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A new transcriptional role for matrix metalloproteinase- 12 in antiviral immunity

David J Marchant1,2, Caroline L Bellac3–5, Theo J Moraes6, Samuel J Wadsworth1, Antoine Dufour3–5, Georgina S Butler3–5, Leanne M Bilawchuk2, Reid G Hendry2, A Gordon Robertson7, Caroline T Cheung1, Julie Ng1, Lisa Ang1, Zongshu Luo1, Karl Heilbron1, Michael J Norris6, Wenming Duan6, Taylor Bucyk2, Andrei Karpov1, Laurent Devel8, Dimitris Georgiadis8,9, Richard G Hegele6, Honglin Luo1, David J Granville1, Vincent Dive8, Bruce M McManus1,10 & Christopher M Overall3–5,10

Interferon-a (IFN-a) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IkBa (encoded by NFKBIA) we show that IFN-a is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IkBa mediates the export of IFN-a from virus-infected cells and that the inability of cells in Mmp12 −/− but not wild-type mice to express IkBa and thus export IFN-a makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate’s gene exons and by MMP-12–mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-a secretion and host protection, extracellular MMP-12 cleaves off the IFN-a receptor 2 binding site of systemic IFN-a, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-a, treatment of coxsackievirus type B3–infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-a levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Interferons1 and macrophages are important in antiviral immunity2. die. We also infected wild-type and Mmp12−/− mice with respiratory

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature MMP-12 (ref. 3; also known as macrophage metalloelastase) is a syncytial virus, an enveloped virus that infects lung airways. As shown secreted protease that degrades extracellular matrix during inflamma- by elevated protein in bronchoalveolar lavage fluid, which reflects the tory tissue destruction4–6. However, like other MMPs that have been host response to viral replication13, Mmp12−/− mice again showed shown to act as regulators of extracellular homeostasis and innate increased morbidity (Fig. 1b). We observed significantly elevated npg immunity7,8—with many new substrates and hence new functions viral loads in Mmp12−/− mice after infection with either coxsackie- in diverse processes9—MMP-12 is also protective, with antitumori- virus (Fig. 1c and Supplementary Fig. 1b) or respiratory syncytial genic10, anti-inflammatory11 and antibacterial activities12. As MMPs virus (Fig. 1d), revealing a deficiency in viral control in the absence regulate macrophage chemotaxis and activation by chemokine cleav- of MMP-12. age7,11, we investigated the role of MMP-12 in antiviral immunity. IFN-α plays a central part in early antiviral responses14. In plasma from wild-type mice, IFN-α levels, but not IFN-β, IFN-γ or inter- RESULTS leukin-1β (IL-1β) levels, were elevated in coxsackievirus infection Viral control in Mmp12−/− mice is impaired (Fig. 1e and Supplementary Fig. 2a). In contrast, in Mmp12−/− mice, We infected Mmp12−/− mice with coxsackievirus type B3, an unen- IFN-α plasma concentrations were unexpectedly low and were no veloped virus that causes systemic infection in mice and humans, and greater than in sham-infected Mmp12−/− and wild-type mouse observed increasing morbidity (Supplementary Fig. 1a) followed by controls (Fig. 1e). This was noteworthy as the coxsackievirus-infected 30% death at 3 d after infection (Fig. 1a). Wild-type mice did not Mmp12−/− mice showed stronger induction of IFN-β, IFN-γ and IL-1β

1Department of Pathology and Laboratory Medicine, UBC James Hogg Research Centre, Institute for Heart + Lung Health, St. Paul’s Hospital/Providence Health Care/University of British Columbia, Vancouver, British Columbia, Canada. 2Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada. 3Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada. 4Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. 5Centre for Blood Research, Life Sciences Institute, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada. 6Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 7Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. 8Commissariat a l’Energie Atomique, Labex LERMIT, Service d’Ingenierie Moleculaire des Proteines, Gif/Yvette, France. 9Present address: Department of Chemistry, University of Athens, Athens, Greece. 10These authors contributed equally to this work. Correspondence should be addressed to B.M.M. ([email protected]) or C.M.O. ([email protected]). Received 10 October 2013; accepted 12 February 2014; published online 28 April 2014 ; doi:10.1038/nm.3508

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–/– –/– –/– WT Mmp12 Mmp12 a WT b Mmp12 e f CVB3 g IFN-α h ) 5 * –/– 2 WT –/– WT–/– WT –/– WT Mmp12 WT 4 Mmp12–/– ) 120 1.0 3 WT OAS1 6 40 0.8 * 100 2 1.11 1.49 1.14 1.31 1.00 1.41 * 80 0.6 IFN- α Sham 1 Pancreas P < 0.001 20 60 0.4 (pg/ml × 10 0 Saline 40 0.2 CVB3 20 OAS1 Survival (%) 0 PFU/ml ( × 10 0 0 1.23 1.00 1.29 1.05 1.20 0.20 BAL protein ( µ g/ l) 1 4 Plasma BAL 1 2 3 4 Time after Mmp12–/– Mmp12–/– P < 0.01 Saline IFN-α Time after CVB3 infection (d) infection (d) WT WT Mmp12–/– i WT Heart Pancreas c d ) 10 Heart Liver 3

–/– ) 0.03 WT Mmp12 4 8 * 8 P < 0.05 0.4 ‡ 6 6 † 0.3 4 RSV 0.02 4 IFN- α P < 0.01 0.2 2 0.01 2 (pg/ml × 10 0 0.1 1 4 RSV BAL titer 0 CVB3 genome 0 0 Lung P = 0.001 (PFU/ml × 10 WT Mmp12–/– Time after RSV infection (d) α Saline IFN-α Saline IFN-

Figure 1 Mmp12−/− mice demonstrate increased viral load, morbidity, mortality and lower IFN-α levels in viral infections. (a) Kaplan-Meier curve showing mortality rates during coxsackievirus type B3 (CVB3) infection. Mmp12−/− mice (n = 16) and wild-type (WT) counterparts (n = 16). (b) Total protein concentrations found in bronchoalveolar lavage (BAL) fluid collected 1 and 4 d after infection from Mmp12−/− (n = 10) and WT (n = 10) mice infected with respiratory syncytial virus (RSV). Analysis of variance (ANOVA) was conducted with Tukey’s test of significance (*P < 0.05). (c) Coxsackievirus RNA genome in the myocardium was detected by in situ hybridization (blue staining of positive-sense viral RNA); morphometric analysis determined the relative quantity of virus staining in heart tissue (n = 16 mice per group, and ten slides per mouse). Scale bar, 50 µm. (d) RSV titer in the BAL fluid 4 d after infection in Mmp12−/− mice compared to WT mice. *P < 0.05, Student’s t-test (n = 20). (e) IFN-α levels in plasma and BAL samples from CVB3-infected (n = 32) and RSV-infected (n = 20) mice, as quantified using ELISA. Data were analyzed by ANOVA with Tukey’s test of significance (*P < 0.05). (f) IFN-α (brown immunostaining) in heart, pancreas and liver from CVB3-infected Mmp12 −/− and WT mice (n = 32, and ten slides per mouse) and in lung from RSV-infected Mmp12−/− and WT mice (n = 20, and ten slides per mouse) (scale bars, 50 µm). Hematoxylin counterstain shows nuclei in blue. Staining was quantified by morphometry using a color segmentation algorithm. Student’s t-test compared the morphometry values between Mmp12−/− and WT mice. (g) Mmp12−/− (n = 24) and WT (n = 24) mice were injected with IFN-α via the tail vein and then infected with CVB3. Three days later, mice were killed. Western blotting of equal protein concentrations of heart tissue detected oligoadenylate synthase (OAS-1) in the hearts of Mmp12−/− mice compared to the hearts of WT mice. (h,i) Plaque assay (h) and quantitative morphometry of in situ hybridization (i) showing the viral load in the hearts of WT (n = 24) and Mmp12−/− (n = 24) mice. Data were analyzed by ANOVA with Tukey’s test of significance. PFU, plaque-forming units. Error bars represent s.d. of the mean. (†P < 0.005; ‡P < 0.001).

