re-evaluated both clinically and in the laboratory.

Results. The baseline serum TWEAK levels and its peripheral mRNA expression in the patients group were significantly lower than those in the control group. After 12 weeks of treatment with methotrexate, the PASI scores were reduced significantly while the serum TWEAK levels and its peripheral expression were significantly elevated.

Conclusion. Enhancing TWEAK expression and elevating its serum levels in psoriasis patients seems to be a newly observed mechanism of action of methotrexate.

Keywords: Methotrexate, psoriasis, TWEAK

Psoriasis is a common chronic papulosquamous inflammatory disorder manifested by erythematous well-defined papules and plaques covered with silvery scales. Despite the continuous research to elucidate the exact pathogenesis of this disease, its actual pathogenic process is not fully clear. Multiple pro-inflammatory molecules and their receptors have been proposed as a key players in psoriasis development, including tumor necrosis factor (TNF)-α and various interleukins such as IL-1, 6, 17, 22, 23.¹

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is considered as a relatively recent member of the TNF superfamily. It is an active molecule that is involved in different vital physiologic body functions such as cell growth, apoptosis, angiogenesis and immune responses. TWEAK exerts these actions via interaction with its receptors, mainly fibroblast growth factor inducible (Fn14).² TWEAK might be involved in the development of different disorders which are inflammatory in nature, such as rheumatoid arthritis, lupus erythematosus, and multiple sclerosis. Moreover, targeting TWEAK has been beneficial in the management of some inflammatory conditions.³ TWEAK and its receptor Fn14 are targets for experimental studies either to agonize or antagonize them according to the role of TWEAK/Fn14 in each disease.⁴ TWEAK has been investigated as a therapeutic target for treating stroke and other central nervous system diseases.⁵ Targeting TWEAK/Fn14 pathway is a novel therapeutic target in treating myotonic dystrophy.⁶ Regarding dermatologic problems, immunotoxins targeting Fn14 receptor have been investigated in malignant melanoma treatment and showed promising results.⁷ Despite this research regarding the role of this active molecule, studies about its role in psoriasis are limited and their results are variable.

Methotrexate is still considered by many physicians as the main treatment option for generalized plaque psoriasis because of its efficacy, affordability, easy administration, and efficacy in managing psoriatic arthritis. It is a folic acid analogue that acts mainly by inhibiting dihydrofolate reductase enzyme with subsequent inhibition of immune cells and keratinocytes proliferation.⁸

The aims of this study were to evaluate the peripheral expression and the serum levels of TWEAK in patients with psoriasis vulgaris before and after receiving methotrexate treatment. 

Methods 

Ethical approval. Participants were selected from the outpatient clinic of Dermatology, Venereology and Andrology Department of Benha University.  The study was approved by the local ethics committee on research involving human subjects of Faculty of Medicine. An informed consent was obtained from all participants before starting the study. 

This was a comparative cross sectional, interventional study. The study included 148 subjects; 58 patients with moderate-to-severe psoriasis vulgaris, in addition to 90 apparently healthy individuals, age, sex and BMI matched to the patients as a control group. 

Patients with active guttate, erythrodermic, pustular psoriasis or psoriatic arthritis were excluded from the study. Using any systemic antipsoriatic therapy or phototherapy within two months or applying topical antipsoriatic therapy within two weeks prior to the study initiation was also considered as an exclusion character. Subjects with concurrent significant medical conditions such as malignancy, diabetes mellitus, hepatic, renal, cardiovascular diseases or other autoimmune or inflammatory cutaneous or systemic diseases, as well as those with any contraindication to methotrexate use were also excluded from this work.

Before starting the treatment course, all patients were subjected to the following: full history taking and clinical examination including body mass index (BMI) assessment and complete cutaneous examination to evaluate the clinical type and severity of psoriasis using PASI score.⁹ All participants were subjected to laboratory investigation (serum TWEAK levels measurement using ELISA technique and evaluation of peripheral TWEAK expression using PCR technique). 

