iNOS inhibition for Eur J Transl Myol 29 (3): 251-260, 2019

Therapeutic effects of iNOS inhibition against vitiligo in an animal model

Hamid Mansourpour (1), Katayoun Ziari (2), Sahar Kalantar Motamedi (3), Amin Hassan Poor (4)

(1) Shahid Beheshti University of Medical Science, Tehran, Iran and AJA University of Medical Science, Tehran, Iran; (2) Department of Pathology, AJA University of Medical Science, Tehran, Iran; (3) Shahid Beheshti University of Medical Science, Tehran, Iran; (4) Tehran University of Medical Science, Tehran, Iran This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract Nitric oxide (NO) is involved in several biological processes, but its role in human melanogenesis and vitiligo need further studies. Previous studies revealed that exposure to UVA and UVB were capable of the inducing nitric oxide production in keratinocytes and melanocytes through the activation of constitutive nitric oxide synthase, whereas only inducible nitric oxide synthase overexpression has been reported to play an important role in hyperpigmentary disorders. The aim of this study was to evaluate iNOS inhibitor aminoguanidine (AG) as a therapeutic agent in our mouse model of vitiligo. In this study, male C57BL/6J Ler-vit/vit mice were purchased to evaluate the effect of iNOS inhibitor (aminoguanidine) (50 anduse 100 mg/kg) and L-arginine (100 mg/kg) in a mouse model of vitiligo induced by monobenzone 40%. Moreover, we used phototherapy device to treat the mice with NBUVB as a gold standard.The findings revealed that monobenzone was capable of inducing depigmentation after 6 weeks. However, aminoguanidine in combination with monobenzone was decrease the effect of monobenzone, while L-arginine play a key role in promoting the effect of monobenzone (P<0.001). Based on the phototherapy, the efficacy of phototherapy significantly increased by adding L-arginine (P<0.05). Taken together, we suggest that iNOS inhibitor can be a novel treatment for the prevention and treatment of vitiligo by combination of NBUVB therapy, furthermore; NO agents like L-arginine could also increase the effectiveness of phototherapy. Taken together, this pilot study showed significant repigmentation of vitiligous lesions treated with iNOS inhibitor plus NBUVB therapy, where other aspect including expression of an inducible iNOS, NO and TNF levels remained to be evaluated in mice model. commercial Key Words: Vitiligo, Nitric oxide, iNOS, Monobenzone, Treatment. Eur J Transl Myol 29 (3): 251-260, 2019 Non itiligo, a skin disease, is a common progressive role in development of disease when detected in V 8 depigmentation of that results in the perilesional skin of actively spreading vitiligo. Several destruction of melanocytes in the depigmented parts of cell lines has been described to play their role in vitiligo the skin, mucous membrane and retinal wall. As a result, development including lymphocytes, keratinocytes, and endothelial cells, involving in melanocyte migration, irregular white patches appear in different areas of the 2, 9-11 body. Vitiligo has been estimated to be one of the most proliferation, and differentiation. Increasing evidence supports that vitiligo could be a disorder of the frequent pigmentary diseases, accounting for about 0.1– 12 2% of the worldwide population.1-3 The pathophysiology entire epidermal unit. Especially, keratinocytes of this disease is not well understood; however, several has been revealed to contribute to the melanocyte homeostasis and its changes affecting melanocyte theories about the pathomechanism of vitiligo have been 1,13,14 previously defined, such as genetic, biochemical, dysfunction in vitiligo epidermis. Vitiligo has been immunological, and environmental theories.4,5 On the defined as a multifactorial disease with autocytotoxic, neural, and autoimmune susceptibilities as well as other hand, increasing evidence indicated that there has a 15,16 correlation between vitiligo and other autoimmune environmental factors. Nitric oxide as an intracellular disease of the endocrine glands,6,7 as well as a messenger plays an important role in the synthesis of an lymphocytic infiltrate was found to play an important enzyme called NO synthase, which consists of three