proteins in the plasma than did infected wild-type mice, consistent elevated levels of IFN-γ or IL-1β in plasma might also contribute. with increased viral replication in Mmp12−/− liver, pancreatic exocrine Expression of oligoadenylate synthase-1, a downstream product of tissue and heart muscle (Fig. 1c and Supplementary Fig. 1b,c). IFN-α signaling, was slightly lower in Mmp12−/− mice during infec- Although elevated plasma IFN-γ and IL-1β protein levels may have tion with coxsackievirus (Fig. 1g), reflecting the low systemic levels of partially compensated for the loss of effective IFN-α–mediated anti- IFN-α. However, to determine whether IFN-α signaling was impaired −/− Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature viral immunity, these levels were ineffective in preventing the high in Mmp12 mice, we administered recombinant IFN-α intravenously, degree of viral replication and titer observed in the coxsackievirus- which elevated cardiac levels of oligoadenylate synthase-1 (Fig. 1g) infected Mmp12−/− mice (Supplementary Fig. 1b,c). IFN-α protein and decreased viral load (Fig. 1h,i and Supplementary Fig. 3). Hence, −/−

npg concentrations were also low in plasma and bronchoalveolar fluid the antiviral defect in Mmp12 mice is not due to impaired IFN-α in Mmp12−/− mice after infection with respiratory syncytial virus cellular responses, but rather to a defect in its secretion. Indeed, (Fig. 1e), a virus that is known to induce IFN-α15. Similar to what IFN-α was retained intracellularly during in vitro coxsackievirus and we observed in coxsackievirus-infected Mmp12−/− mice, IFN-β levels respiratory syncytial virus infection of fibroblasts from Mmp12−/−, were markedly elevated in the plasma of respiratory syncytial virus– but not wild-type mice from 1 to 8 h after infection (Fig. 2a; data are infected Mmp12−/− compared to wild-type mice (Supplementary duplicated as part of a full time course in Supplementary Fig. 4) and Fig. 2b). However, IFN-γ levels were not different between wild- was undetectable in the culture medium of infected Mmp12−/− fibro­ type and Mmp12−/− mice upon respiratory syncytial virus infection blasts (Fig. 2b). This effect was selective, as IFN-β was not retained (Supplementary Fig. 2b), indicating that the elevated IFN-γ response intracellularly in coxsackievirus-infected Mmp12−/− fibroblasts to coxsackievirus infection is virus specific and is not directly linked (data not shown), and, like IFN-γ, IFN-β was secreted to high levels to MMP-12. Thus, Mmp12−/− mice are specifically defective in in the plasma (Supplementary Fig. 2), yet this was insufficient IFN-α–mediated antiviral immunity, which is unexpected. to reduce viral loads in the coxsackievirus-infected Mmp12−/− mice (Fig. 1b–d,f), confirming the important contribution of the IFN-a secretion is defective in Mmp12 −/− mice MMP-12–IFN-α regulation arm in antiviral immunity. Unlike in plasma, there were high IFN-α protein concentrations in MMP-12 transfection into Mmp12−/− fibroblasts rescued IFN-α exocrine pancreas (P < 0.001), heart muscle (P < 0.05) and hepato- secretion (Fig. 2b). As the wild-type cell cultures do not have macro­ cytes (P < 0.01) of coxsackievirus-infected Mmp12−/− mice and in phages as the cellular source of MMP-12, our findings suggest that the lung epithelial lining (P < 0.01) of Mmp12−/− mice during respi- MMP-12 is expressed by mouse fibroblasts. Indeed, western blot- ratory syncytial virus infection (Fig. 1f). These findings supported ting and quantitative RT-PCR (Fig. 2b) confirmed that MMP-12 our hypothesis that a dysfunctional IFN-α pathway may be the is expressed by fibroblasts, which is unexpected, in addition to cause of morbidity and mortality in Mmp12−/− mice, although the monocytes/macrophages in mice.

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a Time after infection (h) WT d e 100 b –/– c 0 1 8 Mmp12 –/– –/– IFN-α/DAPI Mmp12 + MMP-12 WT ‡ 2.0 MW ControlMmp12Control cDNAMMP-12 protein WT 20 (kDa) –/– –/– WT –/– 10 * 15 1.5 75- ‡ 10 ‡ ProMMP-12 ** 50- IFN- α (pg/ml) 5 1.0 37- MMP-12 0 CVB3 IFN- α (pg/ml) p44 ERK 0 8 0.5 37- p42 0

Time after infection (h) Ifna expression total 50- –/– normalized to Hprt IκBα B 0 37- α MW silκ 100- total Control (kDa) –/– WT NF-κB IκBα inh. Nfkbia –/– 15 Ifna2 Ifna9 50- Mmp12 100- Ifna12Ifna13 RSV ERK MAPK inh. T 10 50- β-actin 75- Pro ∆∆ C 5 50- Active Uninfected Virus 4 h 0 37- f IκBα inhibitor siIκBα Nfkbia–/– ( Mmp12 vs. Hprt ) –/– WT 22- IFN-α/DAPI

Figure 2 IFN-α is synthesized but not secreted from Mmp12−/− cells as a result of IκBα deficiency. (a) IFN-α expression in Mmp12−/− heart and lung fibroblasts infected with CVB3 and RSV, respectively (scale bars,10 µm). Red color indicates IFN-α in Mmp12−/− and wild-type (WT) DMSO Scramble WT fibroblasts (these data are duplicated as part of a full time course shown in Supplementary Fig. 4). IFN-α/DAPI Minimal cytoplasmic IFN-α was apparent in wild-type cells where some protein was located in small punctate regions at the cell periphery (arrowheads) before secretion. Data are representative of 3 independent experiments from a total of 6 Mmp12−/− and 6 WT donor mice. (b) Top, IFN-α secretion, as quantified by ELISA of conditioned medium from CVB3-infected WT cardiac fibroblasts, Mmp12−/− cardiac fibroblasts and Mmp12−/− cardiac fibroblasts transfected to express mouse MMP-12. Data were analyzed by ANOVA with Tukey’s test of significance: ‡P < 0.001. Bottom left, quantitative RT-PCR (qRT-PCR) of mRNA isolated from fibroblasts from wild-type and Mmp12−/− mice. Mmp12 mRNA levels were normalized to hypoxanthine-guanine phospho-ribosyl-transferase (Hprt), a housekeeping gene standard. Error bars denote s.d. of the mean. Experiments were repeated once using fibroblasts from two different mice of each genotype in each experiment (n = 4 Mmp12−/− and n = 4 WT). Bottom right, western blotting for MMP-12 in WT and Mmp12−/− fibroblasts. MW, molecular weight. (c) qRT-PCR of mRNA levels for IFN-α genes Ifna2, Ifna9, Ifna12 and Ifna13 at 4 h after infection in Mmp12−/− and WT mouse embryonic fibroblasts infected with RSV. Data are normalized to Hprt. Error bars denote s.d. of the mean. Experiments were repeated three times. (d) Western blot analysis for MMP-12 and candidate regulators of IFN-α expression (ERK p42 and p44 MAP kinase, IκBα and NF-κB) in primary mouse fibroblasts 4 h after CVB3 infection. β-actin was used as a loading control. IκBα was rescued by Mmp12 transfection (cDNA) or addition of recombinant MMP-12 protein to Mmp12−/− cultures 16 h before harvest for western blotting. (e) HeLa cells were pretreated with the IκBα inhibitor Bay11-7082 (IκBα inh.), the ERK MAP kinase inhibitor U0126 or DMSO (control) and then infected with CVB3 (3 independent experiments). Results with Nfkbia−/− NIH 3T3 cells and HeLa cells partially silenced for IκBα expression using RNAi (siIκBα) are each representative of 2 independent experiments). Error bars represent the s.d. of the mean. Significance was determined by ANOVA with Tukey’s test of significance: *P < 0.05; **P < 0.01; ‡P < 0.001. (f) HeLa cells (WT) were infected with CVB3, fixed and immunostained for IFN-α (green) and nuclei (blue) and imaged by confocal microscopy. Cells were treated with Bay11-7082 (IκBα inhibitor) or DMSO control, and transfected with siIκBα oligonucleotides or scrambled controls. Nfkbia−/− NIH 3T3 and IκBα silenced HeLa cell results are each representative of 2 independent experiments. Scale bars, 10 µm. Downward arrows represent no detectable signal. Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature IFN-a expression requires IkBa coxsackievirus-infected HeLa cells (Fig. 2e). We confirmed this using The regulation of IFN-α secretion is not completely elucidated. There coxsackievirus-infected Nfkbia−/− cells (Fig. 2e). Consistent with ongo-

npg was no significant difference in expression of Ifna12 and Ifna13 in ing IFN-α synthesis and a lack of secretion in these cells, we observed Mmp12−/− versus WT cells (Fig. 2c), indicating a post-transcriptional IFN-α staining in the cytosol (1 and 4 h after infection, respectively) defect in IFN-α expression in Mmp12−/− cells. As there was no differ- (Fig. 2f), which is similar to what we found in coxsackievirus- ence in Ifna gene expression in Mmp12−/− versus wild-type fibroblasts infected Mmp12−/− fibroblasts (Fig. 2a). Thus, IFN-α secretion during coxsackievirus infection (Fig. 2c), we analyzed expression of depends on IκBα and is impaired in Mmp12−/− cells that have reduced extracellular signal–regulated kinase (ERK), nuclear factor-κB (NF-κB) IκBα expression. and IκBα, which are key mediators of innate immunity in inflamma- tion and viral infection16. IκBα expression was lower in Mmp12−/− MMP-12 traffics to the nucleus compared to wild-type fibroblasts during coxsackievirus infection and Next, we investigated the association of MMP-12 with viral infec- was restored by MMP-12 transfection or addition of MMP-12 protein tion in humans. Among the 32 human myocardium biopsies we (Fig. 2d). In contrast, there was no difference in NF-κB or ERK expres- tested, MMP-12 staining was elevated in the eight individuals who sion in Mmp12−/− compared to wild-type fibroblasts during coxsackie- had enteroviral myocardial infection but was low in the absence of virus infection or upon exogenous MMP-12 treatment (Fig. 2d). infection in the other 24 samples with different cardiovascular dis- IκB kinase (IKK) phosphorylates and activates IκBα, initiating eases (Fig. 3a). Myocardial cells do not typically express MMP-12. NF-κB activation by releasing the inhibitory IκBα subunit for However, we found MMP-12, which has a signal sequence and is eventual proteasomal degradation. NF-κB then translocates to the secreted, in the nucleus of the infected human myocardial cells (Fig. 3b). nucleus in virus-infected cells17, where it activates more than 500 Confirming this unexpected result, we found MMP-12 in both the immune response genes16. We treated coxsackievirus-infected HeLa nuclear and cytosolic fractions purified from coxsackievirus-infected cells with an inhibitor of IKK, Bay11-7082, which decreases IκBα mouse fibroblasts (Fig. 3c). Correct histone H1 and Cu/Zn super phosphorylation, activation and proteasomal degradation, and found oxide dismutase segregation validated the fractions’ integrity (Fig. 3d). that IFN-α was not secreted (Fig. 2e). After IκBα knockdown by Although MMPs are extracellular and intracellular MMP-12 has not RNA interference, IFN-α secretion was also strongly reduced in previously been described, MMP-14 was recently found in the nucleus18