Patients received intramuscular methotrexate (Unitrxate 50mg/2mL vial containing 25mg/mL methotrexate with benzyl alcohol [Hikma Pharmaceuticals; London, England]) weekly starting with a test dose of 7.5mg followed six days later with a complete blood count and liver function tests evaluation. Then the dose was increased gradually to the usual weekly dose (up to 30mg per week). After 12 weeks of treatment, the patients were clinically evaluated by PASI scores to assess the clinical response. They were also re-evaluated by ELISA for serum TWEAK levels measurement and by qPCR for peripheral TWEAK expression evaluation.

Blood sampling. Five millimeters of venous blood were withdrawn from each participant under complete aseptic condition, and each blood sample was divided into two parts; the first part (2mL) was collected into EDTA vacutainers, immediately stored at -20°C for later analysis of TWEAK gene expression using real time PCR.

The second part of the blood sample (3mL)were loaded into a serum separating tube. The tube was kept at room temperature for 30 minutes until coagulation, and centrifuged (at 1500 rpm for 15 minutes). Serum was separated and the resultant serum was used for assessment of TWEAK serum levels using ELISA technique by special ELISA kits (human TWEAK ELIZA kit cat. No: E-01576hu [Cloud-Clone Corp; Katy, Texas]. Sensitivity of assay 1.0 pg/mL.

Relative quantitation of TWEAK gene expression levels using quantitative RT-PCR (qRT-PCR). First, extraction of mRNA was performed using Thermo Scientific™ GeneJET RNA Purification Kit, cat. No. #K0731, #K0732 (Thermo Fisher Scientific, Waltham, Massachusetts) according to manufacturer’s instructions. RNA quantity (1µg) was measured by NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts).

Next, the reverse transcription and cDNA synthesis was done using RevertAid First Strand complementary deoxyribonucleic acid (cDNA) Synthesis Kit [Thermo Fisher Scientific, Waltham, Massachusetts]) according to manufacturer’s instructions. The tubes were placed in Bio-Rad T100 Thermal Cycler (Bio-Rad Laboratories; Hercules, California) for cDNA synthesis according to the following program: incubation at 42ºC for 60 minutes, followed by incubation at 70ºC for five minutes. The cDNA was immediately kept at -20ºC for later qRT-qPCR assay.

Next, qRT-qPCR for quantification of TWEAK gene expression was done using QuantiTect SYBR Green PCR Kit according to the manufacturer’s instructions. 

The PCR reaction mix was prepared in a total volume of 25μl / (12.5μl of 2x QuantiTect SYBR Green PCR Master Mix, 2.5μl of 10x QuantiTect Primer Assay, 2μl of Template cDNA, 8μl of RNase-free water Variable). PCR tubes were placed in the stepOne plus PCR System, (Applied Biosystem, Singapore) and the run was started according to the following program: initial activation step: for 15 minutes at 95ºC. Then cycling step (40 cycles): denaturation at 94ºC for 15 seconds followed by annealing at 55ºC for 30 seconds then extension at 72ºC for 30 seconds. After the end of the program the TWEAK gene expression levels in each sample were finally determined.

The assay was performed using Hs_TNFSF12_1_SG QuantiTect Primer Assay (TWEAK gene primer assay) (Qiagen, Germany) and B-actin as target and endogenous control genes, respectively. The primer sequence for B actin gene was as follows:

• Forward: 5′-TGA CGT GGA CAT CCG CAA AG-3′ 
• Reverse: 5′-CTG GAA GGT GGA CAG CGA GG-3′

The relative fold gene expression TWEAK in different samples was expressed after normalization by B actin gene using ∆∆Ct method.

Statistical analysis. The collected data was revised, coded, tabulated and introduced to a PC using Statistical package for Social Science (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, New York: IBM Corp.). Data were presented and suitable analysis was done according to the type of data obtained for each parameter.

Results

The study included 58 patients with active moderate to severe generalized plaque psoriasis. The control group included 90 apparently healthy individuals who were psoriasis free and had no family history of psoriasis. There was insignificant difference between patients and control subjects regarding the demographic and anthropometric data (Table 1). 

The mean duration of psoriasis in the study sample was 11.93±10.6 years and the mean PASI score was 19.1±7.4. The baseline serum TWEAK levels and its peripheral mRNA expression in the patients group were significantly lower than those in the control group (Table 2). There was a significant positive correlation between the baseline serum TWEAK levels and its baseline peripheral expression and there was a significant negative correlation between both of them and the disease severity (PASI scores) (Table 3). 