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Fig 1. Monobenzone 40% induced depigmentation in exposed regions. isoforms: inducible NOS (iNOS), neuronal NOS the pathophysiologyonly of this disease using a (nNOS), and endothelial NOS (eNOS).17-30 Nitric oxide pharmacological inhibition of the iNOS, where a mouse has been suggested as a potential factor in inhibiting model of vitiligo was induced by monobenzone. cell proliferation, differentiation, and apoptosis. This free radical gas can be considered as a great challenge, when Materialsuse and Method a key role plays in pathogenesis of a lot of kind of Ethics Approvals autoimmune diseases such as vitiligo. Additionally, it is All procedures were performed in accordance with the likely to be involved, not only in melanocyte dysfunction National Institutes of Health (NIH) Guide for the Care and/or destruction, but also in the accumulating toxic and Use of Laboratory Animals (NIH publication # 80- intermediates of melanin synthesis and in elevating 23) and institutional guidelines for animal care and use reactive oxygen species.17-20,22-27 A growing body of (Department of Pharmacology, School of Medicine, evidence highlights that exposure to UVA and UVB may TUMS). Each experimental group consisted of 6 to 8 be capable of producing nitric oxide, especially in animals. keratinocytes and melanocytes, where it is involved, not only in increasing tyrosinase activity via activating Animals constitutive NOS, but also in the synthesiscommercial melanin.21, In this study, male C57BL/6J Ler-vit/vit mice were 31 The use of NBUVB can improve treatment of vitiligo obtained from Pasteur Institute, Tehran, Iran (weighing by stimulating the proliferation and migration of 25-30 g). Animals were then housed in Plexiglas boxes melanocytes, as well as stimulating the differentiation of (25 × 25 × 15 cm), under standard conditions melanocyte stem cells. UVA and NBUVB have been (temperature: 22 ± 2 °C, humidity: 50 ± 10%, 12-h light– introduced as common typesNon of phototherapy in vitiligo. dark cycle, and free access to food and water). Despite the similar effect of both interventions, Phototherapy device narrowband ultraviolet B (NBUVB) phototherapy can be more effective for patients suffering from vitiligo due to BIOSKIN®, a phototherapy device is equipped with a the lack of side effects of Psoralen.32,33 The topical use of short arc generator that provides a beam of visible and immunosuppressive agents, including glucocorticoids ultraviolet radiations, and is also filtered by an and calcineurin inhibitors, have been introduced as the interference filter for obtaining UV-B narrow band. The first line treatment, while the use of phototherapy has operator is capable of regulating emission time, and plays been considered as the second line for treating these its role in regulating emission time by accompanying a patients.32 iNOS is involved in the production of NO, time-controlled shutter. Furthermore, BIOSKIN® is where promotor polymorphisms of iNOS gene and NO equipped to prepare a spectrum of intensity up to 400 2 overproduction has been found to be associated with the mW/cm , where provide coverage from 300 to 320 nm pathogenesis of vitiligo, leading to melanocyte for emission spectrum, with an emission peak of 311 nm. destruction.34-38 Based on above-mentioned data, the Mice were then irradiated three times a week for 4 weeks. current study was aimed to demonstrate the association The narrow band UV-B (NBUVB) irradiation was of vitiligo with iNOS in phototherapy and to investigate performed using a BIOSKIN® device. It is worth noting

