Nicotinamide N-Methyltransferase in the Inflammatory Pathogenesis of Graves’ Orbitopathy

Abstract

Purpose: Nicotinamide N-methyltransferase (NNMT) has been implicated in inflammatory autoimmune disease pathogenesis, although its pro-inflammatory role in Graves’ orbitopathy (GO) is unclear. Therefore, we investigated the influence and mechanisms of NNMT in GO inflammation.

Methods: We evaluated NNMT mRNA expression in GO and non-GO orbital tissues via reverse transcription-quantitative PCR analysis. A pro-inflammatory process was induced in primary cultured orbital fibroblasts via interleukin (IL)-1β treatment, and NNMT expression was assessed by Western blotting. To further investigate the role of NNMT in GO inflammation, we inhibited NNMT expression and activity using small interfering RNA (siRNA) and pharmacologic antagonists, respectively. The production of inflammatory cytokines and intracellular signaling molecules were analyzed via Western blotting and enzyme-linked immunosorbent assay analysis.

Results: NNMT mRNA expression levels were higher in GO orbital tissues than in healthy orbital tissues. Tissues from patients with type Ⅱ GO showed higher NNMT expression than those with type Ⅰ GO. Pro-inflammatory stimulation induced NNMT expression in dose- and time-dependent manners. NNMT siRNA and antagonists attenuated the expression of pro-inflammatory cytokines (IL-6, IL-8, and monocyte chemotactic protein-1), cyclooxygenase-2, and prostaglandin E2 in orbital fibroblasts. NNMT silencing downregulated the active forms of intracellular signaling molecules (extracellular signal-regulated kinase, c-Jun-terminal kinase, and p38).

Conclusions: Our results demonstrate that NNMT was associated with the inflammatory mechanisms of GO. Inhibiting NNMT, either through mRNA silencing or pharmacologic antagonism, markedly reduced pro-inflammatory reactions. These findings suggest that targeting NNMT is a promising therapeutic strategy for managing inflammation in GO.

Graves’ orbitopathy (GO) is an inflammatory autoimmune disease occurring in patients with autoimmune thyroid diseases, such as Graves’ disease.¹ Many studies have been conducted to clarify GO pathogenesis. Although the exact pathology remains unclear, orbital fibroblasts are considered to play a key role in this disease process.^2,3 Thyroid-stimulating hormone receptor (TSHR) is an autoantigen shared by orbital fibroblasts and thyroid gland cells,⁴ and TSHR transcription and immunoreactivity are significantly elevated in GO orbital fibroblasts.⁵ GO progression involves immune cell infiltration as well as orbital fibroblast activation and proliferation due to T cell-dependent inflammation. These changes result in autoantibody secretion by plasma cells, fibroblast differentiation into adipocytes and myofibroblasts, and hyaluronic acid synthesis, leading to clinical symptoms.⁶ Crosstalk between TSHR and insulin-like growth factor 1-receptor (IGF-1R) has been associated with GO pathogenesis. A drug targeting crosstalk between TSHR and IGF-1R (teprotumumab) was approved in 2020 for use in the United States by the US Food and Drug Administration as a first-in-class medication for GO.⁷ 

Nicotinamide N-methyltransferase (NNMT) is a human enzyme encoded by the NNMT gene.⁸ NNMT methylates nicotinamide to produce methylnicotinamide, with S-adenosyl-methionine (SAM) acting as the methyl donor, whereby SAM is converted to S-adenosyl-1-homocysteine, which is rapidly converted into homocysteine by S-adenosyl-homocysteine hydrolase.⁹ NNMT is expressed at the highest levels in the liver, and lower expression levels are found in most other organs.^8,10 The roles of NNMT in various disorders and diseases are being investigated. The role of NNMT as a key regulator of nicotinamide adenine dinucleotide (NAD⁺) and homocysteine-metabolism pathways have been studied in various diseases, including cancer,^11–14 neurodegenerative disorders,^15,16 diabetes,¹⁷ and cardiovascular conditions.¹⁸ NNMT has also been studied for its roles in autoimmune diseases, such as inflammatory bowel disease (IBD) and multiple sclerosis (MS). Transcriptome analysis of samples from patients with IBD revealed that treatment-related changes in NAD⁺ levels correlated with altered NNMT expression, and the evidence suggested that inhibiting NNMT could reduce NAD⁺-dependent pro-inflammatory signaling.¹⁹ The results of several studies on MS have also shown that regulating NNMT and NAD⁺ levels is a key for attenuating MS severity.^20,21 

Some findings have suggested that nicotinamide (an NNMT substrate) can potentially serve as an antioxidant treatment for GO.^22–24 However, to the best of our knowledge, no studies have focused on the role of NNMT in GO pathogenesis. This study aimed to investigate the role of NNMT in the inflammatory mechanisms of GO in vitro. 

