The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells

International Journal of Molecular Sciences

Article The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells

Byungki Jang 1, Akihito Ishigami 2 ID , Yong-Sun Kim 1,3 and Eun-Kyoung Choi 1,4,*

1 Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do 14066, Korea; [email protected] (B.J.); [email protected] (Y.-S.K.) 2 Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan; [email protected] 3 Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Gangwon-do 24252, Korea 4 Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon, Gangwon-do 24252, Korea * Correspondence: [email protected]; Tel.: +82-31-380-1893

Received: 7 September 2017; Accepted: 17 October 2017; Published: 27 October 2017

Abstract: The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.

Keywords: Cl-amidine; peptidylarginine deiminase; inducible nitric oxide synthase; dendritic cells; inﬂammatory diseases

1. Introduction Peptidylarginine deiminase (PAD) enzymes mediate the citrullination process—which involves the conversion of arginine to citrulline on a protein or peptide but not to free arginine—through

Int. J. Mol. Sci. 2017, 18, 2258; doi:10.3390/ijms18112258 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2258 2 of 14 the generation of an active site cleft via conformational changes in the presence of high calcium concentrations both in vivo and in vitro [1]. Five PAD (1–4 and 6) enzymes have been identified in humans and rodents and share 70–95% amino acid identity; the calcium binding sites show very high levels of conservation, with the exception of those of PAD6 [1,2]. Citrullination, a class of post-translational modifications, plays key roles in chromatin remodeling, gene transcription, conformational stability, the generation of autoantibodies to self-proteins, and neutrophil extracellular trap (NET) formation. Consequently, citrullination has been linked to autoimmune diseases, inflammatory diseases, cancer, and neurodegeneration [2–5]. For example, citrullination of myelin basic protein (MBP), a major protein in the myelin sheath, results in open confirmation, rapid degradation by proteases, and less compact protein-lipid interactions, which are associated with a disorganized myelin structure. Notably, citrullinated MBP accounts for 45% of the total MBP in multiple sclerosis (MS) patients and for more than 80% in fulminant MS patients, whereas 20% of MBP is citrullinated in healthy brain and other neurological disorders [2]. Therefore, citrullinated MBP has been suggested as a hallmark of central nervous system demyelination in MS. Rheumatoid arthritis (RA) is characterized by a chronic inflammation of synovial joints with high amounts of citrullinated proteins and infiltration of inflammatory cells, including PAD2/PAD4-expressing macrophages, neutrophils, and dendritic cells [2,6]. Citrullinated proteins are a source of autoantigens that are responsible for the production of anti-citrullinated protein antibodies (ACPAs). ACPAs are detected in 60–80% of RA patients with a specificity of 85–99%; therefore, they are a useful diagnostic marker for RA. Moreover, immune complexes containing ACPA and citrullinated proteins stimulate a cytokine release, and glycosylation of ACPA leads to the differentiation of osteoclasts and bone loss [6,7]. A variety of PAD inhibitors have been shown to directly bind the enzyme active site. Currently, PAD inhibitors are widely studied as potential anti-cancer and anti-inflammatory drugs, and they can dramatically attenuate the disease course, severity, and clinical manifestations in many disease models, including collagen-induced arthritis [8], MS [9], colitis [10,11], atherosclerosis [12], lupus [13], kidney injury [14], hypoxic ischemia [15], and cancer [11,16]. The pan-PAD inhibitor Cl-amidine was designed to irreversibly inhibit PADs through covalent modification at the enzyme active site [17], and its therapeutic potential has been demonstrated in the aforementioned disease models. Recently, Cl-amidine was also shown to modulate dendritic cell (DC) functions, such as cytokine production, antigen presentation, and T-cell proliferation capacity, in vitro and in vivo [18]. In the present study, we found that Cl-amidine acts as a suppressor of inducible nitric oxide synthase (iNOS), leading to decreased NO (represented without a dot for the unpaired electron) production in DCs. NO is a gaseous free radical and is derived from free L-arginine catalyzed by the NOS family, which is divided into three types: calcium (Ca2+)-dependent constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and Ca2+-insensitive iNOS. Constitutive NOS induces low levels of NO, whereas iNOS produces high levels of NO for prolonged periods of time. The expression of iNOS primarily depends on pro-inflammatory molecules (signals) and pathogens, and this enzyme is therefore generally associated with the immune system [19,20]. NO modulates a number of physiological and pathophysiological processes, including inflammation, neurotransmission, platelet aggregation, vascular relaxation, and defense against pathogens [19,21]. Therefore, iNOS and NO have been linked to many inflammatory diseases, including cardiovascular diseases, septic shock, asthma, RA, and chronic obstructive pulmonary disease [21–23]. For these reasons, selective inhibition of iNOS is a promising approach for the treatment of various inflammatory diseases. Our findings suggest that the suppression of NO generation underlies the therapeutic effects of Cl-amidine in a number of diseases, and that Cl-amidine may have therapeutic benefits for the treatment of iNOS/NO-associated disorders. Int. J. Mol. Sci. 2017, 18, 2258 3 of 14

2. Results

2.1. Cl-Amidine Suppresses NO Generation in Lipopolysaccharide-Stimulated Dendritic Cells During LPS-induced DC maturation, we determined NO levels in the absence or presence of Cl-amidine. In DCs treated with Cl-amidine (50, 100, or 200 µM), LPS-induced NO production was signiﬁcantlyInt. J. Mol. reduced Sci. 2017, 18, 2258 by up to 30%, 60%, and 85%, respectively (Figure1A).3 of The 14 pan-NOS G inhibitor N -methyl-significantlyL -argininereduced by up (L to-NMMA) 30%, 60%, and was 85%, used respectively as a positive (Figure 1A). control The pan-NOS and completely inhibitor inhibited NO production.NG-methyl- We alsoL-arginine found (L-NMMA) that the was decreased used as a positive NO production control and completely induced inhibited by Cl-amidine NO occurs at an early timeproduction. point (6We h)also and found in that a time-dependent the decreased NO pr manneroduction induced (Figure by 1Cl-amidineB). These occurs results at an suggest that early time point (6 h) and in a time-dependent manner (Figure 1B). These results suggest that Cl- Cl-amidine actsInt.amidine J. asMol.a Sci.acts suppressor 2017 as, a18 suppressor, 2258 of NOof NO production. production. 3 of 14

significantly reduced by up to 30%, 60%, and 85%, respectively (Figure 1A). The pan-NOS inhibitor NG-methyl-L-arginine (L-NMMA) was used as a positive control and completely inhibited NO production. We also found that the decreased NO production induced by Cl-amidine occurs at an early time point (6 h) and in a time-dependent manner (Figure 1B). These results suggest that Cl- amidine acts as a suppressor of NO production.