The re-evaluation of the patients after 12 weeks of treatment with methotrexate revealed a significant clinical improvement expressed by the significant PASI scores reduction (the PASI scores improvement percentage was 72.4±10.6). The serum TWEAK levels and its peripheral expression were elevated significantly following treatment (Table 4). There was insignificant correlation between the percentage of improvement and other study variables (Table 5).

Discussion

The cornerstone in psoriasis pathogenesis is the inflammatory cascade which causes the disturbed epidermal cells differentiation and proliferation. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been shown to be involved in controlling the processes of cellular proliferation and differentiation.¹⁰ Although TWEAK levels have been evaluated in various inflammatory and autoimmune diseases, limited studies about TWEAK behavior in psoriasis patients have been reported. 

In the present study, baseline serum TWEAK levels were significantly lower than the levels in the controls. This comes in line with Engin et al¹¹ and Peternel et al¹² who reported a decrease in epidermal TWEAK expression in psoriasis biopsies when compared to the healthy skin control biopsies. They suggested that reduction in TWEAK expression induces a disturbed keratinocytes differentiation and an enhanced keratinocytes proliferation. That’s why they proposed that lower TWAEK levels may trigger psoriasis. 

In contrast, other previous studies showed that the serum TWEAK levels were significantly increased in cases of plaque psoriasis^13,14 and psoriatic arthritis¹⁵ when compared with the control subjects’ levels. Moreover, Sidler et al¹⁶ showed that subcutaneous injection of TWEAK in animals leads to a localized skin inflammation which mimics the histopathological and molecular features of psoriasis, suggesting that elevated TWEAK levels may be involved in psoriasis pathogenesis. 

In fact, those previous findings are a little bit confusing. While Kumar et al¹⁷ and Cheng et al¹⁸ concluded that increased TWEAK expression exerts a pro-inflammatory effect and enhances keratinocytes proliferation, Michaelson et al¹⁹ suggested that the effect of TWEAK on keratinocytes differentiation and proliferation is dose dependent. They speculated that the lower TWEAK levels may be a cell proliferation trigger and a differentiation inhibitor. However, the elevated levels can promote apoptosis of the abnormal epidermal cells. Moreover, Jakubowski et al²⁰ showed that the effect of TWEAK on angiogenesis is complex too. It can exert a proangiogenic effect via enhancing the proliferation and migration of endothelial cells under the effect of basic fibroblast growth factor (FGF), and on the other hand, it can play an antiangiogenic role by suppressing endothelial cell morphogenesis induced by vascular endothelial growth factor (VEGF). 

Regarding the relation between baseline TWEAK serum levels as well as its expression and the different study variables, both had a significant negative correlation with the mean baseline PASI scores. This was in agreement with Mys´liwiec et al^13,21 who showed that lower PASI scores were associated with higher serum TWEAK levels. However, Xia et al¹⁵ detected that disease activity scores of psoriatic arthritis are positively correlated with serum TWEAK levels. 

Methotrexate is still the backbone of the treatment of moderate to severe psoriasis due to multiple advantages; the high efficacy, low cost, easy administration and usefulness in concomitant psoriatic arthritis. Following three months of treatment with systemic methotrexate, our patients improved significantly. This was manifested by the significant reduction in the mean PASI scores of the studied patients. 

In the current work, serum TWEAK levels and its peripheral expression showed significant elevation following treatment with methotrexate. In a study conducted by Engin et al¹¹, serum TWEAK levels were increased significantly after treatment with conventional (methotrexate, cyclosporine and systemic acitretin) or anti-TNF treatments with insignificant difference between both treatment groups. Moreover, treatment with NB-UVB or even topical dithranol also caused a significant increase in serum TWEAK.^{13, 21} However, it was not clarified whether these drugs elevate TWEAK level by increasing its production or by enhancing its release from the treated skin like, for example, the enhanced release of TNF-α from the NB- UVB exposed skin; both psoriatic and healthy.²² 

To the best of our knowledge, this is the first study to report the direct effect of methotrexate on the peripheral expression of TWEAK levels. Methotrexate enhances the peripheral TWEAK expression with subsequent elevation in its serum levels. This increase in TWEAK concentration would promote apoptosis of the impaired keratinocytes with a possible beneficial therapeutic effect for psoriasis patients.¹⁹ Sabour Alaoui et al²³ used confocal microscopy to study the mechanism by which TWEAK enhances apoptosis. They concluded that TWEAK initiates caspase-independent apoptosis via enhancing cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. Moreover, they reported that TWEAK arrests the cell growth cycle at the G2/M phase. 