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Figure 3 Nuclear localization of MMP-12 in human and mouse cells. a b (a) Morphometric quantification of MMP-12 in n = 32 individual biopsies (CR1–CR8 shown) from patients with cardiomyopathies. CR4 and CR8 0.4 0.3 are biopsies from patients diagnosed with viral myocarditis. Significance * * 0.2 MMP-12/DAPI was determined by ANOVA with Tukey’s test of significance: *P < 0.05. 0.1 Virus (b) Top, confocal imaging of biopsies stained with a monoclonal antibody 0 for MMP-12 (shown in red) and with DAPI stain for nuclei. Bottom, Proportion of image stained for MMP-12 immunohistochemical staining for VP1 enterovirus capsid protein CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 Human cardiac biobank samples (brown staining) in human biopsy samples from individuals infected with coxsackievirus. Data are representative of 10 slides of one biopsy per c d e patient. Scale bar, 50 µm. (c) Western blot for MMP-12 in subcellular MW fractions of mouse cardiac fibroblasts. Data are representative of (kDa) Nuclei Cytosol MW (kDa) Anti-prodomainAnti-catalyticAnti-linkerAnti-hemopexin 2 independent experiments. (d) Western blot analysis of cytoplasmic 37 MW Cu/Zn 62 ProMMP-12 Cu/Zn super oxide dismutase (SOD) and nuclear marker histone H1 49 (kDa) Nuclei Cytosol 26 SOD MMP-12 to determine purity of nuclear and cytoplasmic fractions in the mouse 50 38 19 28 fibroblasts. We found that MMP-12 cleaves tubulin (Supplementary 40 26 MMP-12 14 Table 1), so we did not use tubulin as a cytosolic marker. Data are 30 Histone linker Pro Cat Hem representative of 2 independent experiments. (e) Western blot analysis 19 H1 ProMMP-12 of MMP-12 in HeLa cell nuclear lysates using six different antibodies MMP-12 (four shown) against the four domains of human MMP-12: prodomain and f MMP-12 g catalytic (Cat), linker and hemopexin (Hem) domains. This experiment was repeated twice. (f) Recombinant MMP-12 was added to HeLa cells MW CytosolNucleiCytosolNuclei cat in which MMP-12 was silenced by shRNA, and MMP-12 protein was 0 h 1 h Control (4 h) (kDa) Con MMP-12 imaged by confocal immunofluorescence microscopy at the indicated MMP-8 40 time points. Recombinant MMP-8 control is shown in red. The MMP-8 30 4-h time point is duplicated and shown with the full time courses for 20 MMP-8 and MMP-2 negative controls and MMP-14 as a positive control 2 h 4 h 4 h β-actin are shown in Supplementary Figure 5. Data are representative of 1HAE bronchial h HL1 cardiomyocytes i 3 independent experiments. Scale bar, 15 µm. (g) Western blotting of epithelial cells MMP-12 in subcellular fractions of shRNA MMP-12–silenced HeLa cells 1HAE HL1 0 h 48 h 0 h 24 h MW Coculture time (h) cat XZ treated with recombinant MMP-12 catalytic domain only (MMP-12 ) (kDa) 0 24 48 0 24 48 70 showing localization to the nucleus. Con, untreated control cells. Data MMP-12/DAPI MMP-12/DAPI XY are representative of 2 independent experiments. (h) 1HAEo− bronchial 50 MMP-12 epithelial cells and HL-1 cardiomyocytes were cocultured with M1 30 macrophages, and MMP-12 was detected at the indicated times of PARP coculture using confocal immunofluorescence microscopy. Top images show the xz plane. Data represent 4 independent experiments. Scale bar, 15 µm. (i) Western blot analysis of MMP-12 in the nuclear fractions of 1HAEo– and HL-1 cells 24 h and 48 h after macrophage co-culture. The PARP loading control is shown. Data are representative of 4 independent experiments.

and MMP-2 is expressed in the cytosol of cardiomyocytes19, yet in both the soluble form of MMP-14, lacking its stalk, transmembrane and cases the intracellular transport and nuclear targeting mechanisms of cytoplasmic domains, did (Supplementary Fig. 5).

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature these proteins are unknown, as are their intracellular roles. Cells of the monocyte-macrophage lineage are the largest source To increase our confidence that the intracellular localization of of MMP-12 in vivo, and classically activated M1 macrophages, which MMP-12 was real and not an artifact, and due to the unavailability are known to secrete MMP-12, are important for viral defense20.

npg of multiple mouse MMP-12–specific antibodies, we used six differ- We postulated that MMP-12 secreted from macrophages is taken up ent antibodies specific to different domains of human MMP-12, the by the surrounding tissue cells in trans. We cultured activated M1 prodomain and catalytic, hinge and hemopexin domains, to show mouse macrophages in a coculture system separated from mouse nuclear localization of MMP-12 in infected and uninfected HeLa cardiomyocytes or human bronchial epithelial cells by a permeable cells (Fig. 3e). Edman sequencing of immunoreactive bands from membrane to prevent cell mixing. Activated M1 macrophages the nuclear preparations confirmed the identity of MMP-12. Thus, secreted MMP-12 into the medium (Supplementary Fig. 6a). MMP-12 is clearly intracellular in the cytosol and nucleus in mouse In comparison, MMP-12 mRNA expression was undetectable in and human cells. the cardiomyocytes or bronchial epithelial cells before or during coculture (Supplementary Fig. 6b). However, after coculture, we Macrophage MMP-12 traffics to the nuclei of surrounding cells identified MMP-12 in the nuclei of the cardiomyocytes and bronchial To determine whether extracellular MMP-12 can cross the plasma epithelial cells by confocal microscopy (Fig. 3h) and by western blot- membrane, we added recombinant proMMP-12 protein to MMP-12– ting of nuclear lysates of these cells (Fig. 3i). Thus, MMP-12 secreted silenced HeLa cells, where it rapidly trafficked to the nucleus (Fig. 3f from M1 macrophages is taken up by target cells in trans, where it and Supplementary Fig. 5), despite the absence of a canonical nuclear traffics to the nucleus. localization sequence. Recombinant MMP-12 catalytic domain alone also localized to the nucleus of HeLa cells (Fig. 3g), suggesting that the MMP-12 binds specific DNA sequences hemopexin domain (see Fig. 3e)—which is often autolytically shed We hypothesized that nuclear MMP-12 regulates NFKBIA gene from the enzyme—is not essential for targeting. This may also account expression because Mmp12−/− cells expressed low levels of IκBα for the lower molecular weight forms of MMP-12 that we detected (Fig. 2d). Using chromatin immunoprecipitation (ChIP) combined in the nucleus of HeLa cells (Fig. 3c). To confirm the specificity of with whole-genome sequence analysis (ChIP-Seq) we found MMP-12 MMP-12 intracellular transport, we found that recombinant MMP-2 binding to the NFKBIA promoter after just 1 h of coxsackievirus B3 infec- and MMP-8 did not to cross into the intracellular compartment, but tion in HeLa cells, but not in uninfected cells (Fig. 4a). The MMP-12

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binding site was within 1.2 kb of the TATA box transcription Electrophoretic mobility shift assays using a double-stranded start site of the NFKBIA gene (Fig. 4a and Supplementary Fig. 7). oligonucleotide (oligo 3) containing AAAAAAAA in the identical This was confirmed by ChIP-PCR using oligonucleotide pairs sequence to the MMP-12 binding region of the NFKBIA promoter designed from the NFKBIA gene promoter that amplified only the confirmed direct binding of MMP-12 to the promoter sequence same DNA binding site after 1 h virus infection and that did not by electrophoretic mobility shift assays (Fig. 4c and shown in the amplify this site in DNA from uninfected cells (Fig. 4b). Analysis of context of the whole blot in Supplementary Fig. 7c). DNA bind- multiple binding site peak sequences identified a potential MMP-12– ing specificity was confirmed using competing unlabeled double- binding sequence featuring either a poly(A) or poly(T) tract of 6–8 stranded oligonucleotides (Supplementary Fig. 7c). We found bases (Supplementary Fig. 7a,b). similar mobility shifts using the MMP-12 catalytic domain only

a Chromosome 14 b c d NFKBIA promoter 5 Peak –1200 –900 –600 –300 0 4 height 3Oligo N 11 * 3 Shift Control 300 bp 2 *