Figure 1. Cl-amidineFigure 1. Cl-amidine suppresses suppresses nitric nitric oxide oxide (NO) (NO) generation generation in lipopolysaccharide in lipopolysaccharide (LPS)-treated (LPS)-treated dendritic cells (DCs). (A) DCs (5 × 105 cells) were stimulated with LPS (0.1 μg/mL), with or without 5 dendritic cellsCl-amidine (DCs). ((50,A) 100, DCs or 200 (5 × μM),10 forcells) 24 h. L-NMMA were stimulated (500 μM) was withused as LPS a positive (0.1 µcontrolg/mL), for nitric with or without Cl-amidine (50,oxide 100, synthase or 200 (NOS)µM), inhibition. for 24 (B h.) DCsL-NMMA (1 × 106 cells) (500 wereµM) stimulated was used with LPS asa (0.1 positive μg/mL), controlwith for nitric oxide synthaseor (NOS)without inhibition.Cl-amidine (200 (B )μ DCsM), for (1 the× 10indicated6 cells) lengths were of stimulated time. Quantitative with LPSNO levels (0.1 µ areg/mL), with or represented by bars (mean ± SD; n = 3). *** p < 0.001. without Cl-amidineFigure 1.(200 Cl-amidineµM), suppresses for the indicated nitric oxide lengths (NO) generation of time. in Quantitativelipopolysaccharide NO (LPS)-treated levels are represented by bars (mean2.2. NOSdendritic± ActivitySD; cellsn = Is(DCs). 3).not ***Inhibited (A)p DCs< 0.001. by(5 ×Cl-Amidine 105 cells) were stimulated with LPS (0.1 μg/mL), with or without Cl-amidine (50, 100, or 200 μM), for 24 h. L-NMMA (500 μM) was used as a positive control for nitric oxideNext, synthase we determined (NOS) inhibition. whether (B the) DCs decreased (1 × 106 cells) NO wereproduction stimulated we with observed LPS (0.1 was μg/mL), caused with by Cl- 2.2. NOS Activityamidine-inducedor Is without not Inhibited Cl-amidine inhibition by(200 of Cl-Amidine NOSμM), activity.for the indicated Recombinant lengths NOS of time.enzymes Quantitative (iNOS, eNOS,NO levels and are nNOS) wererepresented pretreated by with bars Cl-amidine, (mean ± SD; nand = 3). we *** pthen < 0.001. me asured NOS activity. As shown in Figure 2, Cl- Next, weamidine determined had no effect whether on the enzymatic the decreasedactivity of any NOS NO enzymes production we tested. we These observed results indicate was caused by 2.2.that NOS the inhibitionActivity Is ofnot NO Inhibited production by Cl-Amidine by Cl-amidi ne likely occurs through the misregulation of an Cl-amidine-inducedupstream inhibitionsignaling pathway of NOS for NO activity. production. Recombinant NOS enzymes (iNOS, eNOS, and nNOS) were pretreatedNext, with we Cl-amidine, determined whether and the we decreased then measuredNO production NOS we observed activity. was caused As shown by Cl- in Figure2, amidine-induced inhibition of NOS activity. Recombinant NOS enzymes (iNOS, eNOS, and nNOS) Cl-amidine hadwere nopretreated effect with on Cl-amidine, the enzymatic and we activitythen measured of any NOS NOS activity. enzymes As shown wein Figure tested. 2, Cl- These results indicate that theamidine inhibition had no effect of NOon the production enzymatic activity by Cl-amidineof any NOS enzymes likely we occurs tested. These through results the indicate misregulation of that the inhibition of NO production by Cl-amidine likely occurs through the misregulation of an an upstreamupstream signaling signaling pathway pathway for for NO NO production.production.

Figure 2. Cl-amidine does not directly inhibit NOS activity. Recombinant NOS (180 ng inducible NO synthase (iNOS); 210 ng endothelial NOS (eNOS); and 420 ng nNOS) was preincubated with or without Cl-amidine (200 μM) for 1 h, and then activity was measured for 1 h. Quantitative levels are represented by bars (mean ± SD; n = 3).

Figure 2. Cl-amidine does not directly inhibit NOS activity. Recombinant NOS (180 ng inducible NO Figure 2. Cl-amidinesynthase (iNOS); does 210 not ng directlyendothelial inhibitNOS (eNOS); NOS and activity. 420 ng nNOS) Recombinant was preincubated NOS with (180 or ng inducible NO synthasewithout (iNOS); Cl-amidine 210 ng (200 endothelial μM) for 1 h, and NOS then (eNOS); activity was and meas 420ured ng for nNOS)1 h. Quantitative was preincubated levels are with or without Cl-amidinerepresented (200 by barsµM) (mean for ± 1 SD; h, n and = 3). then activity was measured for 1 h. Quantitative levels are represented by bars (mean ± SD; n = 3). Int. J. Mol. Sci. 2017, 18, 2258 4 of 14 Int. J. Mol. Sci. 2017, 18, 2258 4 of 14

