{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T10:58:00Z","timestamp":1781607480417,"version":"3.54.5"},"reference-count":54,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2017,10,27]],"date-time":"2017-10-27T00:00:00Z","timestamp":1509062400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education","award":["NRF-2013R1A1A2009822"],"award-info":[{"award-number":["NRF-2013R1A1A2009822"]}]},{"name":"Ministry of Education","award":["NRF-2015R1D1A1A01059584"],"award-info":[{"award-number":["NRF-2015R1D1A1A01059584"]}]},{"name":"Ministry of Health &amp; Welfare","award":["HI16C0965"],"award-info":[{"award-number":["HI16C0965"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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-\u03baB p65 (NF-\u03baB 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.<\/jats:p>","DOI":"10.3390\/ijms18112258","type":"journal-article","created":{"date-parts":[[2017,10,27]],"date-time":"2017-10-27T11:33:28Z","timestamp":1509104008000},"page":"2258","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells"],"prefix":"10.3390","volume":"18","author":[{"given":"Byungki","family":"Jang","sequence":"first","affiliation":[{"name":"Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do 14066, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7875-7482","authenticated-orcid":false,"given":"Akihito","family":"Ishigami","sequence":"additional","affiliation":[{"name":"Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong-Sun","family":"Kim","sequence":"additional","affiliation":[{"name":"Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do 14066, Korea"},{"name":"Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Gangwon-do 24252, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9251-8442","authenticated-orcid":false,"given":"Eun-Kyoung","family":"Choi","sequence":"additional","affiliation":[{"name":"Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do 14066, Korea"},{"name":"Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon, Gangwon-do 24252, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,10,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1038\/nsmb799","article-title":"Structural basis for Ca2+-induced activation of human PAD4","volume":"11","author":"Arita","year":"2004","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.1002\/bies.10357","article-title":"PAD, a growing family of citrullinating enzymes: Genes, features and involvement in disease","volume":"25","author":"Vossenaar","year":"2003","journal-title":"Bioessays"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1083\/jcb.200806072","article-title":"Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation","volume":"184","author":"Wang","year":"2009","journal-title":"J. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"42","DOI":"10.4161\/pri.22380","article-title":"Peptidylarginine deiminase and protein citrullination in prion diseases: Strong evidence of neurodegeneration","volume":"7","author":"Jang","year":"2013","journal-title":"Prion"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"700","DOI":"10.2174\/1389450116666150202160954","article-title":"Protein Arginine Deiminases and Associated Citrullination: Physiological Functions and Diseases Associated with Dysregulation","volume":"16","author":"Witalison","year":"2015","journal-title":"Curr. Drug Targets"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1016\/j.autrev.2016.03.003","article-title":"Autoantibodies in inflammatory arthritis","volume":"15","author":"Conigliaro","year":"2016","journal-title":"Autoimmun. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1007\/s00281-017-0627-z","article-title":"The role of autoantibodies in the pathophysiology of rheumatoid arthritis","volume":"39","author":"Derksen","year":"2017","journal-title":"Semin. Immunopathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4396","DOI":"10.4049\/jimmunol.1001620","article-title":"N-\u03b1-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis","volume":"186","author":"Willis","year":"2011","journal-title":"J. Immunol."},{"key":"ref_9","first-page":"467","article-title":"Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis","volume":"6","author":"Moscarello","year":"2013","journal-title":"Dis. Model. Mech."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"G929","DOI":"10.1152\/ajpgi.00435.2010","article-title":"Suppression of colitis in mice by Cl-amidine: A novel peptidylarginine deiminase inhibitor","volume":"300","author":"Chumanevich","year":"2011","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"36053","DOI":"10.18632\/oncotarget.5937","article-title":"Molecular targeting of protein arginine deiminases to suppress colitis and prevent colon cancer","volume":"6","author":"Witalison","year":"2015","journal-title":"Oncotarget"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1161\/CIRCRESAHA.114.303312","article-title":"Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis","volume":"114","author":"Knight","year":"2014","journal-title":"Circ. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2981","DOI":"10.1172\/JCI67390","article-title":"Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus","volume":"123","author":"Knight","year":"2013","journal-title":"J. Clin. Investig."