The current results show significant positive correlations between baseline serum TWEAK levels and patients’ age and between baseline TWEAK expression and the disease duration. To the best of our knowledge, these correlations have not been explained before, and they need more studies to be clarified.

The TWEAK/Fn14 pathway has become a potential therapeutic target lately. There is a growing evidence suggesting that TWEAK inhibition by anti-TWEAK monoclonal antibodies (mAb) can ameliorate inflammatory reactions and tissue damage in several autoimmune and inflammatory diseases, such as rheumatoid arthritis via reducing the expression of proinflammatory cytokines.^24-26 Moreover, topical application of recombinant TWEAK seems to be of therapeutic value in the management of burn ulcers in an animal model. This topical preparation modified the tissue cytokines, differentiation markers, inflammatory cell infiltration, and extracellular matrix production.²⁷

However, in psoriasis, the view is not that clear. The variability of the serum levels of TWEAK and its expression levels in psoriasis patients in different studies, and its dose dependent effects shade light on the complex role of TWEAK in psoriasis. This necessitates more studies to clarify this mystery in order to be able to use or target this molecule in psoriasis treatment appropriately. We totally agree with Engin et al¹¹ that a “threshold value” of TWEAK concentration should be determined accurately. 

Conclusion 

The behavior of TWEAK in psoriasis is not clear yet. However, the significant change in serum levels of TWEAK in response to psoriasis therapy suggests a role of TWEAK in psoriasis pathogenesis and proposes it as a novel molecule to be involved in psoriasis treatment.