2.3. Cl-Amidine Suppresses iNOS Expression 2.3. Cl-Amidine Suppresses iNOS Expression In DCs, iNOS is responsible for NO production. Therefore, we measured iNOS expression In DCs, iNOS is responsible for NO production. Therefore, we measured iNOS expression during during DC maturation induced by LPS in the absence or presence of Cl-amidine. In the DCs that were DC maturation induced by LPS in the absence or presence of Cl-amidine. In the DCs that were treated with 50 μM, 100 μM, and 200 μM of Cl-amidine, LPS-induced iNOS expression was markedly treated with 50 µM, 100 µM, and 200 µM of Cl-amidine, LPS-induced iNOS expression was markedly suppressed by ~30%, 60%, and 80%, respectively (Figure 3A), in a dose-dependent manner. To suppressed by ~30%, 60%, and 80%, respectively (Figure3A), in a dose-dependent manner. To confirm confirm whether decreased iNOS expression was due to the cell death, we analyzed cell viability whether decreased iNOS expression was due to the cell death, we analyzed cell viability using Cell using Cell Counting Kit-8 (CCK-8) staining. No significant decrease in viability was observed in our Counting Kit-8 (CCK-8) staining. No significant decrease in viability was observed in our experimental experimental conditions (Figure 3B). Similar to our NO production results, iNOS expression was conditions (Figure3B). Similar to our NO production results, iNOS expression was suppressed at suppressed at an early time point (6 h) by Cl-amidine (Figure 3C). These results indicate that the an early time point (6 h) by Cl-amidine (Figure3C). These results indicate that the down-regulation of down-regulation of NO production by Cl-amidine treatment is caused by suppression of iNOS NO production by Cl-amidine treatment is caused by suppression of iNOS expression. expression. iNOS Next,Next, we we usedused RT-PCRRT-PCR toto investigateinvestigate whetherwhether Cl-amidine affects affects the the production production of of iNOS mRNAmRNA transcripts.transcripts. As As shown shown in in Figure Figure3D, 3D, Cl-amidine Cl-amidine affected affected transcription transcription of theof theiNOS iNOSgene gene at an at earlyan early time pointtime (3point h). Taken(3 h). together,Taken together, these findings these findings suggest su thatggest the that pan-PAD the pan-PAD inhibitor inhibitor Cl-amidine Cl-amidine is a candidate is a forcandidate suppression for suppression of iNOS. of iNOS.

FigureFigure 3.3. Cl-amidine suppresses suppresses iNOS iNOS induction induction in inLPS-treated LPS-treated DCs. DCs. LPS LPS(0.1 (0.1μg/mL)-treatedµg/mL)-treated or - 6 oruntreated -untreated DCs DCs (5 × (5 10×6 cells)10 cells) were werecultured cultured in the inabsence the absence or presence or presence of Cl-amidine of Cl-amidine (50, 100, (50, or 200 100, orμM) 200 forµM) 24 forh. (A 24) iNOS h. (A expression) iNOS expression was detected was detectedby Wester byn blotting Western with blotting an anti-iNOS with an antibody. anti-iNOS antibody.Anti-glyceraldehyde Anti-glyceraldehyde 3-phosphate 3-phosphate dehydrogenase dehydrogenase (GAPDH) (GAPDH) was used wasas the used loading as the control. loading Bottom control. Bottompanel: relative panel: relative intensity intensity of iNOS of in iNOS upper in panels upper wi panelsth loading with loadingcontrols controlsusing three using individuals three individuals of each ofgroup. each group.Error bars Error represent bars represent SD. ** p SD. < 0.05, ** p ***< 0.05,p < 0.001. *** p <(B 0.001.) Cell viability (B) Cell viabilitywas measured was measured using a CCK- using 6 a8 CCK-8 staining. staining. (C) In the (C )absence In the absenceor presence or presence of Cl-amidine of Cl-amidine (200 μM), (200 DCsµ (4M), × 10 DCs6 cells) (4 × were10 cells)incubated were incubatedwith LPS with(0.1 μ LPSg/mL) (0.1 forµ g/mL)the indicated for the lengths indicated of time. lengths iNOS of time.expression iNOS was expression detected was by Western detected byblotting Western with blotting an anti-iNOS with an antibody. anti-iNOS β antibody.-actin was βused-actin as was the usedloading as thecontrol. loading (D) control.In the absence (D) In theor 6 absencepresence or of presence Cl-amidine of Cl-amidine (200 μM), (200 DCsµ M),(5 × DCs106 cells) (5 × 10werecells) incubated were incubated with LPS with (0.1 LPSμg/mL) (0.1 µforg/mL) the forindicated the indicated lengths lengths of time. of mRNA time. mRNA levels of levels iNOS of andiNOS GAPDHand GAPDH were analyzedwere analyzed by RT-PCR. by RT-PCR.