Methods

Reagents and Chemicals

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and gentamicin were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). An NNMT small-interfering RNA (siRNA; #111655) and a negative-control siRNA (siCon; #4390843) were obtained from Ambion/Applied Biosystems (Austin, TX, USA). Recombinant human interleukin (IL)-1β was purchased from R&D Systems (Minneapolis, MN, USA). We purchased 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay solution from Promega Corporation (Madison, WI, USA). Antibodies against IL-6, IL-8, monocyte chemotactic protein (MCP)-1, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cyclooxygenase (COX)-2, phosphorylated signal transducer and activator of transcription (STAT)-3 (p-STAT3), total STAT3 (t-STAT3), p-nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), t-NF-κB, p-protein kinase B (p-Akt), t-Akt, p-extracellular signal-regulated kinase (p-ERK), t-ERK, p-c-Jun-terminal kinase (p-JNK), t-JNK, p-p38, and t-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA). Two small molecule NNMT inhibitors, namely 5-amino-1-methylquinolinium iodide (5-amino-1MQ, SML2832) and 1-methylnicotinamide chloride (1-MNA, SML0704), were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 

Subjects, Tissue, and Cell Preparation

Orbital adipose connective tissues were obtained as surgical waste during orbital-decompression surgery from 19 patients with GO (7 men and 12 women; aged 21–76 years; Supplementary Table S1). All patients were in a stable euthyroid state, with clinical-activity scores of ≤3. None of the patients had received steroids, other types of immunomodulatory treatments, or radiotherapy for >3 months before surgery. We compared NNMT mRNA expression levels in orbital tissues from patients with type I or type II GO, two clinical subtypes of GO that have been discussed previously.^25,26 Among the 19 patients with GO, 9 had type Ⅰ (lipogenic) GO (without restrictive myopathy and diplopia), whereas the remaining 10 had type Ⅱ (myogenic) GO (with restrictive myopathy and diplopia). Pre-operative computed tomography (CT) was used to confirm GO subtypes. Healthy control tissues were harvested from the post-septal areas of 19 individuals with no history or clinical evidence of GO (12 men and 7 women; aged 28–81 years). The study was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (approval number 4-2024-0581), and written informed consent was obtained from all participants following a detailed explanation of the nature and possible consequences of the study. This study adhered to the tenets of the Declaration of Helsinki. 

The methods for culturing primary orbital fibroblasts were established in our previous studies.^27–30 Briefly, minced tissues were placed directly in a 1:1 mixture of DMEM and F12 medium containing 20% FBS and antibiotics. When monitoring fibroblast cell growth, monolayers were passaged serially with trypsin/ethylenediaminetetraacetic acid solution, and cultures were maintained in DMEM with 10% FBS and antibiotics. Primary cultured cell strains were stored in liquid nitrogen until further analysis, and cells between the third and fifth passages were used in our experiments. 

Reverse Transcription-Quantitative PCR

NNMT expression in orbital tissues from individuals with or without GO were assessed via reverse transcription-quantitative PCR (RT-qPCR), following a previously described method.²⁸ RNA was isolated from orbital tissues (type Ⅰ GO, n = 10; type Ⅱ GO, n = 9; and non-GO, n = 19) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The sequences of primers targeting the NNMT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes are shown in Supplementary Table S2. Complementary DNA was synthesized using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA). PCR was performed using TaqMan Universal PCR Master Mix and a QuantStudio 3 Real-Time PCR thermocycler (Applied Biosystems, Carlsbad, CA, USA). The PCR results were evaluated using the 2^−ΔΔCt method to determine relative exponential changes by comparing threshold cycle (Ct) values, normalized to the Ct value for GAPDH. Each RT-qPCR experiment was conducted in duplicate. 

siRNA Transfection

Orbital fibroblasts from individuals with or without GO were cultured to approximately 80% confluency in 100 mm plates. Then, the orbital fibroblasts (approximately 1 × 10⁵ cells/well) were transfected with siNNMT or siCon using the TransIT-siQUEST reagent (Mirus, PanVera, Madison, WI, USA), and the cells were maintained in growth medium supplemented with 10% FBS and antibiotics. After transfection, the cells were incubated with or without IL-1β stimulation for further experiments. 