References

1. Boehncke WH. Etiology and Pathogenesis of Psoriasis. Rheum Dis Clin North Am. 2015 Nov;41(4):665–675.
2. Mendez-Barbero N, Yuste-Montalvo A, Nuñez-Borque E, J, et al. The TNF-like weak inducer of the apoptosis/fibroblast growth factor-inducible molecule 14 axis mediates histamine and platelet-activating factor-induced subcutaneous vascular leakage and anaphylactic shock. J Allergy Clin Immunol. 2020 Feb;145(2):583–596.e6.
3. Qi X, Li Z, Li H, et al. MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection. Front Immunol. 2020 Jan 23;10:3066.
4. Salzmann S, Seher A, Trebing J, et al. Fibroblast growth factor inducible (Fn14)-specific antibodies concomitantly display signaling pathway-specific agonistic and antagonistic activity. J Biol Chem. 2013 May 10;288(19):13455–13466.
5.  Winkles JA and Yepes MS (2011):Washington, DC: U.S. Patent and Trademark Office.U.S. Patent No. 7, 939–990
6. Yadava RS, Foff EP, Yu Q, et al. TWEAK/Fn14, a pathway and novel therapeutic target in myotonic dystrophy. Hum Mol Genet. 2015 Apr 1;24(7):2035–2048.
7. Zhou H, Ekmekcioglu S, Marks JW, et al. The TWEAK receptor Fn14 is a therapeutic target in melanoma: immunotoxins targeting Fn14 receptor for malignant melanoma treatment. J Invest Dermatol. 2013 Apr;133(4):1052–1062.
8. Menter A, Korman NJ, Elmets CA, et al. Guidelines of care for the management of psoriasis and psoriatic arthritis. Section 3. Guidelines of care for the management and treatment of psoriasis with topical therapies. J Am Acad Dermatol. 2009;60(4):643–659. 
9. Fredriksson T, Pettersson U. Severe psoriasis–oral therapy with a new retinoid. Dermatologica. 1978;157(4):238–244.
10. Wang X, Cheng D, Hu G, et al. Tumor Necrosis Factor (TNF) Receptor Expression Determines Keratinocyte Fate upon Stimulation with TNF-Like Weak Inducer of Apoptosis. Mediators Inflamm. 2019 Dec 5;2019:2945083.
11. Engin B, Tanakol A, Bulut H, et al. Changes in serum TNF-like weak inducer of apoptosis (TWEAK) levels and Psoriasis Area Severity Index (PASI) scores in plaque psoriasis patients treated with conventional versus anti-TNF treatments. Int J Dermatol. 2020 Feb;59(2):207–215. 
12. Peternel S, Manestar-Blažić T, Brajac I, et al. Expression of TWEAK in normal human skin, dermatitis and epidermal neoplasms: association with proliferation and differentiation of keratinocytes. J Cutan Pathol. 2011 Oct;38(10):780–789.
13. Myśliwiec H, Kiluk P, Myśliwiec P, et al. Influence of narrowband ultraviolet B phototherapy on serum tumour necrosis factor-like weak inducer of apoptosis (TWEAK) in patients with psoriasis. Clin Exp Dermatol. 2017 Oct;42(7):786–790.
14. Bilgiç Ö, Sivrikaya A, Toker A, et al. Serum levels of TWEAK in patients with psoriasis vulgaris. Cytokine. 2016 Jan;77:10–13.
15. Xia L, Shen H, Xiao W, et al. Increased serum TWEAK levels in Psoriatic arthritis: relationship with disease activity and matrix metalloproteinase-3 serum levels. Cytokine. 2011 Mar;53(3):289–291.
16. Sidler D, Wu P, Herro R, et al. TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis. Nat Commun. 2017 May 22;8:15395. 
17. Kumar M, Makonchuk DY, Li H, et al. TNF-like weak inducer of apoptosis (TWEAK) activates proinflammatory signaling pathways and gene expression through the activation of TGF-beta-activated kinase 1. J Immunol. 2009;182(4):2439–2448. 
18. Cheng H, Zhan N, Ding D, et al. HPV Type 16 Infection Switches Keratinocytes from Apoptotic to Proliferative Fate under TWEAK/Fn14 Interaction. J Invest Dermatol. 2015 Oct;135(10):2427–2436.
19. Michaelson JS, Cho S, Browning B, et al. Tweak induces mammary epithelial branching morphogenesis. Oncogene. 2005 Apr 14;24(16):2613–2624. 
20. Jakubowski A, Browning B, Lukashev M, et al. Dual role for TWEAK in angiogenic regulation. J Cell Sci. 2002 Jan 15;115(Pt 2):267–274.
21. Myśliwiec H, Myśliwiec P, Baran A, et al. Dithranol treatment of plaque-type psoriasis increases serum TNF-like weak inducer of apoptosis (TWEAK). Adv Med Sci. 2016 Sep;61(2):207–211.
22. Rossi MT, Venturini M, Zanca A, et al. Serum levels of tumor necrosis factor-alpha in patients with psoriasis before, during and after narrow-band UVB phototherapy. G Ital Dermatol Venereol. 2018 Feb;153(1):1–4.
23. Sabour Alaoui S, Dessirier V, de Araujo E, Alexaki VI, et al. TWEAK affects keratinocyte G2/M growth arrest and induces apoptosis through the translocation of the AIF protein to the nucleus. PLoS One. 2012;7(3):e33609. 
24. Wisniacki N, Amaravadi L, Galluppi GR, et al. Safety, tolerability, pharmacokinetics, and pharmacodynamics of anti-TWEAK monoclonal antibody in patients with rheumatoid arthritis. Clin Ther. 2013 Aug;35(8):1137–1149. 
25. Chen T, Guo ZP, Fu LX, et al. Anti-TWEAK monoclonal antibodies reduce vascular damage and leucocyte infiltration in a mouse model of cutaneous reverse passive Arthus reaction. Clinical and Experimental Dermatology. 2016;41(8):871–877.
26. Meulendijks D, Lassen UN, Siu LL, et al. Exposure and Tumor Fn14 Expression as Determinants of Pharmacodynamics of the Anti-TWEAK Monoclonal Antibody RG7212 in Patients with Fn14-Positive Solid Tumors. Clin Cancer Res. 2016 Feb 15;22(4):858–867.
27. Liu J, Peng L, Liu Y, et al. Topical TWEAK Accelerates Healing of Experimental Burn Wounds in Mice. Front Pharmacol. 2018 Jun 21;9:660.