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2.4. Cl-Amidine Does not Affect Mitogen-Activated Protein Kinases (MAPKs), Nuclear Factor-κB p65 (NF- κ2.4.B p65) Cl-Amidine Signaling, Does or Hypoxia-Inducible not Affect Mitogen-Activated Factor 1α (HIF-1 Proteinα) KinasesExpression (MAPKs), but Instead Nuclear Attenuates Factor- κSTATB p65 Activation(NF-κB p65) Signaling, or Hypoxia-Inducible Factor 1α (HIF-1α) Expression but Instead Attenuates STAT Activation The expression of iNOS is regulated by multiple upstream signaling pathways, including MAPK,The NF- expressionκB, phosphatidylinositol of iNOS is regulated 3-kinase by multiple (PI3K), upstream STAT-1, signaling and AP-1 pathways, [23–25]. including To determine MAPK, whetherNF-κB, phosphatidylinositolthe MAPK, NF-κB, or 3-kinasePI3K pathways (PI3K), are STAT-1, involved and AP-1in NO [ 23generation,–25]. To determine we first assessed whether NO the productionMAPK, NF- afterκB, orexposure PI3K pathways to SB203580 are involved (a p38 ininhibitor), NO generation, U0126 we(an firstMAPK/extracellular assessed NO production signal- regulatedafter exposure kinase to (ERK)1/2 SB203580 inhibitor), (a p38 inhibitor), SP600125 U0126 [a C-Jun (an N-terminal MAPK/extracellular kinase (JNK) signal-regulated inhibitor], BAY kinase 11- 7085(ERK)1/2 (an NF- inhibitor),κB, IκBα SP600125 inhibitor), [a C-Junand PI-103 N-terminal (a PI3K kinase inhibitor). (JNK) inhibitor],NO generation BAY 11-7085was significantly (an NF-κB, attenuatedIκBα inhibitor), by the and p38, PI-103 JNK, (aNF- PI3KκB, inhibitor).and PI3K inhibitors NO generation (~47%, was ~22%, significantly ~60%, and attenuated ~18%, respectively), by the p38, whereasJNK, NF- κtheB, andinhibitor PI3K inhibitors U0126 (~47%,enhanced ~22%, NO ~60%, generation and ~18%, (Figure respectively), 4A). Unchanged whereas the inhibitorMAPK phosphorylation,U0126 enhanced NOnuclear generation translocation (Figure4 ofA). NF- UnchangedκB p65 and MAPK phosphorylation phosphorylation, of I nuclearκBα by translocation Cl-amidine haveof NF- beenκB p65reported and phosphorylation previously [18]. of Here, IκBα bywe Cl-amidine confirmed have that beenCl-amidine reported had previously no effect [18 on]. Here,the phosphorylationwe confirmed that of Cl-amidine MAPKs or hadNF-κ noB effectp65 (Figure on the 4B). phosphorylation Next, we examined of MAPKs whether or NF- Cl-amidineκB p65 (Figure affects4B). theNext, DNA we binding examined activity whether of NF- Cl-amidineκB p65. Enhanced affects the DNA DNA bi bindingnding activity activity of of NF- NF-κBκ Bp65 p65. by EnhancedLPS was notDNA changed binding by activity Cl-amidine of NF- κtreatmentB p65 by LPS(Figure was not4C). changed Hypoxia-inducible by Cl-amidine factor-1 treatmentα (HIF-1 (Figureα) 4canC). stimulateHypoxia-inducible iNOS expression factor-1, butα (HIF-1 its expressionα) can stimulate was also iNOS unchanged expression, (Figure but 4D). its expressionInterestingly, was LPS- also inducedunchanged STAT1, (Figure STAT2,4D). and Interestingly, STAT3 phosphorylation LPS-induced STAT1, was decreased STAT2, and by Cl-amidine STAT3 phosphorylation treatment for was 2 h and/ordecreased 4 h by(Figure Cl-amidine 4D). Next, treatment we confirmed for 2 h and/or that the 4 h JAK (Figure and4D). STAT Next, inhibitors we confirmed effectively that thedown- JAK regulatedand STAT iNOS inhibitors expression. effectively The down-regulated JAK inhibitor iNOS tofacitinib expression. and The STAT JAK inhibitorinhibitor tofacitinibniclosamide and dramaticallySTAT inhibitor decreased niclosamide LPS-induced dramatically NO decreased producti LPS-inducedon and iNOS NO expression production (Figure and iNOS 4E,F). expression These results(Figure indicate4E,F). These that decreased results indicate STAT that phosphorylat decreased STATion by phosphorylation Cl-amidine prefer byentially Cl-amidine suppressed preferentially iNOS expression,suppressed resulting iNOS expression, in reduce resultingd production in reduced of NO in production DCs. of NO in DCs.

FigureFigure 4. 4. Cont.Cont.

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FigureFigure 4. 4. Cl-amidineCl-amidine does does not not affect affect MAPK/NF- MAPK/NF-κBκ signalingB signaling but but attenuates attenuates STAT STAT activation. activation. (A (A) After) After pretreatmentpretreatment with with SB203580 SB203580 (20 (20 μµM;M; p38 p38 inhibitor), inhibitor), U0126 U0126 (20 (20 μM;µM; ERK ERK inhibitor), inhibitor), SP600125 SP600125 (20 (20 μM;µM; µ κ κ α µ JNKJNK inhibitor), inhibitor), BAY BAY 11-7085 11-7085 (20 (20 μM;M; NF- NF-κB andB and IκB Iα Binhibitor),inhibitor), or PI-103 or PI-103 (5 μM; (5 PI3KM; PI3K inhibitor) inhibitor) for × 5 2 forh, DCs 2 h, (5 DCs × 105 (5 cells)10 werecells) incubated wereincubated with LPS for with 24 h. LPS NO for was 24 measured h. NO was by ELISA measured at 540 by nm ELISA (mean at ± µ ± 540SD; nmn = (mean3). *** p < SD;0.001.n = (B 3).–D ***) Inp the< 0.001. absence (B– Dor) presence In the absence of Cl-amidine or presence (200 of μM), Cl-amidine DCs (4 × (200 106–8M), × × 6 × 6 µ 10DCs6 cells) (4 were10 –8incubated10 cells) with wereLPS (0.1 incubated μg/mL) with for the LPS indicated (0.1 g/mL) lengths for of the time. indicated Expression lengths levels of time. of B α D phosphorylatedExpression levels (p-) of p38, phosphorylated ERK, JNK, p65 (p-) (B p38,), HIF-1 ERK,α, JNK,STAT1, p65 and ( ), STAT3 HIF-1 (D, STAT1,) were probed and STAT3 by Western ( ) were probed by Western blotting with appropriate antibodies. β-actin and GAPDH were used as the loading blotting with appropriate antibodies. β-actin and GAPDH were used as the loading controls. (C) After controls. (C) After DCs were activated with LPS for 1 h, DNA binding activity of NF-κB p65 was DCs were activated with LPS for 1 h, DNA binding activity of NF-κB p65 was measured by ELISA as measured by ELISA as described in the Materials and Methods. (E,F) After pretreatment with tofacitinib described in the Materials and Methods. (E,F) After pretreatment with tofacitinib (E: Tofa; JAK (E: Tofa; JAK inhibitor) or niclosamide (F: Niclo; STAT inhibitor) for 1.5 h, NO production and iNOS inhibitor) or niclosamide (F: Niclo; STAT inhibitor) for 1.5 h, NO production and iNOS expression expression were measured by ELISA (left panel) and Western blotting (right panel). *** p < 0.001. were measured by ELISA (left panel) and Western blotting (right panel). *** p < 0.001.

