MMP-25 Metalloprotease Regulates Innate Immune Response Through NF- Κb Signaling Clara Soria-Valles, Ana Gutiérrez-Fernández, Fernando G

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 29, 2021. Jesús Vázquez, Antonio Fueyo and Carlos López-Otín J Immunol published online 3 June 2016 http://www.jimmunol.org/content/early/2016/06/01/jimmun

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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 June 3, 2016, doi:10.4049/jimmunol.1600094 The Journal of Immunology

MMP-25 Metalloprotease Regulates Innate Immune Response through NF-kB Signaling

Clara Soria-Valles,* Ana Gutie´rrez-Fernańdez,* Fernando G. Osorio,* Dido Carrero,* Adolfo A. Ferrando,† Enrique Colado,‡ M. Soledad Fernańdez-Garcıá,x Elena Bonzon-Kulichenko,{ Jesu´sVa´zquez,{ Antonio Fueyo,‖ and Carlos Lo´pez-Otıń*

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 biological 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 proinflammatory 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: 000–000.

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 29, 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 Biologıá Molecular, Facultad de Medicina, Instituto 12). In this work, and as part of our long-term studies aimed at Universitario de Oncologıá, 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 Hematologıá, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain; xServicio de Anatomıá 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 Fisiologıá, Departamento de Biologıá Funcional, Facultad de Medicina, Instituto Universitario de Oncologıá, 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 Economıá y Competitividad generate Mmp25-heterozygous mice. Mice genotyping was performed by (Spain) and Red Tema´tica de Investigacioń Cooperativa de Centros de Cancer-Spain. PCR with the following oligonucleotides: 59- CCTGATCAAGTTCTTG- The Instituto Universitario de Oncologıá is supported by Fundacioń 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 Investigacioń 309 bp (knockout-allele) and 531 bp (wild-type allele). Cardiovascular–Las Redes Tema´ticas de Investigacioń 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-Otıń, Departamento Eight- to 10-wk-old male mice received an i.p. injection of LPS (20 mg/kg, de Bioquı´mica y Biologıá 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 2 MMP-25 REGULATES INNATE IMMUNITY 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 washing of the peritoneal cavity with RPMI 1640 medium from Addgene. HEK-293T cell line was purchased from the American Type containing 10% FBS and heparin. Determination of nitrite production in Culture Collection and maintained in culture following the provider’s macrophages was performed using Griess Reagent (Molecular Probes) fol- specifications. Transient transfections were performed using Lipofectamine lowing manufacturer instructions. Bone marrow transplantations were per- reagent (Life Technologies), and proteomic analyses were performed 48 h formed as described previously (19). Briefly, recipient mice were treated after transfection. Firefly luciferase under NF-kB promoter was transduced with 25 mg/kg/day busulfan (Sigma-Aldrich) for 4 d, followed by injection in HEK-293T cell line either with pcDNA-MMP25 or pcDNA3. of 200 mg/kg cyclophosphamide (Sigma-Aldrich). Twenty-four hours after by guest on September 29, 2021 last injection, bone marrow was collected from femurs and tibias of donor RNA and protein analysis mice by flushing the dissected bones with HBSS. Cell suspension was fil- Nuclear extracts and EMSA were performed as described previously (22). m 3 6 tered through 100- m filters and counted. A total of 2 10 cells were Briefly, NF-kB consensus oligonucleotides (Promega) were radiolabeled resuspended in HBSS and then injected in recipient animals via the jugular by T4 polynucleotide kinase (New England Biolabs) in the presence of vein. Eight weeks later, the transplanted animals received an i.p. injection of g-[32P]ATP. Labeled oligonucleotides were incubated with 15–20 mgnuclear LPS (20 mg/kg), and survival was evaluated. Hydrodynamic injection of a extracts in 13 binding buffer (43 glycerol, 20 mM Tris-HCl [pH 8], 60 mM plasmid encoding firefly luciferase under NF-kB promoter was carried out as NaCl, 5 mM MgCl2, 1 mM DTT, and 13 protease inhibitor mixture) in the described previously (20). For histological analysis, tissues were fixed in 