MMP-25 Metalloprotease Regulates Innate Immune Response through NF- κB Signaling Clara Soria-Valles, Ana Gutiérrez-Fernández, Fernando G. Osorio, Dido Carrero, Adolfo A. Ferrando, Enrique Colado, This information is current as M. Soledad Fernández-García, Elena Bonzon-Kulichenko, of September 27, 2021. Jesús Vázquez, Antonio Fueyo and Carlos López-Otín J Immunol 2016; 197:296-302; Prepublished online 3 June 2016; doi: 10.4049/jimmunol.1600094 Downloaded from http://www.jimmunol.org/content/197/1/296 Supplementary http://www.jimmunol.org/content/suppl/2016/06/01/jimmunol.160009 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 41 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/197/1/296.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! Every submission reviewed by practicing scientists • 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. The Journal of Immunology MMP-25 Metalloprotease Regulates Innate Immune Response through NF-kB Signaling Clara Soria-Valles,* Ana Gutie´rrez-Ferna´ndez,* Fernando G. Osorio,* Dido Carrero,* Adolfo A. Ferrando,† Enrique Colado,‡ M. Soledad Ferna´ndez-Garcı´a,x Elena Bonzon-Kulichenko,{ Jesu´sVa´zquez,{ Antonio Fueyo,‖ and Carlos Lo´pez-Otı´n* Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune- related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose bio- logical function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proin- flammatory molecules. Moreover, these immune defects can be tracked to a defective NF-kB activation observed in Mmp25-deficient Downloaded from leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases. The Journal of Immunology, 2016, 197: 296–302. nflammation is part of the complex biological response of their structure in secreted and membrane-anchored proteases. Among innate immunity, which confers immediate defense against in- the membrane-type (MT)-MMPs, there are three different groups I fection protecting multicellular organisms from pathogens (1). depending on the way these proteases interact with the membrane. http://www.jimmunol.org/ Septic shock, induced by the LPS of Gram-negative bacteria, is a This interaction may be through a transmembrane domain, consequence of a disproportionate stimulation of host immune cells. through a GPI moiety, or through an N-terminal signal anchor (6). Leukocytes have a pivotal role on sensing the pathogens that invade The metalloprotease MT6-MMP or MMP-25 is anchored to the cell the organism and initiate the inflammatory response (1). However, membrane through a GPI moiety and is predominantly expressed in their activation must be precisely controlled to regulate the duration leukocytes, lung, and spleen (7, 8). Previous works have also shown and extension of the inflammation, and therefore protect the host that both classical and alternative activation of macrophages in- from tissue damage. crease MMP-25 steady-state mRNA levels (9). In vitro functional Matrix metalloproteinases (MMPs) are a family of zinc-dependent experiments have revealed that this metalloprotease is able to clear by guest on September 27, 2021 endopeptidases that cleave extracellular matrix components with wide components of the extracellular matrix such as type IV collagen, substrate specificity (2, 3) and are involved in multiple pathological fibronectin, fibrin, and gelatin, and its activity is tightly regulated by processes (4, 5). These enzymes have been classified according to tissue inhibitor of metalloproteinase-1 (10). MMP-25 has also been linked with some pathological processes such as multiple sclerosis or cancer, but its biological role has remained largely unknown (11, *Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Medicina, Instituto 12). In this work, and as part of our long-term studies aimed at Universitario de Oncologı´a, Universidad de Oviedo, 33006 Oviedo, Spain; †Institute for generating mouse models of protease deficiency (5, 13–16), we Cancer Genetics, Columbia University, New York, NY 10032; ‡Servivio de Hematologı´a, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain; xServicio