Western Blotting

Western blotting was performed on orbital fibroblasts to evaluate changes in NNMT expression after inflammatory stimulation and to assess the inhibition of pathogenic GO-inflammation mechanisms following NNMT silencing. Only orbital fibroblasts from patients with type Ⅱ GO were used as GO cells in all Western blot experiments. Western blotting was performed as previously described.^28,31 Orbital fibroblasts treated with different reagents were rinsed with phosphate-buffered saline and then lysed in cell-lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF, 20 mM HEPES (pH 7.2), 10% glycerol, 0.1 mM dithiothreitol, 1 mg/mL leupeptin, 1 mg/mL pepstatin, 10 mM Na₃VO₄, and 1% Triton X-100 for 30 minutes on ice. Cell lysates were centrifuged at 13,000 g to obtain homogeneous cell fractions, which were subsequently boiled in lysis buffer. Proteins were resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Immobilon; Millipore Corp., Billerica, MA, USA), and incubated overnight with primary antibodies in Tris-buffered saline with Tween-20. Immunoreactive bands were detected using a horseradish peroxidase-conjugated secondary antibody. The bound peroxidase was visualized via chemiluminescence (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA) and exposed to X-ray film (Amersham Pharmacia Biotech, Inc.). The relative amounts of protein in each band were quantified via densitometry after normalization to the β-actin levels in the same sample. 

Enzyme-Linked Immunosorbent Assay

Supernatants from the culture media of orbital fibroblasts from individuals with or without GO (n = 3 each) were collected and diluted 1:10, and ELISAs were performed to quantify the levels of secreted pro-inflammatory cytokines. Only orbital fibroblasts from patients with type Ⅱ GO were used as GO cells. We used commercially available ELISA kit (Milliplex MPA kit; Millipore, Billerica, MA, USA) and followed a previously described method.^28,29 Absorbance was measured at 405 nm to assess the binding percentage for each sample, and a standard binding curve was drawn to determine the concentrations. The average of three replicate assays was used for statistical analysis. 

Cell Viability Assay

We conducted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to evaluate the effects of siNNMT and 2 different NNMT inhibitors on the viability of orbital fibroblasts. For siNNMT, siNNMT- and siCon-transfected orbital fibroblasts from individuals with or without GO were incubated for 24, 48, or 72 hours in medium supplemented with 10% FBS and antibiotics. Thereafter, MTT solution was added and each plate was incubated again for 4 hours under the same conditions. For NNMT inhibitors, primary cultured orbital fibroblasts were seeded on 24-well culture plates and treated with various concentrations of each NNMT inhibitor for 24 hours. Subsequently, MTS solution was added, and the plate was incubated for an additional 4 hours under identical conditions. Dye absorbance was measured at 490 nm using an ELISA plate reader (EL 340 Biokinetics Reader; Bio-Tek Instruments, Winooski, VT, USA). Cell viabilities were calculated as a percentage relative to untreated control cells. 

Statistical Analysis

All experiments were performed using cell samples from at least three different individuals, which were assayed in duplicate. The results are expressed as mean ± standard deviation. Differences in continuous data among the three groups were assessed using Kruskal-Wallis test with post hoc comparison using Dunn's method, performed with IBM SPSS Statistics for Windows, version 29.0 (IBM Corp., Armonk, NY, USA). Differences in continuous data between the experimental and control groups were assessed using either Student's t-test or Wilcoxon's rank-sum test, performed with R software version 3.1.2 (R Foundation, Vienna, Austria). Any P < 0.05 was considered statistically significant for all analyses. 