Free oligo induction 3 Fold I κ B α 1 0 h 0 h 300 bp 0 TNF-α + – 1 MMP-12 – + Virus 300 bp 2 1 h Virus 1 h e Con Si f Si + MMP-12 50 )

3 40 1 20 ‡ Con Si ) 3 30 15 MMP-12 20 * 10 10 units ( × 10 Relative light NFKBIA Actin 5 ** 0 units ( × 10 Relative light 0 30 Con TNF 100 300 0 –1200 MMP12 (ng/ml) g h i j 30 * 4 8 100 ) ) ) 3 3 3 80 † † 3 6 20 60 2 4 ( × 10 ( × 10 10 * 40 520 520 * 1 2 MMP-12 20 activity (%) A A units ( × 10 Relative light MMP-12 activity 0 MMP-12 activity 0 0 0 cat – 1 1 MMP-12 MMP-12 10 10 Con TNF 100 300 100 300 Nuclei MW (kDa) Cytosol MMP12 IκBα (oligo 3) Control (oligo N) DNase I RXP470.1 75 (pg/ml) MMP-12/DNA 50 20 Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature Figure 4 Nuclear MMP-12 binds the human NFKBIA promoter during viral infection, activating transcription. (a) ChIP-Seq analysis for MMP-12 bound to the NFKBIA promoter in HeLa cells infected with coxsackievirus for 1 h and in uninfected control cells. Data are representative of 2 independent experiments that were sequenced independently. (b) ChIP-PCR using primers to the NFKBIA promoter to determine the location of MMP-12 binding npg to the NFKBIA promoter in virus-infected HeLa cells. Data are representative of 3 independent experiments. (c) Binding of MMP-12 to 50-mer DNA oligonucleotides, as measured by electromobility shift assay. MMP-12 mediated a shift of oligo 3, which contains a sequence of the NFKBIA promoter found to bind MMP-12 in a and b. No shift occurred with the negative control (N) oligonucleotide. There was also an MMP-12–mediated shift of oligonucleotide 11, which contains a sequence from the largest exon of SPARCL1, which was also bound by MMP-12 in ChIP-Seq. Data are representative of 3 independent experiments and are duplicated in the context of the whole blot shown in Supplementary Figure 7c. (d) Human MMP-12 was co-transfected into HeLa cells with a 4.3-kb region of the NFKBIA promoter cloned into a pGL4 luciferase reporter plasmid. Renilla was co-transfected into cells in all experiments as a normalization input/transfection control, and luciferase expression levels were normalized using co-transfected Renilla results. Fold induction was calculated by dividing TNF-α–treated or MMP-12–transfected signal by empty vector signal. Significance was determined by Student’s t-test: *P < 0.05. Data are representative of 5 independent experiments. (e) Left, western blot for MMP-12 in HeLa cells silenced for MMP-12 expression with β-actin loading control. Right, luciferase expression from the 4.3-kb NFKBIA promoter in MMP-12– silenced HeLa cells (Si) and in vector controls (Con) and MMP-12–silenced HeLa cells transfected with a codon optimized MMP-12 (Si + MMP-12); significance was determined by ANOVA with Tukey’s test of significance: **P < 0.01. Experiment was repeated twice. (f) Luciferase expression from NFKBIA promoter activity (4.3-kb construct) in the presence of increasing transfection doses of MMP12-encoding plasmid in HeLa cells. Con, control transfection of empty vector; TNF, stimulation of control cells with 100 ng/ml TNF-α. Significance was determined by ANOVA with Tukey’s test of significance: *P < 0.05; ‡P < 0.001. This experiment was repeated twice and error bars denote s.d. of the mean. (g) Top, full-length MMP-12 or MMP-12 catalytic domain (MMP-12cat) proteins were added (0.01, 0.1 and 1.0 µg) to MMP-12–silenced HeLa cells transfected with the 4.3-kb NFKBIA promoter luciferase and Renilla plasmids. Con, cells transfected with empty vector alone; TNF, 100 ng ml−1 TNF-α. Renilla levels expressed by all treatment groups were similar. Significance was determined by ANOVA with Tukey’s test of significance: *P < 0.05; †P < 0.005. Data are representative of 2 independent experiments. Error bars represent the s.d. of the mean. Bottom, western blotting for MMP-12 in nuclear lysates of the MMP-12 treated HeLa cells confirmed that recombinant MMP-12 and MMP-12 catalytic domain were transported to the nucleus. Data are representative of 2 independent experiments. (h) Sensolyte MMP-12 assay to detect MMP-12 activity in cytoplasmic and nuclear fractions. Error bars denote s.d. of the mean. Data are representative of 6 different experiments. (i) Sensolyte MMP-12 assay to detect MMP-12 activity in the presence of oligo 3 and oligonucleotide N, in increasing concentrations. The specific MMP-12 inhibitor RXP470.1 (100 ng ml−1) was used as a positive control. Error bars represent the s.d. of the mean. (j) MMP-12 was treated with 30 pg of double-stranded DNA oligo 3 and then treated with DNase I. Data are expressed as the percentage inhibition of MMP-12. Error bars denote the s.d. of the mean. This experiment was repeated twice.

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and nuclear extracts of coxsackievirus-infected HeLa cells, which con- (five peaks) genes (Fig. 5a,b). We confirmed DNA binding in vitro by tain endogenous MMP-12 (Supplementary Fig. 7c). Hence, both the electromobility shift assay with double-stranded oligonucleotide 11, nuclear localization and the DNA-binding properties of MMP-12 which contains the sequence from the MMP-12–binding site in the reside in the catalytic domain, which specifically binds the NFKBIA fifth SPARCL1 exon and notably includes the exon’s 6-mer tract of gene promoter only upon viral infection. poly(T) and, similar to that found in the NFKBIA promoter, a 6-mer tract of poly(A) (Supplementary Fig. 7c). To determine the effect of Promoter binding by MMP-12 increases NFKBIA transcription MMP-12 on gene transcription of PSME3 and SPARCL1 upon exon To assay for MMP-12 regulation of the NFKBIA gene, we trans- binding by MMP-12, we silenced endogenous MMP-12 expression fected HeLa cells with a plasmid containing 4.3 kb of the NFKBIA in HeLa cells, which elevated PSME3 and SPARCL1 mRNA (Fig. 5c) promoter in tandem with a luciferase expression cassette. Following and protein (Fig. 5d) expression in uninfected cells. In humans, addition of recombinant MMP-12 protein to the cells, luciferase ~50% of coxsackievirus B3 infections and all respiratory syncytial activity doubled compared with the luciferase activity elicited by virus infections occur via the respiratory tract. We examined mouse classical tumor necrosis factor-α (TNF-α) stimulation (Fig. 4d). bronchial cuboidal epithelium, where all three proteins (MMP-12, HeLa cells that were stably transfected with shRNA to silence PSME3 and SPARCL1) are expressed. PSME3 (Fig. 5e) and SPARCL1 endogenous MMP-12 expression and then transfected with the (Fig. 5f) protein expression was markedly higher in Mmp12−/− lung NFKBIA reporter constructs showed reduced NFKBIA promoter than in wild-type lung after coxsackievirus infection, supporting an activity that was restored upon transfection of MMP-12 (Fig. 4e) in vivo role for MMP-12 in blocking PSME3 and SPARCL1 expression in an MMP12-encoding plasmid concentration–dependent manner upon exon binding. (Fig. 4f). There was no loss of cell viability, as evidenced by stable The expression of low levels of MMP-12 by mouse cells other than Renilla expression (Fig. 4f). We used the transfected NFKBIA macrophages prompted us to screen for MMP-12 expression in other reporter construct to show promoter activity by MMP-12 and TNF-α cells. MMP-12 was expressed in the 1HAEo– human lung epithelial cell because the promoter is present and accessible in the cytosol and line (Fig. 5c,d), and it was also expressed in primary human bronchial not potentially condensed and inaccessible in the nucleus of the epithelial cells from two separate donors (Supplementary Fig. 9). uninfected HeLa cells. Similarly, adding full-length recombinant To further establish that MMP-12 acts as a transcriptional regulator MMP-12 protein to mouse Mmp12−/− fibroblasts (Fig. 2d) or add- in human cells, we found that silencing MMP12 in 1HAEo– cells ing either full-length MMP-12 or MMP-12 catalytic domain to the significantly enhanced PSME3 and SPARCL1 mRNA (Fig. 5c) and MMP12-silenced HeLa cells also increased NFKBIA promoter activity protein (Fig. 5d) expression, consistent with their increased protein of a transfected luciferase reporter construct that contains 4.3 kb levels in coxsackievirus-infected Mmp12−/− mice in vivo (Fig. 5e,f). of the NFKBIA promoter (Fig. 4g) and IκBα protein expression Likewise, NFKBIA mRNA (Fig. 5c) and protein (Fig. 5d) expression (Fig. 2d) in a concentration-dependent manner. was reduced in uninfected 1HAEo– and HeLa cells upon MMP-12 We confirmed that MMP-12 and the MMP-12 catalytic domain silencing, consistent with the mouse analyses. alone trafficked to the nucleus in HeLa cells by western blotting of nuclear lysates (Fig. 4g). Thus, extracellular MMP-12 can localize Proteogenomic analysis reveals MMP-12–regulated substrates to the nucleus of mouse and human cells where it binds to a specific In addition to being transcriptionally downregulated by MMP-12, sequence in the NFKBIA promoter. We hypothesize that when this both SPARCL1 and PSME3 were also cleaved by MMP-12 (Fig. 5g).