de Anatomı´a describe the generation of Mmp25-deficient mice and evaluate the Patolo´gica, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain; {Laboratorio biological function of MT6-MMP in innate immunity. de Proteo´mica Cardiovascular, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain; and ‖A´ rea de Fisiologı´a, Departamento de Biologı´a Funcional, Facultad de Medicina, Instituto Universitario de Oncologı´a, Universidad de Oviedo, 33006 Oviedo, Materials and Methods Spain Generation of Mmp25 knockout mice ORCIDs: 0000-0001-7869-838X (D.C.); 0000-0001-8675-8207 (E.C.); 0000-0003- 0852-7520 (E.B.-K.); 0000-0003-1461-5092 (J.V.). Mmp25-targeted ESCs clones from Texas A&M Institute for Genomic Medicine were microinjected into C57BL/6 mouse blastocysts to produce Received for publication January 19, 2016. Accepted for publication May 2, 2016. chimeric mice that were then subsequently crossed with C57BL/6 mice to This work was supported by grants from Ministerio de Economı´a y Competitividad generate Mmp25-heterozygous mice. Mice genotyping was performed by (Spain) and Red Tema´tica de Investigacio´n Cooperativa de Centros de Cancer-Spain. PCR with the following oligonucleotides: 59- CCTGATCAAGTTCTTG- The Instituto Universitario de Oncologı´a is supported by Fundacio´n Cajastur-Asturias. CTTGC-39;59-ATGGCTCGGAGTCTTTAAAC-39; and 59-CCAATAA- This work was also supported through the generous support of J.I. Cabrera. C.L-O. is an investigator of the Botin Foundation supported by Banco Santander through its Santander ACCCTCTTGCAGTTGC-39. The PCR products consisted in fragments of Universities Global Division. E.B.-K. and J.V. are supported by La Red de Investigacio´n 309 bp (knockout-allele) and 531 bp (wild-type allele). Cardiovascular–Las Redes Tema´ticas de Investigacio´n Cooperativa en Salud, Fondo de Investigaciones Sanitarias, and Instituto de Salud Carlos III. Mice procedures Address correspondence and reprint requests to Dr. Carlos Lo´pez-Otı´n, Departamento Eight- to 10-wk-old male mice received an i.p. injection of LPS (20 mg/kg, de Bioquı´mica y Biologı´a Molecular, Facultad de Medicina, Universidad de Oviedo, serotype O55:B5; Sigma-Aldrich) dissolved in saline buffer, and survival 33006 Oviedo, Spain. E-mail address: [email protected]. was evaluated. Isolation of neutrophils from mouse bone marrow was The online version of this article contains supplemental material. performed as described previously (17). Briefly, after flushing bone marrow cells from mice tibias, they were layered on a Percoll gradient Abbreviations used in this article: HA, hemagglutinin; MMP, matrix metalloprotease; 3 MT, membrane-type; SLAM, signaling lymphocyte activation molecule; TRAF6, (80:65:50%), centrifuged for 30 min at 500 g, and recovered from the TNFR-associated factor 6; UEV1A, ubiquitin-conjugating enzyme E2 variant 1A. third layer of the gradient. For the isolation of peritoneal macrophages, mice were injected with 1 ml 10% Brewer thioglycollate medium as de- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 scribed previously (18). Three days after injection, peritoneal macrophages www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600094 The Journal of Immunology 297 Downloaded from FIGURE 1. Generation of Mmp25-deficient mice. (A) Schematic representation of Mmp25-targeted allele. Genotyping strategy is indicated as well as the position of the inserted cassette. (B) PCR analysis using Mmp25-specific oligonucleotides, genomic DNA from Mmp25+/+, Mmp25+/2 and Mmp252/2 mice were used as template. (C) Representative picture of 6-mo-old wild-type and Mmp25-deficient mice. Original magnification 30.3. (D) Longevity analysis of +/+ 2/2 E wild-type and Mmp25-deficient mice. Kaplan–Meier survival plot of Mmp25 (n = 10) and Mmp25 (n = 10) mice. ( ) Quantitative RT-PCR of Mmp25 http://www.jimmunol.org/ in bone marrow, lung, and spleen from wild-type (n = 4) and Mmp25-deficient (n = 4) mice. Mean relative mRNA levels are shown. *p , 0.05, **p , 0.01, two-tailed Student t test. were obtained by