2.5.2.5. Cl-Amidine Cl-Amidine Impairs Impairs AP-1 AP-1 Family Family Member Member Activity Activity Next,Next, we we examined examined whether whether Cl-amidine Cl-amidine can can affect affect the the activity activity of of the the AP-1 AP-1 family, family, a acandidate candidate regulatoryregulatory factor factor for for iNOS iNOS expression. expression. When When AP-1 AP-1 dimers dimers are are activated, activated, they they bind bind to to a aspecific speciﬁc 0 0 double-strandeddouble-stranded DNA DNA sequence sequence containing containing the the TPA-responsive TPA-responsive element element (5 (5′-TGAGTCA-3-TGAGTCA-3′),), and and this this AP-1AP-1 family family activity alsoalso regulates regulates iNOS iNOS expression expression [24 ].[24]. Therefore, Therefore, we examined we examined whether whether Cl-amidine Cl- amidineregulates regulates LPS-induced LPS-induced activation activation of AP-1 usingof AP-1 nuclear using extract.nuclear As extract. shown As in shown Figure 5in, theFigure expression 5, the expressionlevels of the levels LPS-induced of the LPS-induced AP-1 family AP-1 members family (c-Fos, members JunD, (c-Fos, p-c-Jun, JunD, and p-c-Jun, JunB) wereand JunB) signiﬁcantly were significantlydecreased by decreased Cl-amidine by treatment.Cl-amidine treatment. InIn addition, addition, we we determined determined whether whether the the inhibition inhibition of of AP-1 AP-1 activity activity can can decrease decrease NO NO production production andand iNOS iNOS expression. expression. As As shown shown in in Figure Figure 5B,5B, treatment treatment with with the the AP-1 AP-1 inhibitor inhibitor T-5224 T-5224 (20 (20 μµM)M) slightlyslightly inhibited inhibited NONO productionproduction (~11%) (~11%) but but not not iNOS iNOS expression. expression. Moreover, Moreover, the other the AP-1 other inhibitor, AP-1 inhibitor,SR113201, SR113201, had no effecthad no on effect NO production.on NO producti Theseon. resultsThese results indicate indicate that AP-1 that activityAP-1 activity may notmay be notinvolved be involved in the in regulation the regulation of iNOS of iNOS expression expression in LPS-activated in LPS-activated DCs. DCs.

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Int. J. Mol. Sci. 2017, 18, 2258 7 of 14

Figure 5.FigureCl-amidine 5. Cl-amidine impairs impairs LPS-induced LPS-induced AP-1 AP-1 familyfamily activity. activity. (A ()A Nuclear) Nuclear proteins proteins (10 μg) (10 isolatedµg) isolated 7 from DCsfrom (1 DCs× 10 (17 ×cells) 10 cells) were were stimulated stimulated with with LPSLPS (0.1 μµg/mL)g/mL) with with or without or without Cl-amidine Cl-amidine (200 μM) (200 µM) for 1 h, and then c-Fos, JunD, p-c-Jun, JunB, and Fra-1 activity was measured as described in the for 1 h, and then c-Fos, JunD, p-c-Jun, JunB, and Fra-1 activity was measured as described in the Materials and Methods (mean ± SD; n = 3). * p < 0.05. NS, not significant. (B) After DCs (5 × 105 cells Figure 5. Cl-amidine impairs LPS-induced AP-1 family activity. (A) Nuclear proteins (10 μg) isolated 5 Materials and Methods6 (mean ± SD; n = 3). * p < 0.05. NS, not signiﬁcant. (B) After DCs (5 × 10 cells fromor 5 × DCs 10 cells)(1 × 10 were7 cells) incubated were stimulated with the AP-1with LPSinhibitor (0.1 μT-5224g/mL) forwith 2 h,or cells without were Cl-amidine stimulated (200with μ LPSM) or 5 × 106 cells) were incubated with the AP-1 inhibitor T-5224 for 2 h, cells were stimulated with LPS forfor 124 h, h, and and then then c-Fos, NO production JunD, p-c-Jun, (left JunB,panel) and and Fra-1 iNOS activity expression was (measuredright panel) as weredescribed assessed in the by for 24 h,MaterialsELISA and and then and Western NOMethods production blotting. (mean ** ± pSD; ( left< 0.01. n panel)= 3). * p < and 0.05. iNOS NS, not expression significant. ((rightB) Afterpanel) DCs (5 were × 105 assessedcells by ELISA andor 5 × Western 106 cells) blotting.were incubated ** p < with 0.01. the AP-1 inhibitor T-5224 for 2 h, cells were stimulated with LPS 2.6. Ineffectivefor 24 h, and Regulation then NO of production Cyclooxygenase (left panel) 2 (COX-2) and iNOS Expression expression and Prostaglandin(right panel) were E2 (PGE2) assessed by Production due to Cl-Amidine Treatment 2.6. IneffectiveELISA Regulation and Western of Cyclooxygenaseblotting. ** p < 0.01. 2 (COX-2) Expression and Prostaglandin E2 (PGE2) Production due to Cl-AmidinePGE2 is Treatment a major inflammatory mediator along with NO and is derived from cyclooxygenase 2 2.6.(COX-2). Ineffective Inflammatory Regulation ofstimuli Cyclooxygenase elicit the 2synthesis (COX-2) Expressionof iNOS and and COX-2 Prostaglandin proteins E2 over (PGE2) similar time PGEProductioncourses,2 is a majorand due these to inﬂammatoryCl-Amidine two proteins Treatment also mediator interact [26 along–28]. withTo investigate NO and whether is derived Cl-amidine from affects cyclooxygenase COX- 2

(COX-2).2 expression InﬂammatoryPGE2 is aand major PGE stimuliinflammatory2 production, elicit wemediator the measured synthesis along COX-2 wi ofth iNOSexpressi NO and andon is by derived COX-2 Western from proteins blotting cyclooxygenase with over an similar anti- 2 time courses,(COX-2).COX-2 and theseantibody Inflammatory two and proteins PGE stimuli2 levels alsoelicit using interactthe ELISA synthesis [in26 LPS-stimulated– of28 ].iNOS To and investigate COX-2DCs in proteinsthe whether absence over or Cl-amidinesimilar presence time of affects Cl-amidine. Expression of the COX-2 protein was down-regulated by ~20% in the presence of Cl- COX-2courses, expression and these and two PGE proteins2 production, also interact we [26 measured–28]. To investigate COX-2 expression whether Cl-amidine by Western affects blotting COX- with 2amidine expression treatment and PGE compared2 production, with wethe measuredLPS alone COX-2treatment expressi (Figureon 6A,B),by Western concomitant blotting with with a an similar anti- an anti-COX-2 antibody and PGE2 levels using ELISA in LPS-stimulated DCs in the absence or presence COX-2reduction antibody in PGE and2 formation PGE2 levels (Figure using 6C). ELISA This result in LPS-stimulated indicates that Cl-amidineDCs in the absencealso exerts or apresence mild effect of of Cl-amidine. Expression of the COX-2 protein was down-regulated by ~20% in the presence of Cl-amidine.on COX-2 induction Expression and of PGE the2 COX-2production. protein was down-regulated by ~20% in the presence of Cl- Cl-amidineamidine treatment treatment compared compared with with the LPS LPS alone alone treatment treatment (Figure (Figure 6A,B),6A,B), concomitant concomitant with a withsimilar a similar reductionreduction in PGE in2 PGEformation2 formation (Figure (Figure6C). 6C). This This result result indicates that that Cl-amidine Cl-amidine also alsoexerts exerts a mild aeffect mild effect on COX-2on COX-2 induction induction and and PGE PGE2 production.2 production.