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature site is made accessible early in viral infection, this event will lead to Therefore, we searched for other MMP-12 intracellular and secreted increased NFKBIA transcription and levels of IκBα protein, which is substrates. To do so we used a proteomic approach, terminal amine necessary for IFN-α release from virus-infected cells. isotopic labeling of substrates (TAILS)21, to simultaneously identify

npg Proteolytic activity of endogenous MMP-12 in HeLa nuclear lysates both the substrate and the MMP-12 cleavage site by mass spectrometric was lower than in the cytosol (Fig. 4h) despite higher amounts of the identification of MMP-12–cleaved neo–N-terminal peptides (with N enzyme in the nucleus (Fig. 3c). We found that a double-stranded termini different from the mature proteins) purified from digested DNA oligonucleotide matching the sequence of the MMP-12–binding proteomes12. HeLa and Mmp12−/− mouse embryonic fibroblast (MEF) site in the NFKBIA promoter (oligo 3) inhibited MMP-12 enzymatic secretomes and cell lysates were incubated with human and mouse activity in a concentration-dependent manner (Fig. 4i). We did not MMP-12, respectively. In total, we identified 328 new mouse and observe any inhibition with a negative control oligonucleotide (oligo human cellular substrates by iTRAQ (isobaric tags for relative and N) (Fig. 4i) that was not bound by MMP-12 in ChIP-Seq or electro- absolute quantitation) labeling of all N-terminal peptides purified phoretic mobility shift assays and that lies adjacent to the MMP-12 by TAILS comprising original protein and neo–N-terminal peptides binding site in the NFKBIA promoter (Fig. 4c and Supplementary (Fig. 5h and Supplementary Tables 2–4). Fig. 7c). Finally, inhibition of MMP-12 by oligo 3 was abrogated after We devised a new proteogenomic analysis to identify those MMP-12 digestion of MMP-12–oligo 3 complexes with DNase I (Fig. 4j). Thus, substrates that are also transcriptionally regulated by MMP-12. To the MMP-12 catalytic domain binds a specific DNA sequence in the do so, we combined the identities of the cleaved protein substrates NFKBIA promoter, increasing its transcription and reversibly inhibit- that were present at more than 7.3-fold excess in MMP-12–digested ing proteolytic activity. proteome samples (as determined from the iTRAQ ratios of the neo–N-terminal peptides purified by TAILS) with the ChIP-Seq MMP-12 reduces gene transcription upon binding gene exons gene IDs so as to integrate these large data sets. We found that 177 As it is unprecedented for a protease to function as a transcription MMP-12 substrates identified by TAILS also showed gene binding factor, we sought confirmation by identifying other genes that by ChIP-Seq (Fig. 5h and Supplementary Tables 2–4), whereas MMP-12 can bind in a human genome–wide ChIP-Seq analysis 151 did not (Fig. 5h), suggesting that proteolysis and transcription (Supplementary Fig. 8). The highest MMP-12 DNA-binding peaks repression by MMP-12 were selective. The clearance of SPARCL1 were those over exons of the PSME3 (nine peaks) and SPARCL1 and PSME3 proteins, which are downregulated by MMP-12 exon

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a Chromosome 17 b Chromosome 4 c d Peak Peak 2 PSME3 height height HeLa 1HAEo- MW

R 1 ConSi Con Si (kDa) 0 40 172 MMP-12 45 ConSi Con Si 3.0× 4.0× 1 1 HeLa 1HAEo- PSME3 30

2 SPARCL1 PSME3 (Reg-γ) SPARCL1

R 1 –/– WT Mmp12 –/– 0 e f WT Mmp12 g SPARCL1 100 MMP-12 – + Con Si Con Si 98 SPARCL1 HeLa 1HAEo- 62 MW 49 (kDa) 38 28 2 NFKBIA

R 1 0.25× 0.5× MMP-12 – + IκBα 35 32 PSME3 0 80 80 24 Con Si Con Si β-actin 45 60 60 ** MW 17 HeLa 1HAEo- (kDa) 13 40 * 40 7 PSME3 20 20 SPARCL1 0 0 stained area (%) stained area stained area (%) stained area –/– –/– WT WT

Mmp12 Mmp12 Gene Peak iTRAQ 9-18 6 5837 510876 3 h 15.0 5 GO Protein name IPI name height ratio 98 7 6 4 2 1 3 2 1 4 9 4 Proteasome 12.5 1 1 26S protease regulatory subunit 6A IPI00405114 PSMC3 42 15 19 2 Proteasome subunit α type 5 IPI00122562 PSMA5 22 9.2 3 Proteasome subunit α type 1 IPI00283862 PSMA1 12 15 10.0 20 4 Proteasome subunit β type 2 IPI00128945 PSMB2 11 14.37 10 2 5 26S proteasome non-ATPase regulatory subunit 1 IPI00267295 PSMD1 10 15 21 7.5 non regulated substrates 6 26S proteasome non-ATPase regulatory subunit 14 IPI00113262 PSMD14 10 15 22 23 7 Proteasome subunit α type 3 IPI00331644 PSMA3 8 15 8 26S protease regulatory subunit 6A IPI00133206 PSMC3 6 15 5.0 9 Proteasome subunit α type 6 IPI00131845 PSMA6 6 15 (MMP-12/control) 10 26S proteasome non-ATPase regulatory subunit 11 IPI00222515 PSMD11 7 8.1 24 2.5 11 11 Proteasome subunit β type 7 IPI00136483 PSMB7 6 0.336

N-terminal peptide iTRAQ ratio N-terminal peptide iTRAQ 11 Cytoskeleton 0 1 Tropomyosin α-4 chain IPI00421223 TPM4 22 15 2 Coronin-1C IPI00124820 CORO1C 21 15 0 5 10 15 20 25 30 35 40 45 50 3 Talin-1 IPI00465786 TLN1 21 15 ChIP-SeQ peak height 4 Actin-related protein 3 IPI00115627 ACTR3 16 15 5 Tropomyosin 3; γ IPI00169707 TPM3 16 15 Figure 5 MMP-12 exon binding represses expression of PSME3 6 Tubulin-specific chaperone A IPI00222972 TBCA 12 15 7 Isoform 2 of dynamin-1–like protein IPI000172221 DNM1L 11 15 and SPARCL1 genes, and MMP-12 cleaves PSME3 and SPARCL1 8 Microtubule-associated protein 4 IPI00408119 MAP4 11 15 protein. (a,b) University of California–Santa Cruz (UCSC) Genome 9 F-actin-capping protein subunit α-1 IPI00330063 CAPZA1 10 15 10 14-3-3 protein η IPI00227392 YWHAH 9 15 Browser output of peaks from the MMP-12 ChIP-Seq Illumina-Solexa 11 Actin-related protein 2 IPI00177038 ACTR2 7 15 data set showing the highest binding peaks found were over the 12 Myosin-9 IPI00123181 MYH9 7 15 13 Fascin IPI00353563 FSCN1 6 15 Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature exons of PSME3 (a) and SPARCL1 (b) human genes. The highest 14 Vinculin IPI00405227 VCL 6 15 MMP-12 ChIP-Seq peak signals were 172 and 40, respectively. 15 Radixin IPI00308324 RDX 9 15 16 Actin-related protein 2/3 complex subunit 1B IPI00125143 ARPC1B 8 15 (c) PSME3, SPARCL1 and NFKBIA mRNA levels, as measured by 17 α-actinin-4 IPI00118899 ACTN4 8 15 qRT-PCR in MMP-12–specific shRNA–silenced (Si) HeLa and human 18 Plastin-3 IPI00115528 PLS3 7 15 19 -actinin-1 npg – α IPI00380436 ACTN1 10 11.9 1HAEo cells compared to vector-transduced controls (Con). 20 14-3-3 protein γ IPI00230707 YWHAG 7 10.04 Arrows indicate no qRT-PCR signal. Data are representative of 2 21 Microtubule-associated protein RP/EB family member 1 IPI00117896 MAPRE1 10 8.3 22 Kinesin-1 heavy chain IPI00124954 KIF5B 9 7.06 independent experiments and reported as relative quantification (R) 23 Talin 2 isoform 1 IPI00229647 TLN2 18 7.3 of the target gene versus an HPRT housekeeping control gene. 24 F-actin-capping protein subunit α-2 IPI00111265 CAPZA2 11 3.49 (d) Expression of PSME3, SPARCL1, IκBα and β-actin protein, Diverse 1 T-complex protein 1 subunit ε IPI00116279 CCT5 40 13.11 as determined by western blotting in MMP-12–specific shRNA– 2 Transitional endoplasmic reticulum ATPase IPI00622235 VCP 33 15 silenced (Si) HeLa and 1HAEo– cells and empty vector control 3 cAMP-dependent protein kinase IPI00227900 PRKACA 32 15 4 Puromycin-sensitive aminopeptidase IPI00130000 NPEPPS 25 15 cells (Con). Quantification numbers presented were averages 5 Eukaryotic translation initiation factor 3 subunit F IPI00120914 EIF3F 26 14.6 from n = 8 experiments and not from the lanes shown. 6 Isoform long of adenosine kinase IPI00126940 ADK 20 15 7 Sulfated glycoprotein 1 IPI00321190 PSAP 19 15 (e,f) Immunohistochemistry analysis (brown) of SPARCL1 (e) and 8 Pyruvate kinase isozymes M1/M2 IPI00407130 PKM2 18 15 PSME3 (f) proteins in sections of formalin-fixed, wax-embedded 9 Septin 8 IPI00331497 SEPT8 18 14.66 10 Amyloid β A4 protein precursor IPI00114389 APP 17 15 mouse lung tissue. Scale bars, 50 µm. Staining was quantified by 11 Phosphoglucomutase-1 IPI00555140 PGM1 22 3.1 color segmentation using ImagePro Plus software (bottom graphs), and significance was determined by Student’s t-test: *P < 0.05; **P < 0.01. Results are representative of 5 sections cut from 16 mice per genotype that were stained. Error bars are representative of s.d. of the mean. (g) SDS-PAGE and silver staining analysis of SPARCL1 and PSME3 reaction products after incubation with MMP-12. Substrate (black arrowheads) and digestion products (open arrowheads) are shown. Data are representative of 2 independent experiments. (h) Proteogenomic identification of MMP-12–regulated substrates by correlating TAILS with ChIP-Seq. Correlation of MMP-12 substrates identified using TAILS, with ChIP-Seq peak heights of MMP-12 gene binding 1 h after coxsackievirus type B3 infection. Dots represent genes and proteins identified in both data sets (n = 594). Gene ontology (GO) annotation, inferred from international protein index (IPI) number, gene binding peak height and MMP-12–cleaved neo–N-terminal peptide ratio of substrates of selected proteins and genes shown by numbered dots. iTRAQ ratios (MMP-12 cleaved/uncleaved) of cleaved neo–N-terminal peptides >7.3 or <0.13 are high-confidence MMP-12 substrates (P < 0.05) (n = 407). Genes with peak heights >5 are bound by MMP-12 (P < 0.001) (n = 256). Regulated substrates (n = 177) are high-confidence MMP-12 substrates with strong gene binding by MMP-12 and include components of the proteasome (red) and the cytoskeleton (green). Nonregulated substrates (n = 151) are high-confidence, high iTRAQ ratio substrates without gene binding (blue frame).