Results

GO Tissues Showed Increased NNMT Expression

To evaluate NNMT expression, whole orbital tissue explants were obtained from patients with type Ⅰ GO, type Ⅱ GO, and from individuals without GO, and the relative NNMT mRNA expression levels were analyzed via RT-qPCR (Fig. 1). NNMT transcript levels in type Ⅱ GO tissues were significantly higher than those in non-GO control tissues (P < 0.001) and type Ⅰ GO tissues (P = 0.021). 

Figure 1.

 

NNMT mRNA expression in GO orbital tissues. Orbital tissues were obtained from patients with GO (n = 19) and healthy subjects without GO (n = 19). Patients with GO from whom orbital tissues were obtained were divided into two groups based on CT scans: those with type Ⅰ GO (non-infiltrative type and lipogenic type, n = 9) and those with type Ⅱ GO (infiltrative type and myogenic type, n = 10). The mRNA-expression levels of NNMT were compared via RT-qPCR. Differences among the three groups were assessed using Kruskal-Wallis test (χ² [2, n = 38] = 17.512, P < 0.001), with post hoc analyses using Dunn's method (*P < 0.05, **P < 0.01). The results are presented as mean ± standard error (SE).

Inflammatory Conditions Induced NNMT Upregulation in GO and Non-GO Orbital Fibroblasts

NNMT expression in GO and non-GO orbital fibroblasts was assessed via Western blotting after IL-1β treatment. The results displayed increased NNMT protein levels in a time-dependent manner in both GO and non-GO fibroblasts (Fig. 2A). After 24 hours of IL-1β treatment, NNMT protein levels increased in a dose-dependent manner in both GO and non-GO fibroblasts (Fig. 2B). 

Figure 2.

 

IL-1β induced NNMT upregulation in GO and non-GO orbital fibroblasts. Orbital fibroblasts from individuals with (n = 3, black columns) or without GO (n = 3, white columns) were obtained and cultured. (A) After treatment with 10 ng/mL IL-1β, Western blot analysis was performed to evaluate how NNMT levels changed over time. NNMT-expression levels were measured at 0, 24, 48, and 72 hours after IL-1β treatment. (B) After 24 hours of treatment with IL-1β at varying doses (0.1, 1, and 10 ng/mL), NNMT levels were evaluated via Western blotting. The assays were performed in triplicate with cells from three different individuals with GO and three different individuals without GO. The results are presented as the mean relative density ratio and SD (**P < 0.01 versus untreated controls).

Knocking Down NNMT Reduced the Expression of Pro-Inflammatory Cytokines and COX-2 in GO and Non-GO Orbital Fibroblasts

Before evaluating the effect of NNMT on the pathogenesis of GO through NNMT silencing, we analyzed the effects of siNNMT on the viabilities of GO and non-GO orbital fibroblasts. MTT assay showed that siNNMT transfection did not decrease the cell viability of orbital fibroblasts to below 95% (Supplementary Fig. S1). We investigated whether NNMT contributed to inflammatory response in GO and non-GO orbital fibroblasts via Western blotting (Fig. 3). The protein-expression levels of NNMT in GO and non-GO fibroblasts demonstrated that siNNMT successfully silenced NNMT expression relative to siCon. The protein levels of pro-inflammatory cytokines (IL-6, IL-8, and MCP-1), and COX-2 were examined via Western blotting. We found that siNNMT transfection substantially decreased IL-1β-induced IL-6, IL-8, MCP-1, and COX-2 protein expression in GO and non-GO orbital fibroblasts. Cells that were not treated with IL-1β showed no noticeable differences pre- and post-siNNMT treatment. 

Figure 3.

 

Inhibitory effect of NNMT silencing on the expression of pro-inflammatory cytokines in GO and non-GO orbital fibroblasts. Orbital fibroblasts from (A) healthy control and (B) patients with GO (n = 3, each) were transfected with 10 nM siNNMT or siCon, cultured for 48 hours, and then either treated with IL-1β (10 ng/mL) for 72 hours or left untreated. NNMT and pro-inflammatory cytokines such as IL-6, IL-8, MCP-1, and COX-2 were analyzed via Western blotting. The assays were performed in triplicate with cells from three different individuals. The results showed the mean and SD of the relative density ratios for NNMT and pro-inflammatory cytokines (**P < 0.01 versus siCon transfectants).