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Fig 2. Effect of aminiguanidine (100 mg/kg) plus monobenzone 40% on depigmentation that vitiligo patches were next irradiated by excluding investigated. In the sixthonly group, injectable NO inducer mucous membrane and genital part for each micro- was daily used at dose of 100 mg/kg (L-arginine) and the phototherapy session. Although BIOSKIN® equipment combined effect of this drug was investigated by is able to provide a large spectrum of intensity (0–400 phototherapy. As a matter of fact, the effects of L- mW/cm2), the intensity of 50 mW/cm2 was employed in arginine anduse AG on development of vitiligo were the present study for all animals during all sessions. The assessed by the vitiligo-inducing compound initial dose of irradiation was 20% less than minimum monobenzone. Optimal treatment dose of erythema dose (MED) that was applied to vitiligo area at aminoguanidine was determined by implementing a least 3 days before the beginning of the treatment. During dose-response model, assessing increasing doses (50 mg the subsequent sessions, the dose in each session and 100 mg). All drugs were purchased from Sigma, St increased to 20% until the development of erythema. Louis, MO, USA and afterward dissolved in saline. Additional dose of the next session was diminished by Animals received drugs intraperitoneally (i.p.), except 20% only in the erythematous area when erythema was for monobenzone. developed. Results Experimental design commercialMonobenzone induced vitiligo In the present study, sex-matched mice were divided in 6 groups of 10 mice. In the first part of this study we In the first step of this study, we tried to evaluate emploied the vitiligo-inducing agent monobenzone in the monobenzone 40%-induced depigmentation from 1 to 6 mice model, as previously described by Zhu et al.,39 weeks after exposure. Analysis revealed that highlightening that monobenzoneNon-induced vitiligo is a monobenzone 40% can induce vitiligo with a mean of valid mouse model of vitiligo. In the first group (control 95% depigmentation after 6 weeks, which was associated group), mice received only monobenzone cream 40% on with the increasing trend of vitiligo. One-way ANOVA the mouse tail and skin on the back of the mouse and analysis revealed that monobezone could significantly thereafter skin hypo-pigmentations were investigated induce depigmentation after 6 weeks (F [6, 35] = 218.2, every week as compared to intact part. In the second P<0.001, Fig1). Post Tukey’s analysis showed group, the mice receive phototherapy (BIOSKIN® depigmentation of exposed regions using color cell compression, as given by a computer software after 6 device; 50mW/cm2) in combination with the monobenzone, which was considered a positive control. weeks (P<0.001). As shown in figure 1, we suggested an In the third group of mice, the role of iNOS in increased trend of depigmentation in initiation phase on development of vitiligo was then evaluated. Therefore, exposed area. beginning the first day after vitiligo induction animals Co-treatment of monobenzone with iNOS inhibitor were treated with aminoguanidine (AG), an injectable Following mentioned experiment, we provide a co- iNOS inhibitor, at daily dose of 50 and 100 mg/kg (i.p. treatment of iNOS inhibitor (aminoguanidine at 100 6x weekly) to clarify the NO pathway. In group 5, the mg/kg) with monobenzone 40% to clarify the interaction combined effect of this inhibitor with phototherapy was between nitrergic system and vitiligo. Statistical analysis

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only use

Fig 3. Effect of L-arginine (100 mg/kg) plus monobenzone 40% on depigmentation. revealed that co-treatment of aminoguanidine (100 Dose dependency of aminiguanidine on inducing vitiligo mg/kg i.p) with monobenzone 40% could significantly decrease vitiligo-inducing effect of monobenzone (F [12, In the next step, we tried to show the dose dependency of 65] = 127.1; P<0.001; Fig 2). Post hoc analysis aminiguanidine 50 and 100 mg/kg as previously demonstrated a significant difference 4 weeks after indicated by our experiments. In order to evaluate the treatment (P<0.001). In addition, the difference between dose dependency of iNOS inhibitor, administration of groups was determined as about 50% of depigmentationcommercial aminoguanidine (50 and 100 mg/kg) with monobenzone after 6 weeks. Overall, aminoguanidine (100 mg/kg) 40% were applied in the current study (F [5, 30] = 273.0; decreased 50% of depigmentation after about 6 weeks. P<0.001; Fig 4). One-way ANOVA analysis suggested that administration of aminoguanidine (100 mg/kg) Co-treatment of monobenzone with NO agent resulted in induction of depigmentation after 6 weeks as In the next step, we use NOSNon agent (L -arginine at 100 compare to aminoguanidine (50 mg/kg), (P<0.001). This mg/kg) in combination with monobenzone 40% to experiment showed that aminoguanidine dose evaluate the interaction between increasing NO level and dependently could be linked to decreased vitiligo (F [12, 65] = 181.2; P<0.001; Fig3). One-way depigmentation-inducing effect of monobenzone ANOVA analysis demonstrated that a co-treatment of L- following 6 weeks treatment (Figure 4). arginine (100 mg/kg i.p.) with monobenzone 40% was Effect of iNOS inhibitor on phototherapy significantly associated with an increased level of vitiligo-inducing effect of monobenzone. Additionally, Finally, we evaluated the phototherapy by using NBUVB post hoc analysis revealed a significant difference as the common treatment of vitiligo in combination with between groups after 3 weeks of treatment (P<0.05). In L-arginine to clarify the effect of NO level on NBUVB addition, the difference between groups comprised of induced pigmentation (F [2, 15] = 10.12; P<0.001; Fig 5). 35% of depigmentation. Furthermore, a significant Analysis revealed that a combination therapy, consisting difference was only observed after 4 weeks of treatment L-arginine and NBUVB was significantly capable of (P<0.001). Overall, L-arginine (100 mg/kg) significantly enhancing the pigmentation of the exposed area, when accelerated the depigmentation after about 3 weeks compared with NBUVB treatment (P<0.01). Duration of (Figure 3). NBUVB radiation therapy is about 4 weeks, where a