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Figure 6 Targeted drug inhibition of a b c Infected – + extracellular MMP-12 during virus infection –/– Time (h) 0 1 6 18 18 Mmp12 + IFN-α WT WT enhances plasma levels of IFN-α, significantly WT + IFN-α MMP-12 – + + + + MW (kDa) reducing viral load and morbidity. (a) Mouse –/– IFN-α + + + + – WT, Mmp12 + saline 50 −/− IFN-α was administered to WT and Mmp12 20 kDa 1CD... MMP-12 40 mice infected with coxsackievirus type B3 for 1L161↓R162 39 17 kDa 37 4 d. WT and saline-treated mouse data were L157↓Q155 38 * 105 pooled because they demonstrated no MMP-12 1.0 0 1.0 0.1 ‡ 37 104 difference in temperature (n = 12 per group). IFN-α – + + + 3 36 ‡ 10

Error bars show s.d. of the mean. Significance Body temp ( ° C) 2 35 10 was determined by ANOVA with Tukey’s test 20 kDa (pg/ml) 1 0 1 48 72 96 IFN-α 10 ‡

of significance: *P < 0.05; P < 0.001. Time after IFN- Plasma MMP-12 0 α 10 (b) In vitro cleavage of IFN-α-2A by MMP-12 treatment (h) 0 3 9 30 (1:50 enzyme/substrate, 1–18 h) showing Time after two C-terminal cleavages. Cleavage sites were infection (d) d n = 20 1010 e f Saline RXP470.1 determined by MALDI-TOF MS and Edman * 0.5 Saline 120 * 20 RXP470.1 degradation (Supplementary Fig. 10a). 0.4 100 ** The full electrophoretic gel is presented 80 0.3

1 60 (+) sense uncut in Supplementary Figure 10b. CD 0.2 40 is the N-terminal sequence of the intact IFN-α ‡ Proportion of 0.1

20 image stained starting at position 1. The effect on IFN-α 0 Body weight (g) 15 0 upon digestion with decreasing ratios Plasma IFN- α (pg/ml)

(enzyme/substrate) of MMP-12 is shown by Saline Saline (–) sense (+) sense (–) sense the western blot at the bottom. Labels to the Uninfected RXP470.1 RXP470.1 right of the silver-stained gel denote the cleavage product that each band size represents. (c) MMP-12 was detected in plasma during coxsackievirus infection by western blotting at day 3 of infection and quantified during infection up to 30 d by ELISA (bottom graph). Significance was determined by ANOVA with Tukey’s test of significance: ‡P < 0.001. Error bars denote the s.d. of the mean. Results were derived from the MMP-12 plasma levels from 5 individual mice at each time point. (d) A/J mice infected with coxsackievirus type B3 (n = 20) were treated with MMP-12 inhibitor RXP470.1 (n = 10) or saline (n = 10) by continuous minipump infusion for 7 d. ELISA detected plasma IFN-α levels after 96 h of virus infection. Uninfected control animals (n = 20). Significance was determined by ANOVA with Tukey’s test of significance: *P = 0.05. Error bars denote the s.d. of the mean. (e) Effect of extracellular MMP-12 inhibition on body weight during coxsackievirus type B3 infection. The difference in body weight between the treatment groups (n = 10 for each group) is statistically significant at *P = 0.04, whereas there was no weight difference between the groups before coxsackievirus B3 infection. (f) Coxsackievirus replication, as detected by in situ hybridization of the viral RNA genome (+ sense stained blue) and replication intermediates (− sense) in pancreas from infected mice. Scale bar, 50 µm. Morphometric quantification (n = 20 mice, ten slides per mouse) shows less virus in the RXP470.1-treated mice. Significance was determined by ANOVA with Tukey’s test of significance: **P < 0.01; ‡P < 0.001. Error bars represent s.d. of the mean.

binding and also by MMP12 proteolysis is unprecedented, and so we An MMP-12 inhibitor reduces viral infection term this new group of proteins as dual-regulated substrates. As we showed that extracellular MMP-12 cleaves and inactivates all iso- forms of IFN-α, we sought to establish whether extracellular MMP-12

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature Systemic IFN-a is inactivated by extracellular MMP-12 inhibition could be used as a broad-spectrum antiviral therapy by Infusion of IFN-α, which is pyrogenic22, into Mmp12−/− mice boosting systemic IFN-α levels. However, targeting pleiotropic protein decreased viral load (Fig. 1h,i) but was associated with a significant activities that are outside of their usual function is a therapeutic chal- 23,24

npg initial elevation in body temperature followed by profound hypother- lenge , and broad-spectrum MMP inhibitors have unacceptable mia after 4 d (Fig. 6a). This indicated there was a failure in attenuation side effects25. We addressed this challenge using an MMP-12–specific of IFN-α–induced responses in these mice. phosphinic-peptide inhibitor (RXP470.1) which, because of its double Consistent with an in vivo role for extracellular MMP-12 in clearing negative charge, is membrane impermeable26,27 and so spares the ben- systemic IFN-α, MMP-12 efficiently cleaved the C terminus of IFN-α eficial intracellular activity of MMP-12. Wild-type A/J mice are the at two sites (between Leu157 and Gln158 and between Leu161 and most susceptible strain to model coxsackievirus infection. We treated Arg162), as shown by MALDI-TOF mass spectrometry and Edman infected mice by continuous infusion of RXP470.1 using minipumps sequencing (Fig. 6b and Supplementary Fig. 10a–c), with a kcat/Km over 7 d, resulting in highly elevated plasma IFN-α levels (Fig. 6d), of 221 ± 14 M−1 s−1. Twelve IFN-α isoforms are susceptible to cleav- reduced morbidity and a significant retention of body weight that we age at both conserved cleavage sites, whereas IFN-α-21 was cleaved attribute to reduced degradation of systemic IFN-α (Fig. 6e). Viral at only the most C-terminal of the two sites. Notably, these lie in the replication was also abrogated at day 7, resulting in an ~50% reduction IFN-α receptor-2 binding region, the cleavage of which eliminates in viral load (Fig. 6f) that we expect would further decrease with time IFN-α receptor-2 activation. MMP-12–mediated cleavage of IFN-α following continued dampening of viral replication. is specific, as IFN-β was not cleaved (Supplementary Fig. 10c). At higher MMP-12 to IFN-α ratios (1:10) in vitro IFN-α is completely DISCUSSION degraded (Fig. 6b). Notably, MMP-12 expression is elevated 100-fold We demonstrate that MMP-12 is a protease with highly unexpected in the plasma of wild-type mice 3 d after coxsackievirus infection, and previously unknown transcriptional activity. HeLa cells, which consistent with an in vivo role for MMP-12 in forming a negative are commonly used to model virus infection, constitutively express feedback loop to regulate systemic levels of IFN-α (Fig. 6c). Hence, low concentrations of MMP-12. Here, the secreted MMP-12 is taken we propose two distinct roles for MMP-12: (i) MMP-12 promotes up by the cells in cis and is localized to the nucleus. Whereas human IFN-α export during viral infection and (ii) MMP-12 later attenuates and mouse cardiomyocytes do not produce MMP-12 and bronchial the antiviral response by clearing systemic IFN-α. epithelial cells produce only low levels of MMP-12, macrophage-