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"F1052","DOI":"10.1152\/ajprenal.00243.2014","article-title":"Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury","volume":"307","author":"Ham","year":"2014","journal-title":"Am. J. Physiol. Ren. Physiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1111\/jnc.12744","article-title":"Peptidylarginine deiminases: Novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates","volume":"130","author":"Lange","year":"2014","journal-title":"J. Neurochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"25941","DOI":"10.1074\/jbc.M112.375725","article-title":"Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity","volume":"287","author":"Wang","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"11727","DOI":"10.1021\/bi061180d","article-title":"Inhibitors and inactivators of protein arginine deiminase 4: Functional and structural characterization","volume":"45","author":"Luo","year":"2006","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1189\/jlb.3A0314-142RR","article-title":"Peptidylarginine deiminase inhibition impairs Toll-like receptor agonist-induced functional maturation of dendritic cells, resulting in the loss of T cell-proliferative capacity: A partial mechanism with therapeutic potential in inflammatory settings","volume":"97","author":"Jang","year":"2015","journal-title":"J. Leukoc. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1089\/107999003322277838","article-title":"Regulatory role of nitric oxide on monocyte-derived dendritic cell functions","volume":"23","author":"Corinti","year":"2003","journal-title":"J. Interferon Cytokine Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1189\/jlb.1010550","article-title":"Nitric oxide and redox mechanisms in the immune response","volume":"89","author":"Wink","year":"2011","journal-title":"J. Leukoc. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.imlet.2007.04.013","article-title":"Nitric oxide, chronic inflammation and autoimmunity","volume":"111","author":"Nagy","year":"2007","journal-title":"Immunol. Lett."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1016\/j.pharmthera.2013.07.004","article-title":"Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: Possible therapeutic targets?","volume":"140","author":"Rochette","year":"2013","journal-title":"Pharmacol. Ther."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.it.2015.01.003","article-title":"Nitric oxide synthase in innate and adaptive immunity: An update","volume":"36","author":"Bogdan","year":"2015","journal-title":"Trends Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1016\/j.ejphar.2004.07.030","article-title":"Regulation of the expression of inducible nitric oxide synthase","volume":"500","author":"Kleinert","year":"2004","journal-title":"Eur. J. Pharmacol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"17736","DOI":"10.1074\/jbc.M601896200","article-title":"Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: Critical dimerization of inducible nitric-oxide synthase","volume":"281","author":"Sakai","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/0165-5728(95)00091-F","article-title":"Mediation of inflammation by encephalitogenic cells: Interferon gamma induction of nitric oxide synthase and cyclooxygenase 2","volume":"61","author":"Misko","year":"1995","journal-title":"J. Neuroimmunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/S1054-3589(08)60274-4","article-title":"Induction of cyclo-oxygenase and nitric oxide synthase in inflammation","volume":"35","author":"Appleton","year":"1996","journal-title":"Adv. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1966","DOI":"10.1126\/science.1119407","article-title":"Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2","volume":"310","author":"Kim","year":"2005","journal-title":"Science"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1853","DOI":"10.1084\/jem.20100239","article-title":"PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps","volume":"207","author":"Li","year":"2010","journal-title":"J. Exp. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1136\/ard.51.11.1219","article-title":"Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases","volume":"51","author":"Farrell","year":"1992","journal-title":"Ann. Rheum. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1002\/cyto.a.21178","article-title":"Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential","volume":"81","author":"Keshari","year":"2012","journal-title":"Cytometry"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1177\/0394632016651273","article-title":"A case for IL-6, IL-17A, and nitric oxide in the pathophysiology of Sj\u00f6gren\u2019s syndrome","volume":"29","author":"Benchabane","year":"2016","journal-title":"Int. J. Immunopathol. Pharmacol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1186\/ar3045","article-title":"Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus","volume":"12","author":"Nagy","year":"2010","journal-title":"Arthritis Res. Ther."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1038\/nm.4294","article-title":"An emerging role for neutrophil extracellular traps in noninfectious disease","volume":"23","author":"Jorch","year":"2017","journal-title":"Nat. Med."