4% presence of poly(deoxyinosinic-deoxycytidylic) acid. Complexes were run buffered paraformaldehyde solution. Paraffin sections were stained with H&E. in nondenaturing 6% acrylamide gels and exposed to X-ray detector Fuji All of the animal experiments were performed in accordance with the guidelines phosphorimager (Fujifilm Global). Total RNA from cells and tissue samples of the Committee for Animal Experimentation of the Universidad de Oviedo. were extracted using TRIzol reagent (Life Technologies) and processed FACS and blood analysis through alcohol precipitation. RNA pellets were then washed in cold 75% ethanol and resuspended in nuclease-free water (Life Technologies). The Blood was extracted directly from the mandibular sinus after anesthetizing samples were quantified and evaluated for purity (260/280-nm ratio) with a mice with isoflurane. Analysis of primitive cells (Lin2SCA-1+c-KIT+;LSK+), NanoDrop ND-1000 spectrophotometer. cDNA was synthesized with 1–4 mg committed progenitors (Lin2SCA-12c-KIT+;LSK2), and signaling lym- total RNA with the ThermoScript RT-PCR system (Invitrogen). RT-quantitative phocyte activation molecule (SLAM)-enriched long-term hematopoietic PCR was performed in triplicate for each experimental condition using stem cells (21) was performed by FACS using the following mAbs: PE anti- either TaqMan PCR Mastermix or SYBR Green PCR Master mix (Applied mouse CD117 (105807), PerCP/Cy5.5 anti-mouse CD45 (103132), PE/Cy7 Biosystems), according to the manufacturer’s instructions. To normalize anti-mouse CD150 (115914), allophycocyanin anti-mouse CD127 (135012), mRNA levels, GAPDH or ACTB probes were used. Protein lysates for allophycocyanin/Cy7 anti-mouse CD48 (103432), Brilliant Violet 421 anti- Western blot were prepared in radioimmunoprecipitation buffer; equal mouse Ly-6A/E (Sca-1) (108127), anti-mouse CD34 (11-0341-82; eBio- amounts of total proteins were loaded onto SDS-polyacrylamide gels. science), and Lineage mixture (558074; BD Pharmingen). FACS data were After electrophoresis, gels were electrotransferred onto nitrocellulose acquired using a FACScanto II flow cytometer (BD Biosciences) and ana- membranes and incubated overnight with the different primary Abs used. lyzed using Infinicyt software (Cytognos, Santa Marta, Spain). A represen- Finally, blots were incubated for 1 h with secondary Abs conjugated with tative FACS analysis gating scheme is shown in Supplemental Fig. 1. Blood HRP to develop immunoreactive bands. The primary Abs used in the study proteins separation was carried out using manual agarose gels (Hydragel were as follows: GFP (632592; BD Clontech), TLR4 (sc-293072; Santa K20; Sebia) and quantified by densitometry analysis. Serum IL-1a was Cruz Biotechnology), TNFR-associated factor 6 (TRAF6) (sc-7221; Santa determined by ELISA. A pool of four animal sera per condition was hy- Cruz Biotechnology), ubiquitin-conjugating enzyme E2 variant 1A (UEV1A) bridized onto mouse L308 glass slide arrays (RayBiotech). Data were (ab101476; Abcam), HA (3F10; Roche), IkBa (9242; Cell Signaling Tech- clustered using Cluster 3.0 software, and the heat maps were created using nology), and a-tubulin (T6074; Sigma-Aldrich). All the Abs were used at TreeView 1.1.5 software. 1:1000 dilution. Cell culture and transient transfection Immunoprecipitation MMP25 and TRAF6 cDNAs were amplified by PCR and either cloned into MMP-25 immunoprecipitation experiments were performed in the HEK- pcDNA3 or pEGFP. Hemagglutinin (HA)-ubiquitin plasmid was obtained 293T cell line. To this end, cells were transfected with either pEGFP-MMP25 The Journal of Immunology 3

FIGURE 2. Characterization of Mmp25-deﬁcient mice. (A)Meanrel- ative cell number in bone marrow from wild-type (n = 5) and knockout animals (n =5).(B)LSK+/LSK2 ratio and percentage of SLAM cells in LSK compartment of Mmp25+/+ (n =5)and Mmp252/2 (n =5)mice.(C) Blood proteinogram from Mmp25+/+ (n =4) and Mmp252/2 (n =4)animals.(D) IL-1a levels in peripheral blood from Mmp25+/+ (n =5)andMmp252/2 (n = 6) mice. Values were normal- ized to total viable cellularity. (E) Heat map represents alterations in proinﬂammatory cytokines protein levels between Mmp25+/+ (n =4)and Mmp252/2 (n =4)animals.Dataare Downloaded from displayed as log2-transformed expression signals. Error bars indicate SEM (*p , 0.05, **p , 0.01, two-tailed Student t test). GRA, granulocyte; LYM, lymphocyte; MONO, monocyte. http://www.jimmunol.org/