Figure 6. Mild suppression of COX-2 expression and PGE2 production by Cl-amidine treatment. (A) LPS (0.1 μg/mL)-treated or -untreated DCs (1 × 107 cells) were cultured in the absence or presence of Cl-amidine (50, 100, and 200 μM) for 24 h. COX-2 expression was measured by Western blotting with an anti-COX-2 antibody. β-actin was used as the loading control. (B) Columns represent the relative FigureFigure 6. Mild 6. Mild suppression suppression of of COX-2 COX-2 expression expression and and PGE PGE2 production2 production by Cl-amidine by Cl-amidine treatment. ( treatment.A) 5 (A) LPSLPSintensity (0.1 (0.1µg/mL)-treated μ ofg/mL)-treated COX-2 from orthree -untreated -untreated independent DCs DCs (1 experiments. × (1 10×7 cells)107 cells) were (C) DCs cultured were (1 × cultured 10 in cells)the absence were in the stimulated or absence presence with or of presence LPS (0.1 μg/mL), with or without Cl-amidine (50, 100, or 200 μM), for 24 h. PGE2 production was of Cl-amidineCl-amidine (50, (50, 100, 100, and and 200 200µ μM)M) forfor 24 h. COX-2 COX-2 expression expression was was measured measured by Western by Western blotting blotting with with measured as described in the Materials and Methods (mean ± SD; n = 3). * p < 0.05. an anti-COX-2an anti-COX-2 antibody. antibody.β-actin β-actin was was used used as as thethe loading control. control. (B) (ColumnsB) Columns represent represent the relative the relative 5 intensity of COX-2 from three independent experiments. (C) DCs (1 × 10 cells)5 were stimulated with intensity of COX-2 from three independent experiments. (C) DCs (1 × 10 cells) were stimulated with LPS (0.1 μg/mL), with or without Cl-amidine (50, 100, or 200 μM), for 24 h. PGE2 production was µ µ LPS (0.1measuredg/mL), as withdescribed or withoutin the Materials Cl-amidine and Methods (50, 100, (mean or ± 200 SD; n M),= 3). for* p <24 0.05. h. PGE2 production was measured as described in the Materials and Methods (mean ± SD; n = 3). * p < 0.05.