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only

Fig 4. Effect of dose dependency of aminoguanidine (50 and 100 mg/kg) in comparison monobenzone 40% on pigmentation incidence after 6 weeks. use combination therapy of L-arginine and NBUVB precarious role in several hyperproliferative and increased the effect of NBUVB (Figure 5). autoimmune disorders has been previously conformed.40- 43. L-arginine is defined to be a biological precursor of Discussion NO, a nerve regulator in the brain, by the Nitric Oxide In the current study, we demonstrated that Synthase (NOS) enzyme. The enzymatic family of NOS aminoguanidine could inhibit vitiligo-inducing effect of consists of three isoforms that include inducible NOS monobenzone after about 4 weeks of treatment. In (iNOS), neuronal NOS (nNOS), and endothelial NOS addition, our results verified that L-arginine could (eNOS). All three NOS isoforms play their crucial role in significantly increase the effect of NBUVB treatment in different parts of the brain. Unlike iNOS, nNOS and mice model of vitiligo. Nitric oxide has been defined to eNOS are both calcium / calmodulin-dependent, where be a noteworthy mediator that plays a commercial crucial role in preclinical evidence supporting important role for this numerous biological procedures including isoforms in cell homeostasis, and alteration of immune melanogenesis, microcirculation, and keratinocyte and cytotoxic response 31, 44, 45. It is noteworthy, that response to ultraviolet radiation, cell growth and eNOS and nNOS were originated in numerous tissues, differentiation. Nitric oxideNon is known to be a major which have been identified to synthesize by different cell neurotransmitter that plays a key role in the including keratinocytes, melanocytes, and fibroblasts in pathophysiology of many diseases of the nervous system, skin. Ample evidence suggests that expression of iNOS such as epilepsy, schizophrenia, anxiety, and is under the regulation of different cytokines including autoimmune disease. Nitric oxide is a chemical IL-1, IL-6, IL-23, IL-17, IL-33, IFN-γ and TNF-α,32,46- messenger that can easily pass through cell membranes, 49,50-63 which play a key role in inducing the and unlike other neurotransmitters, it does not depigmentation. In this regard, our study should be accumulate in synaptic vesicles and does not release considered in light of a limitation, where further studies through exocytosis. It seems that nitric oxide plays a are needed to clarify the expression of cytokines such as crucial role in regulating various body responses, in TNF-a in the future. IFN-γ and TNF-α as Th1 cytokines, association with other neurotransmitter systems, such as because increasing evidence supports a role for Th1 norepinephrine, serotonin, dopamine, and glutamate. An response in mediating vitiligo. A study by Taïeb increasing body of evidence indicates a possible role for described vitiligo as an inflammatory disease similar to NO in hair follicle biology, where is not a well-known other skin inflammatory diseases, including psoriasis and regulator in the human hair follicle and the hair-growth atopic dermatitis. This research reported that the cycle.38,39 NO is known as an important mediator of elimination of inflammation can be considered as a inflammatory responses in the body. Moreover, its priority in the treatment of this disease.64

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Vaccaro et al. have shown that UVA and UVB radiation level under L-arginnine treatment. In addition, using