plasmid or pEGFP empty vector. Cells were lysed with coimmunoprecipi- and then incubated with anti-GFP–conjugated dynabeads for 1 h at 4˚C. tation buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, Beads were washed three times with lysis buffer, and bound proteins were 1mMMgCl2, 10% glycerol, and 13 complete protease inhibitors). Protein released from beads by boiling in 23 Laemmli sample buffer. Immuno- extracts were precleared for 2 h at 4˚C with dynabeads (Life Technologies) precipitates and input samples were resolved by SDS-PAGE or subjected to by guest on September 29, 2021

FIGURE 3. Reduced innate immunity in Mmp25-deﬁcient mice. (A) Kaplan–Meier survival plot of Mmp25+/+ (n =8)andMmp252/2 (n =8)malemice after injection with 20 mg/kg LPS. p , 0.01, log-rank/Mantel–Cox test. (B) Representative images from H&E staining of lung sections of LPS- treated and untreated Mmp25+/+ (n = 6) and Mmp252/2 (n = 6) mice. Plot represents inﬂammation and edema histological score in this mice cohort. Scale bar, 50 mm. (C) Peripheral blood levels of glutamate-pyruvate transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatinine, and blood urea nitrogen (BUN) in LPS-treated and untreated Mmp25+/+ and Mmp252/2 mice (n = 6 mice/ group). Mean values are represented. (D) Kaplan–Meier survival plot of wild-type males transplanted with knockout cells (n = 5) and knockout males transplanted with wild-type bone marrow (n = 5) after injection with 20 mg/kg LPS. p , 0.01, log-rank/ Mantel–Cox test. Error bars indicate SEM (*p , 0.05, **p , 0.01, two- tailed Student t test). 4 MMP-25 REGULATES INNATE IMMUNITY Downloaded from