Knocking Down NNMT Reduced Secretory Pro-Inflammatory Cytokines and PGE2 in GO and Non-GO Orbital Fibroblasts

The effects of siNNMT on the secretion of pro-inflammatory cytokines, IL-6, IL-8, MCP-1, and prostaglandin E2 (PGE2) from both GO and non-GO cells were analyzed using ELISA (Fig. 4). Silencing NNMT in GO and non-GO orbital fibroblasts significantly dampened the IL-1β-induced increase in the secretion of IL-6, IL-8, MCP-1, and PGE2. In cells not treated with IL-1β, the protein levels of the cytokines IL-6 and IL-8 decreased in GO cells after siNNMT treatment. 

Figure 4.

 

Inhibitory effect of NNMT silencing on the expression of secretory pro-inflammatory cytokines in (A) non-GO and (B) GO orbital fibroblasts. Orbital fibroblasts from individuals without (n = 3) or with GO (n = 3) were transfected with either 10 nM siNNMT or siCon. After culturing for 48 hours, the cells were either exposed to 10 ng/mL IL-1β for 72 hours or left untreated. Secretory pro-inflammatory cytokines such as IL-6, IL-8, MCP-1, and PGE2 were analyzed in the culture supernatants using ELISA. The assays were performed in triplicate with cells from three different individuals. The results are presented as mean ± SD (*P < 0.05 and **P < 0.01).

NNMT Antagonists Reduced IL-1β-Induced Inflammation

MTT assays were performed to identify non-toxic doses of NNMT inhibitors (5-amino-1MQ and 1-MNA) for orbital fibroblasts. Orbital fibroblasts from patients with or without GO were treated with small-molecule inhibitors for 24 hours. The 0 to 100 µM range of 5-amino-1MQ and the 0 to 5 mM range of 1-MNA did not affect decrease cell viability below 95% in normal and GO orbital fibroblasts (Supplementary Fig. S2). Pro-inflammatory cytokine expression was evaluated in GO orbital fibroblasts treated with an NNMT inhibitor post-IL-1β stimulation. Western blot analysis revealed that treatment with the NNMT inhibitors, 1-MNA and 5-amino-1MQ, significantly reduced the IL-1β-induced expression of IL-6, IL-8, MCP-1, and COX-2, when compared to its levels in untreated cells (Fig. 5). 

Figure 5.

 

Inhibitory effect of small-molecule NNMT inhibitors on the expression of pro-inflammatory cytokines in GO orbital fibroblasts. Orbital fibroblast from individuals with GO (n = 3) were not treated or were pretreated with either 100 µM 5-amino-1MQ or 5 mM 1-MNA for 30 minutes before exposure to 10 ng/mL IL-1β for 24 hours. The expression levels of NNMT and pro-inflammatory cytokines (IL-6, IL-8, MCP-1, and COX-2) were analyzed via Western blotting. The assays were performed in triplicate with cells from three different individuals. The mean and SD of the relative density ratios are shown (**P < 0.01 versus IL-1β treated cultures).

Effect of siNNMT on the Intracellular Signaling Pathways That Induced Pro-Inflammatory Cytokine Production

To identify signaling pathways affected by NNMT, phosphorylation of intracellular signaling molecules were assayed in GO and non-GO orbital fibroblasts (Fig. 6). Under the inflammatory condition induced by IL-1β, the activated forms of signaling molecules were upregulated and phosphorylated forms of intracellular signaling molecules (e.g. ERK, JNK, and p38) were substantially reduced in siNNMT-transfected cells. Decrease in the phosphorylated form of NF-κB was GO-specific. 

Figure 6.

 

Effect of siNNMT on intracellular signaling pathways that induce pro-inflammatory cytokine production. Orbital fibroblasts from (A) healthy control subjects and (B) patients with GO (n = 3, each) were transfected with 10 nM siNNMT or siCon, cultured for 48 hours, and then treated with either 10 ng/mL of IL-1β for 30 minutes or left untreated. We analyzed NNMT and intracellular signaling molecules such as ERK, JNK, p38, and NF-κB, along with their phosphorylated forms, via Western blotting. The assays were performed in triplicate with cells from three different individuals. The mean and SD of the relative density ratios for NNMT and downstream signaling molecules (**P < 0.01 versus siCon transfectants) are shown.