Fig 5. Effect of L-arginine (100 mg/kg) on pigmentation by using NBUVB therapy. were capable of triggering the synthesis of NO (NOS) iNOS inhibitor could decrease the efficacy of enzymes in keratinocytes and melanocytes. They have monobenzone in the presentonly study. Therefore, emerging suggested that an increased level of NO may increase evidence has persuaded us to hypothesize that iNOS and tyrosine kinase activity and then triggered melanin NO levels increased in vitiligo lesions compared to formation, but excessive increased of inos could lead to healthy controls. In particular, the increased activity of processes that result in depigmentation. Aforementioned iNOS mightuse be responsible for the overproduction of NO study exhibited that the NO imbalance play a key role in in vitiligo model. Vitiligo is linked to some factors such the development of vitiligo, where inhibitors of this as increased cytokines expression and iNOS activation factor can potentially play a role in improving this that play pivotal roles in the depigmentation process complication, and recommend the assessment of these even, if NOS overexpression not being considered as the inhibitors as a priority.65 Many studies suggested the use only important factor involved in vitiligo. An increasing of topical corticosteroids as the first line of treatment for body of evidence indicates that NOS has been vitiligo. Moreover, some studies emphasized on the overexpressed in vitiligo lesions in comparison with systemic effectiveness of these drugs for reducing healthy subjects, as well as NO2−/NO3− levels. The inflammation, and symptoms, as well as inducing increased activity of iNOS has been also suggested to be repigmentation.65 On the other hand, immunomodulators potentially linked to the increased level of NO in vitiligo, (e.g., and calcineurin inhibitors)commercial and reducing leading to decrease/death of melanocytes up to the activity of T cells by decreasing the expression of depigmentation.31 Therefore, in the present study, we inflammatory cytokines (e.g., IL-2, IL-3, IL-4, IL-5, used an inhibitor to demonstrate whether iNOS interferon (IFN), and TNF) along with the granulocyte- inhibition, which is associated with a reduction in NO macrophage colony-stimulatingNon factor (GM -CSF) can be production, can lead to lesion healing. It is noteworthy suggested as effective therapeutic strategies for vitiligo that NO increased in vitiligo, as shown preciously by by targeting them that are recently being evaluated.66-67 studies.31 But its types and possible interventions have Previous studies revealed that the production of high NO not been thoroughly investigated as compared to level could be associated with the increased activity of presented findings herein. On the other hand, evidence iNOS, confer self-destruction of melanocytes, leading to from in vivo and in vitro studies supports that the a decrease in De novo attachment of melanocytes at the melanocytes can be never entirely disappeared in the extracellular matrix,65, 68-70 where such evidence can depigmented epidermis, where these melanocytes are follow skin depigmentation.71 Recent mechanism have capable of recovering their function in vivo and in vitro also attributed to initial imbalance of epidermal cytokines hydrogen peroxide removal.74 Moreover, melanocyte at the affected areas in promoting tetrahydrobiopterin, survival after some years has been previously iNOS activation, and NO overproduction, resulting in an demonstrated.75 Although measuring NO and TNF levels increase of self-destruction of melanocytes.65,72,73 In the and viability of skin melanocytes can be useful in present study, vitiligo skin was induced by monobenzone confirming the findings, their assessments are considered 40%, where early findings demonstrated a significant as part of our limitations. Therefore, further evaluation is degree of efficacy with appropriate responses, when L- needed as a future study, which we hope will be done in arginnine was applied, leading to a decrease in the NO the near future. Anyhow, our findings revealed that