FIGURE 4. Defective NF-kB activation in Mmp25-deficient mice. (A) NF-kB EMSA analysis in bone marrow neutrophils from untreated and LPS- 2 2 2 2 treated Mmp25+/+ and Mmp25 / mice (pool of five animals per group). (B) Representative image of bioluminescence signal in Mmp25+/+ and Mmp25 / http://www.jimmunol.org/ mice 3 d after hydrodynamic transfection. Original magnification 30.3. Plot represents bioluminescence signal quantification (n = 4/genotype). Mean values are represented as relative values of photon flux per second and square centimeter. (C–F) Quantitative RT-PCR of Csf2, Tnfa, Cox2, and Il1a in Mmp25+/+ and Mmp252/2 neutrophils after stimulation with LPS (10 mg/ml, 2 h). Mean relative mRNA levels of three replicates are shown. Error bars indicate SEM (*p , 0.05, **p , 0.01, two-tailed Student t test). protein digestion followed by nanoliquid chromatography coupled to mass The fact that Mmp25 is predominantly expressed in WBCs spectrometry for protein identification and quantification by peptide counting prompted us to analyze the hematological system in Mmp25- (23). For ubiquitination experiments, HEK-293T cells were transfected with deficient mice, but no significant differences between Mmp25+/+ HA-ubiquitin, pcDNA3-MMP25 and pEGFP-TRAF6 plasmids, and total ly- 2/2 by guest on September 29, 2021 sates were precipitated with anti-GFP Ab and immunostained with anti-HA Ab. and Mmp25 animals were observed neither in the distribution of blood cell populations (Fig. 2A) or in the hematopoietic stem Statistical analysis cell compartment (LSK+ cells and SLAMs) (Fig. 2B, Supplemental All the experimental data were collected from experiments performed in Fig. 1). However, additional analysis of blood parameters related technical triplicate; each experiment was repeated three times (unless with immune response revealed that knockout animals spontane- noted), and statistically significant results were obtained in independent ously developed hypergammaglobulinemia (Fig. 2C), showed re- biological replicates. Experimental conditions were blinded randomized, and no statistical method was used to predetermine sample size. Differences duced levels of IL-1a (Fig. 2D), an important mediator of the between groups were assayed using Microsoft Excel and SPSS statistical immunological response mainly produced by leukocytes, and package. In all the cases, experimental data assumed t test requirements exhibited a severe reduction of several proinflammatory molecules (normal distribution and similar variance); in those cases where the as- (Fig. 2E). These findings addressed us to evaluate the potential role sumption of the t test was not valid, a nonparametric statistical method was of MMP-25 metalloprotease in immune response. used (e.g., Wilcoxon signed-rank test). Results were expressed as means and error bars indicate the SEM, as indicated in the figure legends. Mmp25-deficient mice show an impaired innate immune response Results On the basis of the above results as well as on previous data describing Mmp25-deficient mice are viable but show alterations related the ability of this metalloprotease to cleave key components of innate with innate immunity immunity such as galectin-1 or CD16 (24, 25), we focused on the To investigate the biological role of MT6-MMP, we have generated analysis of innate immunity in Mmp25-null mice. To this end, we Mmp25-deficient mice using an insertion strategy (Fig. 1A, 1B). The performed an endotoxin shock protocol based on the injection of modified allele generates a null allele through splicing to a trapping Mmp25-wild-type and knockout animals with 20 mg/kg LPS, and we element contained in the targeting cassette (Fig. 1A, 1B). Mmp25- evaluated mice survival, with the finding of a remarkable impairment targeted ESCs were microinjected into C57BL/6 mouse blastocysts in the innate immune response in knockout animals. Thus, whereas the to generate chimeric mice that were subsequently crossed with C57BL/6 survival rate of control mice was ∼23%, the survival rate of Mmp25- mice to generate Mmp25-heterozygous mice. Mmp25-deficient an- deficient mice was .63% (Fig. 3A). Accordingly, mutant mice also imals were born at the expected Mendelian ratio and developed showed reduced signs of lung inflammatory damage and intersticial normally (Fig. 1C); both males and females were fertile and did not edema (Fig. 3B) as well as reduced blood levels of liver and kidney show significant differences in survival rates as compared with the damage markers, such as glutamate-pyruvate transaminase, glutamic wild-type littermates (Fig. 1D). As expected, Mmp25-knockout oxaloacetic transaminase, and blood urea nitrogen (Fig. 3C). In animals did not produce the protease in tissues like bone marrow, agreement with the prominent expression of this metalloprotease in lung, or spleen, where wild-type animals showed the appropriate WBCs (26), reciprocal wild-type to mutant and mutant to wild-type Mmp25 expression levels in all these tissues (Fig. 1E). transplantations resembled the effect of primary donors (Fig. 3D). The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 5. MMP-25 promotes TRAF6 ubiquitination. (A) Bioluminescence signal quantification of HEK-293T cells transduced with NF-kB luciferase reporter gene and either pcDNA3-MMP25 or pcDNA-empty vector. Mean relative values are represented. (B) Western blot analysis of UEV1A in total lysates and anti-MMP25-GFP immunoprecipitates from HEK-293T cells either transfected with pMMP25-GFP or pEGFP-N1. (C) Western blot analyses of TLR4, TRAF6, UEV1A, IkBa, and a-tubulin in LPS-treated (10 mg/ml, 2 h) and untreated wild-type and Mmp25-deficient cells. (D) Anti-HA Western blot analysis of GFP immunoprecipitates from HEK-293T cells transiently cotransfected with GFP-TRAF6 and HA-ubiquitin plus either MMP25 cDNA or empty vector (pcDNA3). Signal intensity was quantified, and mean values from three independent experiments are represented. Error bars indicate SEM (**p , 0.01, two-tailed Student t test).

NF-kB impaired signaling in Mmp25-deﬁcient mice marrow neutrophils isolated from wild-type and knockout ani- by guest on September 29, 2021 NF-kB signaling is a critical mediator of innate immunity upon a mals. As expected, exposure of wild-type mice to LPS resulted in plethora of stress signals (27). Among them, NF-kB’s role in LPS a remarkable activation of NF-kB in neutrophils (Fig. 4A), response has been reported extensively (18, 28, 29). Therefore, we whereas Mmp25-deﬁcient mice had a severely impaired activation decided to study the activity of this signaling pathway in bone upon LPS stimulation (Fig. 4A). Moreover, we studied in vivo the