3. Discussion Recently, PAD enzymes and protein citrullination have been reported to possess a variety of physiological activities, such as tumorigenesis, bactericidal effects, and immunomodulatory Int. J. Mol. Sci. 2017, 18, 2258 8 of 14 effects [5,18,29], suggesting that the inhibition of PAD is a potential therapeutic strategy for the treatment of various disease models. Indeed, the PAD inhibitor Cl-amidine abrogates citrullinated, histone-linked NET formation in vitro and in vivo [3,13], enhances p53-targeted gene expression [5], and regulates the function of immune cells such as T cells and DCs [18]. In the present study, we found that Cl-amidine acts as a potential inhibitor for NO production and iNOS expression (Figures1 and3 ). NO is involved in NET activity, autoimmune diseases, and inflammatory diseases [23,30–33]. Therefore, it is also possible that the suppression of NO production by Cl-amidine, as described here, might attenuate NO/iNOS-involved disease phenotypes. NETs have roles in many infectious and noninfectious diseases, including lupus, RA, and cancer [34,35]. NETs are highly decondensed chromatin structures containing histones, protease, and extracellular DNA, which can result in suicidal NETosis or vital NETosis [29,34,35]. The generation of NETs primarily protects the host from infection, but reducing NETs protects against tissue injury [34,36]. PAD4 deficiency and Cl-amidine treatment block NET formation and its release [29,37]. Moreover, exogenous NO promotes NET release, which is blocked by the reactive oxygen species scavenger N-acetyl cysteine [38]. In addition, NO is produced during NET release, and the iNOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) reduces NET formation [39,40]. Therefore, we hypothesized that reduced NET formation by Cl-amidine treatment not only blocked histone citrullination but also inhibited NO generation by suppressing iNOS expression. In other words, reduced disease activity by Cl-amidine treatment may be attributed to PAD inactivation with reduced NO generation in a number of diseases, such as lupus, MS, and RA [41]. The expression of iNOS is regulated by multiple upstream signaling pathways in mouse macrophages and DCs, including MAPK, NF-κB, STAT, PI3K, HIF-1α, and the AP1 family [23–25]. The inhibition of the MAPK, NF-κB p65, and PI3K signaling pathways inhibited NO production (Figure4A). The p38 (SB203580) and NF- κB p65 (BAY 11-7085) inhibitors suppressed ~50% of NO production compared with controls. The JNK and PI3K inhibitors were less effective, reducing NO production by ~22% and ~18%, respectively. Both a PI3K deficiency and a PI3K inhibitor impaired NO generation in peritoneal macrophages [42]. By contrast, it was found that two PI3K inhibitors, wortmannin and LY294002, actually increased iNOS and NO production in RAW264.7 macrophages [43]. This discrepancy may have been due to the different types of cells used in each set of experiments. Altogether, our observations indicate that multiple signaling pathways are involved in NO generation in DCs. Interestingly, the ERK inhibitor U0126 enhanced NO production (Figure4A). U126 was previously shown to enhance NO production in RAW264.7 macrophages [44]. Therefore, ERK may negatively regulate NO production in LPS-stimulated DCs. Because Cl-amidine had no effect on MAPK and NF-κB p65 signaling pathways (Figure4B) and the DNA-binding activity of NF- κB p65 (Figure4C), these signaling pathways are not likely associated with Cl-amidine-mediated iNOS suppression, although the MAPK and NF-κB p65 signaling pathways are involved in the regulation of iNOS expression. The PAD inhibitor BB-Cl-amidine {N-[(1S)-1-(1H-benzimidazol-2-yl)-4-[(2-chloro-1-iminoethyl) amino]butyl]-[1,10-biphenyl]-4-carboxamide} inhibited LPS-induced expression of interleukin (IL)-1β and tumor necrosis factor α (TNFα) by preventing nuclear translocation of NF-κB p65 in neutrophils [45]. Interestingly, citrullination of NF-κB p65 by LPS-mediated PAD4 enzyme activity enhanced its interaction with importin α3 and nuclear translocation. Similarly, PAD4 promoted nuclear translocation of NF-κB p65 in renal proximal tubules [46]. By contrast, Cl-amidine had no impact on LPS-induced phosphorylation and DNA binding activity of NF-κB p65 (Figure4B,C) and degradation of I κBα in DCs, although Cl-amidine inhibits LPS-induced cytokine production of IL-1β, TNFα, and IL-12p70 [18]. Because PAD activity is effectively inhibited by both BB-Cl-amidine and Cl-amidine, we hypothesized that PAD-targeted proteins and their function are different in each cell type, i.e., histone H3 citrullination is involved in NET formation in neutrophils and regulates gene expression in cancer cells [3,5,13]. Moreover, LPS promotes citrullination [18], and histone H3 is predominantly citrullinated in DCs. It is Int. J. Mol. Sci. 2017, 18, 2258 9 of 14 still unclear whether histone H3 citrullination is important during DC maturation, including gene expression, which should be further investigated in detail. JAK/STAT were implicated in NO generation in a variety of cells [47–49]. As JAK undergoes transphosphorylation and, subsequently, phosphorylate STATs, activated STAT proteins dimerize and translocate into the nucleus, where they activate or repress target gene promoters [50]. In this study, Cl-amidine down-regulated STAT phosphorylation (Figure4D). Involvement of STAT signaling in iNOS expression was also confirmed by treatment with the JAK inhibitor tofacitinib and the STAT inhibitor niclosamide (Figure4E,F). Although AP-1 activity, a potential regulator for iNOS expression, was significantly decreased by Cl-amidine, both iNOS expression and NO production in DCs were not affected by AP-1 inhibition (T-5224) (Figure5). Therefore, Cl-amidine-mediated suppression of iNOS expression may be due to the suppression of STAT phosphorylation but not the MAPK, NF-κB, HIF-1α, and AP-1 signaling pathways. Inflammatory stimuli elicit both NO and PGE2 production through the induction of iNOS and COX-2 over similar time courses. More specifically, NO enhances PGE2 production through S-nitrosylation of COX-2 [28]. Our data showed that LPS-induced COX-2 expression and PGE2 production still maintained ~80% of their normal expression levels by Cl-amidine (Figure6). Therefore, our results indicate that Cl-amidine selectively inhibits iNOS induction rather than COX-2 induction signaling. It is possible that a combination of Cl-amidine with a COX-2 inhibitor may have synergistic therapeutic benefits in inflammatory diseases. Physiologically, NO attacks the thiol groups of cysteines on proteins, forms S-nitrosothiols, − − and reacts with superoxide anions radical (O2 ) to generate peroxynitrite (ONOO ), which can modify proteins (e.g., tyrosine nitration) [23]. These modifications are involved in a number of disease models and cellular processes, such as signal transduction and apoptosis [51,52]. Furthermore, ONOO− secreted from DCs can kill tumor cells [53]. Therefore, further studies are needed to determine whether Cl-amidine can reduce S-nitrosothiol levels and nitration formation and modulate associated signaling pathways. In this study, we determined that Cl-amidine inhibits NO generation by suppressing iNOS transcription in DCs. It is also possible that Cl-amidine may have effects on iNOS expression in other cell types, including macrophages. Indeed, Witalison et al. showed that Cl-amidine suppressed iNOS induction in mouse macrophages [54]. NO also plays an important role in epithelial cells and endothelial cells, and further studies will be needed to determine whether Cl-amidine has an effect on these cell types. In summary, iNOS expression and NO production are reduced in DCs by the PAD inhibitor Cl-amidine through the repression of STAT activities. We hypothesized that Cl-amidine may downregulate NO production via modulation of iNOS expression in the other cell types, including macrophages. Taken together, these findings indicate that Cl-amidine could improve excessive NO-mediated inflammatory responses and damage in certain disease states. Therefore, PAD inhibitors should be further examined to determine whether they have potential therapeutic effect on the disease(s) associated with iNOS activity and/or NO production.

4. Materials and Methods

4.1. Chemicals and Reagents Cl-amidine (Calbiochem, Darmstadt, Germany), the TLR4 agonist lipopolysaccharide (ultrapure LPS; Invivogen, San Diego, CA, USA), SB203580, U0126, SP600125, BAY 11-7085, PI-103 (Adooq, Irvine, CA, USA), Tofacitinib, niclosamide (Tocris, Bristol, UK), and T-5224 (APExBIO, Houston, TX, USA) were purchased commercially. Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ten or twenty millimoles stock solution of Cl-amidine was dissolved in phosphate buffer solution (PBS), and other inhibitors were dissolved in DMSO. PBS or DMSO were used as vehicle controls in each experiment. Int. J. Mol. Sci. 2017, 18, 2258 10 of 14

4.2. Cell Culture C57BL/6 female mice (6–8 weeks of age) were purchased commercially (Orient bio, Seongnam, Korea). Animal experiments and research protocols were approved by the Institutional Animal Care and Use Committee of Hallym University (HMC2015-0-1130-16; 23 December 2015). DCs were cultured as described previously [16]. Briefly, bone marrow cells from the femurs and tibiae of C57BL/6 female mice (6–8 weeks) were cultured in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Biowest, Riverside, MO, USA), 10 mM HEPES buffer (pH 7.4, Sigma-Aldrich), 2 mM L-alanyl-L-glutamine dipeptide (Sigma-Aldrich), 50 µM 2-mercaptoethanol, MEM non-essential complements (Sigma-Aldrich), 20 ng/mL recombinant mouse granulocyte-macrophage colony stimulating factor (JW CreaGene, Seongnam, Korea), 0.5 ng/mL interleukin (IL)-4 (JW CreaGene), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 ◦C in a 5% CO2 incubator. Fresh medium was added on days 4 and 7, and all experiments were performed after 8 days of culture. The samples had more than 80% CD11c+ cells, as determined by flow cytometry.

