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Annexin A1 Natural Protease Inhibitors Are Mediated By Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1 This information is current as Juliana P. Vago, Luciana P. Tavares, Michelle A. Sugimoto, of September 28, 2021. Graziele Letícia N. Lima, Izabela Galvão, Thais R. de Caux, Kátia M. Lima, Ana Luíza C. Ribeiro, Fernanda S. Carneiro, Fernanda Freire C. Nunes, Vanessa Pinho, Mauro Perretti, Mauro M. Teixeira and Lirlândia P. Sousa J Immunol published online 22 January 2016 Downloaded from http://www.jimmunol.org/content/early/2016/01/22/jimmun ol.1500886 Supplementary http://www.jimmunol.org/content/suppl/2016/01/22/jimmunol.150088 http://www.jimmunol.org/ Material 6.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 22, 2016, doi:10.4049/jimmunol.1500886 The Journal of Immunology Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1 Juliana P. Vago,*,†,‡ Luciana P. Tavares,‡ Michelle A. Sugimoto,*,x Graziele Letı´cia N. Lima,*,‡ Izabela Galva˜o,‡ Thais R. de Caux,*,‡ Ka´tia M. Lima,*,†,‡ Ana Luı´za C. Ribeiro,*,‡ Fernanda S. Carneiro,*,‡ Fernanda Freire C. Nunes,* Vanessa Pinho,†,‡ Mauro Perretti,{ Mauro M. Teixeira,‡ and Lirlaˆndia P. Sousa*,†,‡,x Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) Downloaded from protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage as- sociated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-kB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neu- trophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to http://www.jimmunol.org/ increased levels of intact AnxA1 and decreased expression of NF-kB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals–induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis. The Journal of Immunology, 2016, 196: 000–000. he inflammatory process triggered by infection or tissue Proteases are enzymes produced by a variety of phagocytic in- damage is characterized by microscopic events that in- flammatory cells, including neutrophils (3, 4). Neutrophil elastase by guest on September 28, 2021 T clude increased vascular permeability and leukocyte ac- (NE) and proteinase 3 (PR3) are destructive serine proteases with cumulation. Leukocyte recruitment, mainly polymorphonuclear a range of substrates causing impact on cell and tissue function leukocytes (PMN), is triggered by a number of proinflammatory through diverse mechanisms, from degradation of ingested path- mediators generated at the site of inflammation acting as chemo- ogens to favoring cell motility through the extracellular matrix (2). tactic agents (1). Once recruited, PMNs release several granules rich Therefore, it is not surprising that protease activity is tempered in proteases that are important against infection. However, neutrophil by anti-protease molecules, which are secreted to neutralize any products can also be harmful to the host leading to intense tissue excess of these enzymes. Antiproteases are classified as both injury (2). systemic (produced by hepatocytes and distributed through the *Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Farma´cia, L.P.S., M.M.T., and J.P.V. designed research, analyzed data, and wrote the paper; J.P.V., Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Brazil; L.P.T., and M.A.S. performed experiments and analyzed data; M.A.S., G.L.N.L., †Programa de Po´s-Graduac¸a˜o em Biologia Celular, Departamento de Morfologia, K.M.L., T.R.d.C., and F.S.C. performed Western blot analysis; J.P.V. and A.L.C.R. Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, 31270-901 carried out some in vivo experiments; F.F.C.N. helped with apoptosis experiments; Belo Horizonte, Brazil; ‡Laborato´rio de Imunofarmacologia, Departamento de Bio- I.G. and J.P.V. carried out in vitro neutrophil analysis and in vivo experiments on quı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de the murine model of gout; V.P. provided expertise; and M.P. provided essential Minas Gerais, 31270-901 Belo Horizonte, Brazil; xPrograma de Po´s-Graduac¸a˜oem tools and guidance in the study and contributed to manuscript writing. Cieˆncias Farmaceˆuticas, Faculdade de Farma´cia, Universidade Federal de Minas { Address correspondence and reprint requests to Dr. Lirlaˆndia P. Sousa or Dr. Mauro Gerais, 31270-901 Belo Horizonte, Brazil; and William Harvey Research In- M. Teixeira, Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de stitute, Barts and The London School of Medicine, Queen Mary University of Farma´cia, Universidade Federal de Minas Gerais, Avenida Antoˆnio Carlos 6627, Pam- London, London EC1M 6BQ, United Kingdom pulha, 31270-901 Belo Horizonte, Minas Gerais, Brazil (L.P.S.) or Departamento de ORCIDs: 0000-0001-8188-3738 (J.P.V.); 0000-0002-4527-6065 (M.A.S.); 0000- Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de 0002-3243-8424 (T.R.d.C.); 0000-0003-1038-2324 (V.P.); 0000-0003-2068-3331 Minas Gerais, Avenida Antoˆnio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, (M.P.); 0000-0002-6944-3008 (M.M.T.); 0000-0002-1042-9762 (L.P.S.). Minas Gerais, Brazil (M.M.T.). E-mail addresses: [email protected] (L.P.S.) or [email protected] (M.M.T.) Received for publication April 15, 2015. Accepted for publication December 14, 2015. The online version of this article contains supplemental material. This work was supported by grants from Conselho Nacional de Desenvolvimento Abbreviations used in this article: AnxA1, annexin A1; CR, cleavage-resistant; ELA, Cientı´fico e Tecnolo´gico (Conselho Nacional de Pesquisas [Brazil]), Fundac¸ao de Elafin; GC, glucocorticoid; i.pl., intrapleural; Mres, resolution-promoting macro- Amparo a` Pesquisa do Estado de Minas Gerais, Pro´-Reitoria de Pesquisa da Uni- phage; MSU, monosodium urate; NE, neutrophil elastase; PMN, polymorphonuclear versidade Federal de Minas Gerais (Programa de Auxı´lio a` Pesquisa de Doutores leukocyte; PR3, proteinase 3; Ri, resolution interval; SIV, sivelestat; SLPI, secretory Rece´m-Contratados), the European Community’s Seventh Framework Programme leukocyte protease inhibitor. (FP7-2007-2013) under Grant Agreement HEALTH-F4-2011-281608, and the Wil- liam Harvey Research Foundation. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500886 2 PROTEASE INHIBITORS EVOKE INFLAMMATION RESOLUTION circulating) and alarm (synthesized and secreted by local cells to recombinant human SLPI (number 1274-PI-100; R&D Systems) were the site of inflammation) (5, 6). Alarm antiproteases such as se- dissolved in DMSO and diluted further in PBS. Endotoxin level in , m cretory leukocyte protease inhibitor (SLPI) and Elafin are secreted recombinant human SLPI was 1.0 EU/1 g of the protein by the Limulus amebocyte lysate method (as presented in R&D Systems catalog predominantly by the mucosal epithelium, and their levels are number1274-PI-100). Rabbit anti–P-ERK1/2 (number 4377), anti–caspase-3 modulated during multiple pathological conditions (3, 4, 6). Re- (number 9665), anti–Mcl-1 (number 5453), anti-GAPDH (number 3683), cent investigations indicate that SLPI and Elafin are inducible in and mouse anti–P-IkB-a
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