FIGURE 6. Impaired macrophages activation in Mmp25-deﬁcient mice. (A) Relative nitrite production of Mmp25+/+ and Mmp252/2macrophages from four animals of each genotype after stimulation with 10 mg/ml LPS during 24 h. Mean relative units of three replicates are shown. (B)NF-kBEMSAanalysis in macrophages from Mmp25+/+ (n =6) and Mmp252/2 (n = 6) mice incubated or not with 10 mg/ml LPS during 1 h. (C) Quantitative RT-PCR of Csf2, Lif, and Cxcl10 in Mmp25+/+ (n =4)and Mmp252/2 (n = 4) macrophages after stimulation with LPS (10 mg/ml, 6 h). Mean relative mRNA levels of three replicates are shown. Error bars indicate SEM (*p , 0.05, **p , 0.01, two- tailed Student t test). 6 MMP-25 REGULATES INNATE IMMUNITY

NF-kB activity in both mutant and control mice using a reporter- new aspects of the functional relevance of MT6-MMP, demon- based assay and hydrodynamic transfection approaches. Consistent strating a critical role for this metalloproteinase in innate immunity with the above results, we found that Mmp25-deficient animals are through the control of WBCs activation. These results agree with unable to properly activate NF-kB (Fig. 4B). According to NF-kB previous findings describing the upregulation of MMP-25 in clas- defective activation, we observed a reduced expression of several sical and alternative macrophage activation pathways, pointing to key genes associated with inflammatory response, such as Csf2, the essential role of this membrane protease in these processes (9). Tnfa, Cox2,andIl1a,inMmp25-deficient mice (Fig. 4C–F). Furthermore, we have provided in this paper experimental proof about the mechanistic role of MMP-25 in NF-kB activation, MMP-25 promotes TRAF6 ubiquitination to activate NF-kB promoting the ubiquitination of TRAF6 through its direct inter- To get mechanistic insight about MMP-25 regulation of NF-kB action with the E2 ligase UEV1A. Previous works have described signaling pathway activity, we first transduced HEK-293T cells the function of MMPs in different physiological and pathological with a plasmid encoding MMP25 revealing that the expression of situations related with immune system and inflammation, such as MMP-25 in this cell line was associated with an increase in NF-kB responses to infectious and autoimmune diseases, cancer pro- activity, supporting the validity of this experimental model (Fig. 5A). gression, and wound healing (14, 34–37). Although further studies Immunoprecipitation experiments on HEK-293T cells revealed a should address the specific biochemical nature of this cross-talk complex interaction pattern of MMP-25, which included the protein between MMP-25 and NF-kB, this work has described new reg- UEV1A, a critical regulator of TRAF6 ubiquitination that activate ulatory functions for this family of metalloproteinases that could NF-kB signaling pathway (Fig. 5B, Supplemental Table 1) (30–32). be promising for exploring the functions of MMPs on different

We then analyzed some of these key components of LPS-induced mechanisms mediated by NF-kB, including those associated with Downloaded from NF-kB activation pathway, such as TLR4, TRAF6, UEV1A or the modulation of inflammation during aging processes (38–40). IkBa, in wild-type and Mmp25-deficient mice, observing a remark- Moreover, our results describing novel MMP-25 interactions may able increase of TRAF6 expression because of Mmp25 deficiency contribute to identify new specific therapeutic targets in sepsis and (Fig. 5C). These results made us hypothesize about a direct regulation other disorders related to innate immunity impairment that could of MMP-25 over TRAF6 activity and ubiquitination. To test this, we be more effective and avoid the alteration of the beneficial effects

overexpressed a GFP-TRAF6 fusion protein in HEK-293T cells that this MMP may have in different physiological contexts. Fi- http://www.jimmunol.org/ along with HA-tagged ubiquitin, and either with pcDNA-MMP25 or nally, although it is known that leukocytes can modulate the im- pcDNA-empty vector, observing a .2-fold increase in TRAF6 ubiq- munological response by secreting MMPs, the roles of MT-MMPs uitination as a consequence of MMP-25 expression (Fig. 3D). These had not been deeply explored yet (41). In this sense, Mmp25-deﬁcient results ﬁrst describe the precise molecular mechanism through which mice may constitute an extremely valuable experimental model to MMP-25 may regulate NF-kB activation. further study innate immunity regulation through the ability of this MMP-25 regulates macrophage activation through NF-kB metalloprotease to modify WBCs activity. signaling Acknowledgments