Discussion

In this study, we examined the roles of NNMT in GO pathogenesis, especially in inflammation. Our results demonstrated that inflammation in orbital fibroblasts led to increased NNMT expression. Silencing NNMT through siNNMT transfection or inhibiting NNMT activity with small molecule inhibitors significantly reduces pro-inflammatory signals, such as the production and secretion of pro-inflammatory cytokines and phosphorylation of signaling molecules. NNMT silencing also inhibited pro-inflammatory intracellular signaling pathways. These data indicate that NNMT inhibition may have therapeutic potential against GO. 

The main chemical reaction mediated by NNMT is nicotinamide methylation to form methylnicotinamide, with SAM acting as the methyl donor. Therefore, NNMT activity is directly related to nicotinamide levels because NNMT facilitates the removal of excess nicotinamide. Nicotinamide has therapeutic potential for GO, as evidenced by both in vitro and clinical studies. Previous in vitro data showed that nicotinamide treatment inhibited superoxide-induced fibroblast proliferation.²² Superoxide radicals are known to promote orbital fibroblast proliferation and differentiation.^32–34 The results of another study further revealed that nicotinamide inhibited the induction of various cell-surface proteins, such as intercellular adhesion molecule-1 and human leukocyte antigen-DR, which are typically induced by cytokines interferon gamma and tumor necrosis factor alpha in GO fibroblasts. Nicotinamide delayed GO progression in a clinical study.³⁵ Furthermore, data from clinical trials showed that nicotinamide treatment relieved the clinical symptoms of GO, as assessed using the NOSPECS total eye-score system.²⁴ Collectively, the results of these studies have indicated that nicotinamide, an NNMT substrate, can potentially be used as an antioxidant to treat GO. 

NNMT has been extensively studied as a regulator of NAD⁺ levels, especially in the context of autoimmune diseases. In the case of IBD, treatment-related changes in NAD⁺ levels correlated with shifts in NNMT expression.¹⁹ Several previous reports showed that NAD⁺ levels were diminished in patients with MS, and that the extent of this decrease correlated with disease severity.^36,37 In vivo data derived using mice with experimental autoimmune encephalomyelitis (EAE), a well-established animal model of MS, showed that maintaining higher NAD⁺ levels through NNMT inhibition is a beneficial mechanism for current MS treatment.²⁰ The results of another study of mice with EAE showed that NAD⁺ treatment inhibited inflammasome formation, modulated T cell differentiation, reduced the levels of pro-inflammatory factors, and increased expression of anti-inflammatory cytokine, IL-10.²¹ Although the pathways linking NNMT to inflammation may differ slightly across diseases, findings with IBD and MS suggested that inhibiting NNMT to regulate NAD⁺ levels is a promising therapeutic approach. 

To the best of our knowledge, this study is the first to provide insights on the role of NNMT in GO pathogenesis. Previously, we performed RNA sequencing to compare GO and non-GO orbital tissues, revealing a 2.55-fold increase in NNMT gene expression and 1.32-fold decrease in nicotinamide nucleotide adenylyltransferase 3 expression in the GO orbital tissues.³⁸ Therefore, we compared NNMT-expression levels between GO and non-GO tissues and found that NNMT expression was significantly higher in type Ⅱ GO orbital tissues than in non-GO or type I GO orbital tissues. Owing to type Ⅱ GO's association with greater inflammation and more severe outcomes, such as restrictive myopathy and diplopia, NNMT may play an important role in the changes such as inflammation, primary symptoms, and more severe sequelae of type Ⅱ GO. Furthermore, this insight into NNMT is an important clue for identifying unknown factors that might precede changes to NNMT in distinguishing between type Ⅰ and type Ⅱ GO. 

Orbital fibroblasts form patients with GO are continuously exposed to pro-inflammatory stimuli from immune cells infiltrating into orbital tissues. Therefore, treating orbital fibroblast cultures with pro-inflammatory stimulants such as IL-1β is useful for observing their response in an inflammatory condition, similar to in vivo event. IL-1β induces stronger production of pro-inflammatory cytokines and consequent characteristic remodeling in orbital fibroblasts from patients with GO,³⁹ and IL-1β has been used as an effective pro-inflammatory stimulant in many in vitro GO studies.^29–31 Our data showed that the inflammatory condition induced by IL-1β increased NNMT levels in orbital fibroblasts in time- and dose-dependent manners. Thus, we can assume that NNMT contributes to the inflammatory mechanism of GO in orbital tissues. 