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Papayannopoulos, V. (2017). Neutrophil extracellular traps in immunity and disease. Nat. Rev. Immunol.","DOI":"10.1038\/nri.2017.105"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1097\/SHK.0000000000000146","article-title":"Role of neutrophil extracellular traps following injury","volume":"41","author":"Liu","year":"2014","journal-title":"Shock"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"227","DOI":"10.3389\/fimmu.2016.00227","article-title":"Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production","volume":"7","author":"Kusunoki","year":"2016","journal-title":"Front. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/j.niox.2010.01.001","article-title":"Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation","volume":"22","author":"Patel","year":"2010","journal-title":"Nitric Oxide"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1189\/jlb.1010549","article-title":"Rac2 is required for the formation of neutrophil extracellular traps","volume":"90","author":"Lim","year":"2011","journal-title":"J. Leukoc. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"290","DOI":"10.3389\/fimmu.2017.00290","article-title":"Neutrophil Extracellular Traps of Cynoglossus semilaevis: Production Characteristics and Antibacterial Effect","volume":"8","author":"Zhao","year":"2017","journal-title":"Front. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"461","DOI":"10.3389\/fimmu.2016.00461","article-title":"A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination","volume":"7","author":"Konig","year":"2016","journal-title":"Front. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1007\/s11010-011-1023-y","article-title":"Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats","volume":"359","author":"Xia","year":"2012","journal-title":"Mol. Cell. Biochem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"6184","DOI":"10.4049\/jimmunol.162.10.6184","article-title":"Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages","volume":"162","author":"Castrillo","year":"1999","journal-title":"J. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.femsim.2005.03.012","article-title":"Inhibition of extracellular signal-regulated kinase 1\/2 augments nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells","volume":"45","author":"Koide","year":"2005","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"eaal3062","DOI":"10.1126\/sciimmunol.aal3062","article-title":"Citrullination of NF-\u03baB p65 promotes its nuclear localization and TLR-induced expression of IL-1\u03b2 and TNF\u03b1","volume":"2","author":"Sun","year":"2017","journal-title":"Sci. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"F437","DOI":"10.1152\/ajprenal.00254.2016","article-title":"Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion","volume":"311","author":"Rabadi","year":"2016","journal-title":"Am. J. Physiol. Ren. Physiol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"C1050","DOI":"10.1152\/ajpcell.1999.277.6.C1050","article-title":"Involvement of JAK2 and MAPK on type II nitric oxide synthase expression in skin-derived dendritic cells","volume":"277","author":"Cruz","year":"1999","journal-title":"Am. J. Physiol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"9797","DOI":"10.1074\/jbc.M609426200","article-title":"Essential role of the JAK\/STAT1 signaling pathway in the expression of inducible nitric-oxide synthase in intestinal epithelial cells and its regulation by butyrate","volume":"282","author":"Stempelj","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1002\/jnr.1169","article-title":"JAK\/STAT signaling pathway mediates cytokine-induced iNOS expression in primary astroglial cell cultures","volume":"65","author":"Santangelo","year":"2001","journal-title":"J. Neurosci. Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1177\/1947601910397187","article-title":"Jak\/STAT pathways in cytokine signaling and myeloproliferative disorders: Approaches for targeted therapies","volume":"1","author":"Jatiani","year":"2010","journal-title":"Genes Cancer."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.abb.2008.10.034","article-title":"Protein tyrosine nitration\u2014An update","volume":"484","author":"Ischiropoulos","year":"2009","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"2602","DOI":"10.2174\/0929867323666160627114839","article-title":"Emerging Roles of Protein S-Nitrosylation in Macrophages and Cancer Cells","volume":"23","author":"Benhar","year":"2016","journal-title":"Curr. Med. Chem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1876","DOI":"10.4049\/jimmunol.0900831","article-title":"Peroxynitrite-dependent killing of cancer cells and presentation of released tumor antigens by activated dendritic cells","volume":"184","author":"Fraszczak","year":"2010","journal-title":"J. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1124\/jpet.115.222745","article-title":"Inhibiting protein arginine deiminases has antioxidant consequences","volume":"353","author":"Witalison","year":"2015","journal-title":"J. Pharmacol. Exp. Ther."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/18\/11\/2258\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:48:41Z","timestamp":1760208521000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/18\/11\/2258"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,10,27]]},"references-count":54,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2017,11]]}},"alternative-id":["ijms18112258"],"URL":"https:\/\/doi.org\/10.3390\/ijms18112258","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,10,27]]}}}