- 256 - iNOS inhibition for vitiligo Eur J Transl Myol 29 (3): 251-260, 2019 aminoguanidine in combination with monobenzone was References able to decrease the effect of monobenzone. The findings 1. Ala Y, Pasha MK, Rao RN, et al. Association of indicated that iNOS inhibitor could serve as a novel IFN-gamma : IL-10 Cytokine Ratio with effective therapy against vitiligo by combination of Nonsegmental Vitiligo Pathogenesis. Autoimmune NBUVB therapy. Furthermore, NO agents like L- diseases 2015;2015:423490. arginine could markedly increase the effectiveness of 2. Aroni K, Voudouris S, Ioannidis E, et al. Increased phototherapy. Further studies are needed to illustrate angiogenesis and mast cells in the centre compared expression of an inducible iNOS, NO and TNF levels in to the periphery of vitiligo lesions. Arch Dermatol mice model for clarification of their role in the Res 2010;302:601-7. pathogenesis of vitiligo. Another approach is necessary 3. Banerjee P, Venkatachalam S, Mamidi MK, et al. to confirm the iNOS implications in the near future: the Vitiligo patient-derived keratinocytes exhibit use of 1400W. These approaches normalize the "non- characteristics of normal wound healing via specific effects" of aminoguanidine. An in-vivo siRNA epithelial to mesenchymal transition. Exp Dermatol targeting iNOS could be another way to link the iNOS to 2015;24:391-3. vitilogous lesions. Systemic infusion of the drugs could 4. Gupta SC, Patchva S, Aggarwal BB. Therapeutic interfere with immune profile of the animal. Supporting roles of curcumin: lessons learned from clinical data for the immune status would strengthen our results trials. AAPS J 2013;15:195-218. and their interpretations. 5. Camara-Lemarroy CR, Salas-Alanis JC. The role of List of acronyms tumor necrosis factor-alpha in the pathogenesis of vitiligo. Am J Clin Dermatol 2013;14:343-50. IFN – interferon 6. Campanati A, Giuliodori K, Ganzetti G, et al. A GM-CSF - granulocyte-macrophage colony-stimulating patient with psoriasis and vitiligo treated with factor only etanercept. Am J Clin Dermatol 2010;11 Suppl NBUVB - narrow band narrowband ultraviolet B 1:46-8. NO - Nitric oxide 7. Barbaud A. What's new in clinical dermatology? NOS - Nitric Oxide Synthase Annales de dermatologie et de venereologie eNOS - endothelial NOS use 2014;141 Suppl 4:S597-609. iNOS - inducible NOS 8. Grandemange S, Sanchez E, Louis-Plence P, et al. nNOS - neuronal NOS A new autoinflammatory and autoimmune Authors contributions syndrome associated with NLRP1 mutations: NAIAD (NLRP1-associated autoinflammation with KZ, HM, SKM, and AHP conceived and designed the arthritis and dyskeratosis). Ann Rheum Dis experiments, analyzed the data and wrote the manuscript 2017;76:1191-8. and supervised the experiments. All authors read and 9. Vaccaro M, Cicero F, Mannucci C, et al. IL-33 approved the final manuscript. circulating serum levels are increased in patients Acknowledgments None. with non-segmental generalized vitiligo. Arch Dermatol Res 2016;308:527-30. Funding None. commercial10. Yao L, Hu DN, Chen M, Li SS. Subtoxic levels Conflict of Interest hydrogen peroxide-induced expression of interleukin-6 by epidermal melanocytes. Arch The authors declare they have no financial, personal, or Dermatol Res 2012;304:831-8. other conflicts of interest.. Non 11. Yazici AC, Erdal ME, et al. Lack of association Ethical Publication Statement with TNF-alpha-308 promoter polymorphism in We confirm that we have read the Journal’s position on patients with vitiligo. Arch Dermatol Res issues involved in ethical publication and affirm that this 2006;298:46-9. report is consistent with those guidelines. 12. Sandoval-Cruz M, Garcia-Carrasco M, Sanchez- Porras R, et al. Immunopathogenesis of vitiligo. Corresponding Author Autoimmun Rev 2011;10:762-5. Katayoun Ziari, Departemnt of Pathology, AJA 13. Karam RA, Zidan HE, Khater MH. Genetic variants University of Medical Science, Tehran, Iran. Tel: of interferon-gamma and its mRNA expression and 00989122993226 inflammatory parameters in the pathogenesis of Email: [email protected] vitiligo. Biochem Cell Biol 2017;95:474-81. 14. Ning W, Wang S, Dong X, et al. Epigallocatechin- E-mails of co-authors 3-gallate (EGCG) Suppresses the Trafficking of Hamid Mansourpour: [email protected] Lymphocytes to Epidermal Melanocytes via Sahar Kalantar Motamedi: Inhibition of JAK2: Its Implication for Vitiligo [email protected] Treatment. Biol Pharm Bull 2015;38:1700-6. Amin Hassan Poor: [email protected]

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