Cytokines are essential for GO studies, as various types of T cells and the cytokines they release drive the primary immune response in the local orbital environment.^1,2,40,41 The major proteins involved in this process include IL-1β, IL-6, IL-8, MCP-1, COX-2, and PGE2. Our results confirmed that silencing NNMT mRNA with siNNMT reduced the production and secretion of these major inflammation-related proteins including pro-inflammatory cytokines such as IL-6, IL-8, and MCP-1 in fibroblasts. We also investigated changes in the levels of inflammation-related proteins with small molecule NNMT inhibitors, 1-MNA and 5-amino-1MQ. The levels of pro-inflammatory cytokines (IL-6, IL-8, and MCP-1), COX-2, and PGE2 decreased substantially after treatment with NNMT inhibitors, similar to the effects of mRNA silencing. Therefore, inhibiting NNMT activity attenuated IL-1β-induced inflammatory responses of orbital fibroblasts. These results clearly demonstrate the therapeutic potential of NNMT-modulating agents, which is consistent with previous studies showing that NNMT inhibition suppresses inflammation in autoimmune diseases.^19,20 

Previous findings suggest that the p38, ERK, JNK, and NF-κB pathways are key intracellular pathways involved in GO pathogenesis.^6,42,43 We discovered that signaling pathways, including ERK, JNK, and p38 pathways, were inhibited by siNNMT transfection in both GO and non-GO cells. In accordance with these findings, our findings indicate that NNMT inflammatory signaling in GO likely involves the ERK, JNK, and p38 pathways. Therefore, NNMT silencing could potentially be used to control inflammation in confluent GO fibroblasts. 

In this study, we showed that NNMT mRNA expression was elevated in GO orbital tissues and that NNMT inhibition reduced pro-inflammatory cytokines in cultured GO orbital fibroblasts. However, how NNMT specifically contributes to inflammation in GO remains unclear. As GO is a complex autoimmune disease influenced by various genetic and environmental factors, in vitro results may not fully reflect the in vivo situation. Although several NNMT inhibitors have been discovered and studied with other diseases, no attempts have been made to target NNMT for GO treatment. With mounting evidence suggesting that NNMT is a viable therapeutic target for autoimmune diseases, cancer,⁴⁴ and metabolic disorders,^45,46 substantial progress has been made in developing various NNMT inhibitors, including those that compete with SAM, nicotinamide analogs, bisubstrate inhibitors, covalent inhibitors, and natural products.⁴⁷ However, further research is needed before NNMT inhibitors can be applied clinically. These inhibitors hold promise as a potential therapeutic option for GO, and clinical trials are needed to evaluate their effectiveness. 

In conclusion, NNMT appears to be a pivotal regulator of inflammatory processes in orbital fibroblasts. NNMT expression correlates with disease severity, and NNMT inhibition attenuates pro-inflammatory cytokine(IL-6, IL-8, and MCP-1), COX-2, and PGE2 production and suppresses critical intracellular signaling pathways. These findings suggest that targeting NNMT may offer a novel therapeutic strategy for mitigating inflammation in GO. 

Acknowledgments

Supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT, Ministry of Science and ICT; grant number RS-2023-00208570). The Korean government had no role in the design or conduct of the study. 

Portions of this study were presented at the Asia Pacific Society of Ophthalmic Plastic and Reconstructive Surgery (APSOPRS) 2024 Congress (Seoul, Korea). 

Author Contributions: All authors have read the article and approved of its submission for publication. D. Cho: Investigation, Formal analysis, Writing–original draft preparation, Writing–review & editing. S.H. Choi: Methodology, Investigation, Formal analysis. J.S. Yoon: Conceptualization, Methodology, Investigation, Formal analysis, Writing–review & editing. J. Ko: Conceptualization, Methodology, Investigation, Formal analysis, Writing–original draft preparation, Writing–review & editing. 

Disclosure: D. Cho, None; S. H. Choi, None; J. S. Yoon, None; J. Ko, None