4.3. NO Detection and NOS Activity The culture medium was obtained from LPS (0.1 µg/mL)- or vehicle-stimulated DCs (5 × 105 or 1 × 106 cells) in the absence or presence of Cl-amidine (50–200 µM). The levels of NO in the culture medium were measured by ELISA according to the manufacturer’s instructions (Intron Biotechnology, Seongnam, Korea). To assay NOS activity, recombinant NOS (180 ng iNOS; 210 ng eNOS; 420 ng nNOS; Enzo Life Sciences, New York, NY, USA) was incubated with or without Cl-amidine (200 µM) for 1 h, and then activity was measured for 1 h by ELISA, according to the manufacturer’s instructions (Biovision, Milpitas, CA, USA).

4.4. Western Blotting Analysis C57BL/6 DCs were lysed in PRO-PREP lysis buffer (Intron Biotechnology), and protein concentrations were measured using the Bicinchoninic Acid Kit (Thermo Fisher Scientific). Samples (40–50 µg) were loaded into sodium dodecyl sulfate polyacrylamide electrophoresis gels and transferred to polyvinylidene difluoride membranes (Merck-Millipore, Billerica, MA, USA). Rabbit monoclonal anti-phospho-p38 (1:1000), rabbit monoclonal anti-phospho-ERK (1:1000), rabbit monoclonal anti-phospho-JNK (1:1000), rabbit monoclonal anti-phospho-NF-κB p65 (1:1000), rabbit monoclonal anti-phospho-STAT1 (1:1000), rabbit monoclonal anti-phospho-STAT3 (1:1000), rabbit monoclonal anti-STAT1 (1:2000), rabbit monoclonal anti-STAT2 (1:2000), rabbit monoclonal anti-STAT3 (1:2000) (Cell Signaling, Danvers, MA, USA), and rabbit polyclonal anti-phospho-STAT2 antibodies (Merck-Millipore; 1:1000) were purchased and used in the Western blotting experiments. Mouse monoclonal anti-HIF-1α (1:1000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-β-actin (1:10,000; Sigma-Aldrich), mouse monoclonal anti-GAPDH (1:10,000; BETHYL, Montgomery, TX, USA), rabbit polyclonal anti-iNOS (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and goat polyclonal anti-COX-2 (1:1000; Santa Cruz Biotechnology) antibodies were used in the Western blotting experiments. The primary antibodies were detected with appropriate horseradish peroxidase-conjugated goat anti-mouse, rabbit anti-goat, or goat anti-rabbit secondary antibodies (1:5000; Cell Signaling or Merck-Millipore). Chemiluminescent signals were detected on X-ray film (Agfa HealthCare, Mortsel, Belgium) using ECL Western blotting detection reagents (ATTO, Tokyo, Japan).

4.5. Semi-Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) DCs were stimulated with LPS (0.1 µg/mL) with or without Cl-amidine (200 µM) for the indicated times, and then total RNA was extracted using the Ribospin RNA extraction kit following the manufacturer’s instructions (GeneAll, Seoul, Korea). The cDNA synthesis was performed with AMV reverse transcriptase (Promega, Madison, WI, USA) according to the instructions of the manufacturer. RT-PCR was performed using primers speciﬁc to the iNOS (600 bp; Int. J. Mol. Sci. 2017, 18, 2258 11 of 14 forward, 50-GGTATGCTGTGTTTGGCCTT-30 and reverse, 50-GCAGCCTCTTGTCTTTGACC-30) and GAPDH (200 bp; forward, 50-TGGTATCGTGGAAGGACTCATGAC-30 and reverse, and 50-ATGC CAGTGAGCTTCCCGTTCAGC-30) genes with GoTaq DNA polymerase, according to the manufacturer’s instructions (Promega). The resulting RT-PCR products were separated by gel electrophoresis on a 1.0% agarose gel with the SafeView nucleic acid gel stain (ABM, Richmond, BC, Canada) and then visualized using UV light.

4.6. NF-κB p65 Transcription Factor Activity Assay DCs (8 × 106 cells) were stimulated with LPS (0.1 µg/mL), with or without Cl-amidine (200 µM), for 1 h, and then cell lysates (20 µg) were assessed with the NF-κB p65 Transcription Factor Assay Kit, following the manufacturer’s instructions (Abcam).

4.7. Activator Protein-1 (AP-1) Transcription Activity Assay DCs (1 × 107 cells) were stimulated with LPS (0.1 µg/mL), with or without Cl-amidine (200 µM), for 1 h, and then nuclear proteins were isolated using the Nuclear Extraction Kit, following the manufacturer’s instructions (Abcam). To determine AP-1 family member activity, ten micrograms of total nuclear protein used with the AP-1 Transcription Factor Assay Kit, according to the manufacturer’s instructions (Abcam).

4.8. Prostaglandin E2 (PGE2) Assay LPS (0.1 µg/mL)-treated or -untreated DCs (1 × 105 cells) were cultured in the absence or presence of Cl-amidine (50, 100, or 200 µM) for 24 h. PGE2 production was measured by ELISA, according to the manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY, USA).

4.9. Statistical Analysis All data are presented as mean ± standard deviation (SD) values. The probability of statistically signiﬁcant differences between experimental groups was assessed by a two-sample t test or by one-way analysis of variance.

Acknowledgments: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2009822 and NRF-2015R1D1A1A01059584), and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C0965). Author Contributions: Byungki Jang designed the study, performed the experiments, and analyzed the data. Akihito Ishigami, Yong-Sun Kim, and Eun-Kyoung Choi discussed the data and read the manuscript. Byungki Jang and Eun-Kyoung Choi supervised the study and wrote the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

PAD Pepitylarginine deiminase iNOS Inducible nitric oxide synthase DC Dendritic cell LPS Lipopolysaccharide AP-1 Activator protein 1 MAPK Mitogen-activated protein kinases HIF-1 Hypoxia-inducible factor 1 NF-κB p65 Nuclear factor-kappaB p65 PI3K Phosphatidylinositol 3-kinase JNK C-Jun N-terminal kinase IL Interleukin Int. J. Mol. Sci. 2017, 18, 2258 12 of 14

COX-2 Cyclooxygenase 2 PGE2 Prostaglandin E2 JAK Janus kinase STAT Signal transducer and activator of transcription

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