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secreted MMP-12 is taken up by the nuclei of target acceptor cells after supplementary information; S.J.W. provided guidance on MMP biology and the viral infection. Nuclear MMP-12 increases NFKBIA transcription, direction of the project; A.D. performed western blotting, mass spectrometry/ biochemical analyses of IFN-α cleavage assays and biochemical validation of the which is essential for optimal IFN-α secretion and antiviral immunity. MMP-12 inhibitor RXP470.1 on MMP-s in vitro and in vivo; G.S.B. expressed and The antiviral response is tightly regulated by MMP-12 through DNA purified MMP-2, MMP-8 and MMP-14, edited the paper and did IFN-α digestion binding of multiple gene exons, including those encoding PSME3, assays; L.M.B. performed macrophage coculture experiments, ran western blots, the immunoproteasome cap protein, and SPARCL1, which reduces did ELISA for cytokines, did RNAi for IκBα and performed confocal microscopy −/− for IFN-α and MMP-12. R.G.H. performed macrophage coculture experiments their mRNA and protein levels in vitro and in vivo in Mmp12 mice. and ran western blots; A.G.R. analyzed ChIP-PCR and ChIP-Seq data and By concerted downregulation, PSME3 is cleaved intracellularly and identified MMP-12 target genes; C.T.C. performed the first Mmp12−/− in vivo SPARCL1 is cleaved extracellularly by MMP-12, and so they are arche- experiment; J.N. performed ELISAs, confocal microscopy and experiments on typical of a new group of proteins we term ‘regulated substrates’, which human biopsy samples; L.A. performed surgeries and RXP470.1 in vivo treatment are rapidly eliminated from cells by both degradation and reduced experiments; Z.L. performed western blots, ELISAs, in situ hybridization for virus and immunohistochemistry and analyzed data; K.H. performed ChIP-PCR; transcription mediated by intracellular MMP-12. M.J.N. performed in vivo respiratory syncytial virus experiments in Figure 1 and We found significantly elevated viral titers and morbidity in supplementary information ; W.D. performed in vivo respiratory syncytial virus Mmp12−/− mice infected with respiratory syncytial virus, with high experiments in Figure 1 and supplementary information; T.B. performed ELISAs mortality also occurring in coxsackievirus infection. This was asso- and edited the paper; A.K. performed ChIP-PCR and western blots; L.D. and D.G. designed and synthesized RXP470.1; R.G.H. conceived of experiments and ciated with reduced IFN-α levels despite strong IL-1β and IFN-γ edited the manuscript; H.L. provided crucial advice and guidance regarding responses in vivo. Later in infection, extracellular MMP-12 also cleaves the cell culture and cell biology experiments and the mouse experiments; plasma IFN-α in the IFN-α receptor-2–binding site, terminating the D.J.G. performed surgeries and RXP470.1 in vivo treatment experiments; long-term and potentially toxic effects of IFN-α in vivo in a negative- V.D. designed and synthesized RXP470.1; B.M.M. conceived of the in vivo experiments, analyzed data, conducted histopathological analysis and supervised feedback loop. The antiviral activities of MMP-12 that are lifesaving in the project; C.M.O. conceived of in vivo and in vitro experiments, designed coxsackievirus type B3 infection are boosted by specific drug inhibi- proteomics analyses, wrote and edited the paper, analyzed data and supervised tion of just the extracellular activity of MMP-12 by RXP470.1, a poten- and provided support for the project. tial new antiviral drug. When administered intravenously, RXP470.1 COMPETING FINANCIAL INTERESTS improved the outcomes of infected mice by reducing morbidity and The authors declare no competing financial interests. virus titers and replication in infected tissues to achieve whole-animal protection. As the viral load was decreased in the mouse strain that is Reprints and permissions information is available online at http://www.nature.com/ most susceptible to virus infection after inhibition of MMP-12 extra- reprints/index.html. cellular activity, MMP-12 could serve as a new antiviral drug target. As pegylated IFN-α is therapeutically administered to patients with 1. Wang, B.X. & Fish, E.N. The yin and yang of viruses and interferons. Trends serious viral infections28, our results lead us to suggest that clinical Immunol. 33, 190–197 (2012). 2. Brunner, K.T., Hurez, D., Mc, C.R. & Benacerraf, B. Blood clearance of P32-labeled development of selective extracellular MMP-12 inhibitors may have vesicular stomatitis and Newcastle disease viruses by the reticuloendothelial system potential as a broad-spectrum antiviral therapeutic strategy. in mice. J. Immunol. 85, 99–105 (1960). 3. Shapiro, S.D., Kobayashi, D.K. & Ley, T.J. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J. Biol. Methods Chem. 268, 23824–23829 (1993). Methods and any associated references are available in the online 4. Belvisi, M.G. & Bottomley, K.M. The role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature version of the paper. role for inhibitors of MMPs? Inflamm. Res. 52, 95–100 (2003). 5. Liang, J. et al. Macrophage metalloelastase accelerates the progression of Note: Any Supplementary Information and Source Data files are available in the atherosclerosis in transgenic rabbits. Circulation 113, 1993–2001 (2006). online version of the paper. 6. Curci, J.A., Liao, S., Huffman, M.D., Shapiro, S.D. & Thompson, R.W. Expression

npg and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal Acknowledgments aortic aneurysms. J. Clin. Invest. 102, 1900–1910 (1998). We thank C. Smits at St. Paul’s Hospital for technical assistance and expert advice 7. McQuibban, G.A. et al. Inflammation dampened by gelatinase A cleavage of regarding the humane care of animal models used in this study. We thank monocyte chemoattractant protein-3. Science 289, 1202–1206 (2000). 8. Parks, W.C., Wilson, C.L. & Lopez-Boado, Y.S. Matrix metalloproteinases as T. Buroker at Seattle Children’s Hospital for IκBα plasmid promoter constructs −/− modulators of inflammation and innate immunity. Nat. Rev. Immunol. 4, 617–629 and A. Hoffmann at the University of California–San Diego for Nfkbia cells (2004). and advice. HL1 cardiomyocytes were a gift from W. Claycomb (Louisiana State 9. Morrison, C.J., Butler, G.S., Rodriguez, D. & Overall, C.M. Matrix metalloproteinase University). This work was supported by Canadian Institutes of Health Research proteomics: substrates, targets, and therapy. Curr. Opin. Cell Biol. 21, 645–653 grants on MMPs during viral infection (no. 08-0369 (B.M.M., D.J.M.)) and on (2009). MMPs in inflammation (nos. MOP-37937 and MOP-111055 (C.M.O.)) and an 10. Houghton, A.M. et al. Macrophage elastase (matrix metalloproteinase-12) suppresses Infrastructure Grant from the Michael Smith Research Foundation (University growth of lung metastases. Cancer Res. 66, 6149–6155 (2006). of British Columbia Centre for Blood Research) and by the British Columbia 11. Dean, R.A. et al. Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR+ CXC chemokines and generates CCL2, -7, -8, and -13 antagonists: Proteomics Network (C.M.O.); salary support for D.J.M. is provided by a Canada potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Research Chair in Viral Pathogenesis and is supported by research fellowships Blood 112, 3455–3464 (2008). from the US Myocarditis Foundation and the Heart and Stroke Foundation of 12. Houghton, A.M., Hartzell, W.O., Robbins, C.S., Gomis-Ruth, F.X. & Shapiro, S.D. Canada; H.L. is funded by the Heart and Stroke Foundation of British Columbia Macrophage elastase kills bacteria within murine macrophages. Nature 460, and Yukon; salary support for C.M.O. is provided by a Canada Research Chair in 637–641 (2009). Metalloproteinase Proteomics and Systems Biology B.M.M. is funded by the Heart 13. Lambert, A.L., Mangum, J.B., DeLorme, M.P. & Everitt, J.I. Ultrafine carbon and Stroke Foundation of British Columbia and Yukon, Genome Canada/British black particles enhance respiratory syncytial virus–induced airway reactivity, Columbia and the Networks of Centres of Excellence–CECR Centre of Excellence pulmonary inflammation, and chemokine expression. Toxicol. Sci. 72, 339–346 (2003). for Prevention of Organ Failure. 14. Samuel, C.E. Antiviral actions of interferons. Clinical Microbiol. Rev. 14, 778–809 (2001). AUTHOR CONTRIBUTIONS 15. Guerrero-Plata, A., Casola, A. & Garofalo, R.P. Human metapneumovirus induces a D.J.M. conceived of, performed and planned most experiments, analyzed the data profile of lung cytokines distinct from that of respiratory syncytial virus. J. Virol. and wrote the paper. C.L.B. performed TAILS, analyzed proteomic data, performed 79, 14992–14997 (2005). biochemical cleavage assays and expressed and purified MMP-12; T.J.M. planned 16. Vallabhapurapu, S. & Karin, M. Regulation and function of NF-κB transcription and conducted the respiratory syncytial virus in vivo experiments in Figure 1 and factors in the immune system. Annu. Rev. Immunol. 27, 693–733 (2009).