Because MMP-25 is highly expressed in macrophages and these by guest on September 29, 2021 We thank F. Rodrı´guez, A. Moyano, R. Feijoo, S. A´ lvarez, D. A. Puente, cells show an important role in the initiation of the innate im- and C. Garabaya for excellent technical assistance and the Servicio de mune response (33), we also analyzed peritoneal macrophages of +/+ 2/2 Histopatologıá (Instituto Universitario de Oncologıá) for histological Mmp25 and Mmp25 mice. Thus, macrophages from wild- preparations. type and Mmp25-deficient mice were isolated, primed with IFN-g, and exposed to LPS (10 mg/ml). Nitrite production was assessed in Disclosures the cultures to monitor macrophage activity (29), observing that The authors have no financial conflicts of interest. the nitrite production in Mmp252/2 macrophages was 2-fold lower than in wild-type macrophages, which suggests intrinsic defects in macrophage activation capacity (Fig. 6A) caused by NF-kBim- References 2 2 pairment (Fig. 6B). We also observed that Mmp25 / macrophages 1. Newson,J.,M.Stables,E.Karra,F.Arce-Vargas,S.Quezada,M.Motwani,M.Mack, show a compromised production of inflammatory mediators con- S. Yona, T. Audzevich, and D. W. Gilroy. 2014. Resolution of acute inflammation bridges the gap between innate and adaptive immunity. Blood 124: 1748–1764. trolled by NF-kB after LPS stimulation. Thus, we analyzed some 2. Bonnans, C., J. Chou, and Z. Werb. 2014. Remodelling the extracellular matrix macrophage LPS-characteristic inflammatory mediators such as in development and disease. Nat. Rev. Mol. Cell Biol. 15: 786–801. 3. Puente, X. S., L. M. Sańchez, C. M. Overall, and C. Lo´pez-Otıń. 2003. Human and Csf2, Lif,andCxcl10 (Fig. 6C) and found a reduced expression of mouse proteases: a comparative genomic approach. Nat. Rev. Genet. 4: 544–558. these genes in Mmp25-deficient macrophages as compared with 4. Soria-Valles, C., A. Gutie´rrez-Fernańdez, M. Guiu, B. Mari, A. Fueyo, controls. R. R. Gomis, and C. Lo´pez-Otıń. 2014. The anti-metastatic activity of collagenase-2 in breast cancer cells is mediated by a signaling pathway involving decorin and miR-21. Oncogene 33: 3054–3063. 5. Gutie´rrez-Fernańdez, A., C. Soria-Valles, F. G. Osorio, J. Gutie´rrez-Abril, Discussion C. Garabaya, A. Aguirre, A. Fueyo, M. S. Fernańdez-Garcıá, X. S. Puente, and Globally, our results reveal that the lack of the membrane-bound C. Lo´pez-Otıń. 2015. Loss of MT1-MMP causes cell senescence and nuclear metalloproteinase MT6-MMP impairs NF-kB activation, a criti- defects which can be reversed by retinoic acid. EMBO J. 34: 1875–1888. 6. Fanjul-Fernańdez, M., A. R. Folgueras, S. Cabrera, and C. Lo´pez-Otıń. 2010. cal mediator of immune response (1), connecting defective immune Matrix metalloproteinases: evolution, gene regulation and functional analysis in response with reduced cytokine release in WBCs, and therefore mouse models. Biochim. Biophys. Acta 1803: 3–19. explaining the higher survival rate of Mmp25-deficient mice after 7. Velasco, G., S. Cal, A. Merlos-Sua´rez, A. A. Ferrando, S. Alvarez, A. Nakano, J. Arribas, and C. Lo´pez-Otıń. 2000. Human MT6-matrix metalloproteinase: LPS administration. To raise this conclusion, it has been necessary identification, progelatinase A activation, and expression in brain tumors. Cancer to generate this new strain of mutant mice because the mouse model Res. 60: 877–882. 8. Kojima, S., Y. Itoh, S. Matsumoto, Y. Masuho, and M. Seiki. 2000. Membrane- deficient in MMP-25 (MT6-MMP) was one of the very few in vivo type 6 matrix metalloproteinase (MT6-MMP, MMP-25) is the second glycosyl- murine models of MMP deficiency that was not yet available. phosphatidyl inositol (GPI)-anchored MMP. FEBS Lett. 480: 142–146. Similar to most MMP-deficient mice (13, 14), Mmp25-null mice are 9. Huang, W. C., G. B. Sala-Newby, A. Susana, J. L. Johnson, and A. C. Newby. 2012. Classical macrophage activation up-regulates several matrix metalloproteinases fertile and viable and do not exhibit apparent phenotype abnor- through mitogen activated protein kinases and nuclear factor-kB. PLoS One malities. However, it is remarkable that our studies have unveiled 7: e42507. The Journal of Immunology 7

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