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17. Garmaroudi, F.S. et al. Pairwise network mechanisms in the host signaling response 23. Butler, G.S. & Overall, C.M. Proteomic identification of multitasking proteins in to coxsackievirus B3 infection. Proc. Natl. Acad. Sci. USA 107, 17053–17058 unexpected locations complicates drug targeting. Nat. Rev. Drug Discov. 8, (2010). 935–948 (2009). 18. Shimizu-Hirota, R. et al. MT1-MMP- regulates the PI3Kδ.Mi-2/NuRD–dependent 24. Dufour, A. & Overall, C.M. Missing the target: matrix metalloproteinase antitargets control of macrophage immune function. Genes Dev. 26, 395–413 (2012). in inflammation and cancer. Trends Pharmacol. Sci. 34, 233–242 (2013). 19. Wang, X. et al. Matrix metalloproteinase-7 and ADAM-12 (a disintegrin and 25. Overall, C.M. & Kleifeld, O. Tumour microenvironment—opinion: validating matrix metalloproteinase-12) define a signaling axis in agonist-induced hypertension and metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. cardiac hypertrophy. Circulation 119, 2480–2489 (2009). Cancer 6, 227–239 (2006). 20. Mosser, D.M. & Edwards, J.P. Exploring the full spectrum of macrophage activation. 26. Devel, L. et al. Development of selective inhibitors and substrate of matrix Nat. Rev. Immunol. 8, 958–969 (2008). metalloproteinase-12. J. Biol. Chem. 281, 11152–11160 (2006). 21. Kleifeld, O. et al. Isotopic labeling of terminal amines in complex samples identifies 27. Johnson, J.L. et al. A selective matrix metalloproteinase-12 inhibitor retards protein N-termini and protease cleavage products. Nat. Biotechnol. 28, 281–288 atherosclerotic plaque development in apolipoprotein E–knockout mice. Arterioscler. (2010). Thromb. Vasc. Biol. 31, 528–535 (2011). 22. Koyanagi, S., Ohdo, S., Yukawa, E. & Higuchi, S. Chronopharmacological 28. Rizza, P., Moretti, F. & Belardelli, F. Recent advances on the immunomodulatory study of interferon-alpha in mice. J. Pharmacol. Exp. Ther. 283, 259–264 effects of IFN-α: implications for cancer immunotherapy and autoimmunity. (1997). Autoimmunity 43, 204–209 (2010). Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature npg

502 VOLUME 20 | NUMBER 5 | MAY 2014 nature medicine ONLINE METHODS antibodies conjugated with horseradish peroxidase were from Santa Cruz Mouse infection. Five-week-old male MMP-12–knockout mice (Mmp12−/−) Biotechnology. A rabbit polyclonal antibody to IFN-α (ab27715, AbCam) was (n = 8) (stock number 004855) (Jackson Laboratories) and wild-type litter­ used at a 1/500 dilution on tissue sections for immunohistochemistry and at mates (n = 8) were infected with coxsackievirus type B3 (Gauntt strain) 1/2,000 for western blotting. A goat polyclonal antibody was used to detect (1 × 105 plaque forming units (PFU) by intraperitoneal injection and followed IFN-β (ab10740, AbCam) at 1/2,000 dilution. Six different MMP-12–specific for 4 d. In follow-up experiments, n = 24 Mmp12−/− and n = 24 wild-type monoclonal and affinity-purified polyclonal antibodies were purchased from mice were infected with 1 × 104 PFU to minimize morbidity and mortality R&D Systems (catalytic domain, mab919), AbCam (C-terminal hemopexin as per Canadian Council for Animal Care (CCAC) guidelines. Recombinant domain, ab39867) and Triple Point Biologics (pro, catalytic, hinge and hemo- mouse IFN-α or saline vehicle was administered in the anterior tail vein of 8 pexin domains, antibody kit ATK-MMP-12) each at 1/1,000 dilution. Anti-actin Mmp12−/− and 8 wild-type mice, repeated four times daily. Body temperature monoclonal antibody (sc7210) was from Santa Cruz Biotechnology and used was measured using an intrarectal digital probe (Vivo Sonics Technologies) at 1/10,000 dilution. Antibodies for ERK MAP kinase, IκBα and NF-κB (9102, immediately after tail vein injection and 1, 48, 72 and 96 h later. Mice were 9242 and 3035, respectively) were from Cell Signaling Technologies, and each treated as per the CCAC guidelines, and morbidity scores were determined. was used at 1/1,000 dilution. Five-week-old male A/J mice (Jackson Laboratories) were infused with MMP-12 inhibitor RXP470.1 (n = 10) or saline (0.9%, n = 10) using Alzet 1004 osmotic Recombinant proteins. Recombinant human MMP-12, MMP-2, MMP-8 and minipumps (Alzet). Pumps were implanted subcutaneously during isoflurane soluble form of MMP-14 lacking the stalk, transmembrane sequence and cyto- anesthesia and meloxicam analgesia, as per CCAC guidelines. Animals were plasmic C-terminal tail were expressed in Escherichia coli or CHO cells and monitored twice daily. Five days post-implantation, the mice were infected with purified10 or purchased from R&D Systems (mouse MMP-12, 3467-MP-010; 1 × 105 PFU coxsackievirus type B3, weighed daily and euthanized after 4 days. human MMP-12, 917-MP-010). MMP-12 (100 ng µl−1) was activated by 30 µM Blood was obtained via facial vein into heparin tubes and plasma was prepared amino-phenyl mercuric acetate in assay buffer (50 mM Tris-HCl, 1 µM ZnCl2, for MMP-12 assay and IFN-α ELISA (see below). Permission to work with, and 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij 35, pH 7.5), 37 °C. A quenched fluores- oversight of animal work, was provided by the UBC Animal Care Committee. cent peptide substrate (ES010) to assay MMP-12 activity was from R&D Systems. RSV animal studies were approved by the Hospital for Sick Children Animal Recombinant mouse IFN-α (HC1040b) was from HyCult Biotechnology and Care Committee in accordance with the regulation of the CCAC. We obtained IFN-β (12400-1) was from R&D Systems. Human SPARCL1 (AF2728, R&D), female C57 and Mmp12−/− mice, age 5–6 weeks old, from Jackson Laboratories. human PSME3 recombinant protein (Novus Biologicals, H00010197-P01) and Animals were housed in a specific pathogen–free environment and fed food and TNF-α (210-TA-010/CF) were purchased from R&D. water ad libitum. For intranasal instillation, lightly sedated mice (isoflurane) inhaled 100 µl of instillate, which we applied to the nares with a P-200 pipette Inhibitors. The MEK kinase (upstream activator of ERK MAP kinase) inhibitor while their mouths were held closed. On day 0, mice received 5 × 106 PFU U0126 (1144) and IκBα inhibitor Bay11-7083 (1743) were from Tolcris. (100 µl) of respiratory syncytial virus via intranasal instillation. Mice were moni- tored daily, and on day 1 or day 4 they were killed. The right lung was removed, In situ hybridization. The presence of virus (− strand probe) and of active weighed, resuspended in DMEM (100 mg ml−1) and homogenized. We plated virus replication (+ strand probe) in cardiac tissue was assessed by in situ serial dilutions of clarified lung homogenates for viral titer (plaque assays). After hybridization29. removal of the right lung, the left atrium was cut and the right ventricle flushed with 5 ml clean PBS to flush the left lung vasculature. We cannulated the trachea Plaque assay for infectious virus in heart tissue. Infectious virus in the hearts 29 and inflated the lungs with 10% (v/v) formalin at 20 cm H2O pressure. The of coxsackievirus type B3–infected mice was determined by plaque assay . lung was then removed and submerged in formalin for fixation and subsequent Mouse hearts were flash frozen in liquid nitrogen, minced with mortar and histological sectioning. pestle, reconstituted with saline and plated on HeLa indicator cells for plaque determination under agar overlay. Plaques in a limiting dilution series were −1 Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature St. Paul’s Hospital Cardiac Biobank samples. Thirty-two paraffin-embedded counted and reported as PFU ml . autopsy samples of human cardiac tissue with suspected myocarditis, taken from the apical ventricular region of the heart, were obtained from the St. Paul’s ELISA analysis of plasma cytokines and MMP-12. Mouse blood was collected Hospital Cardiac Biobank. Informed consent was provided by the patients for into heparin tubes during euthanasia and centrifuged at 14,000g. Bradford npg their samples to be used for the research purposes. For confidentiality purposes, protein assay (BioRad) of plasma was determined. For ELISAs, 100 ng total the samples presented in this study were recoded as Cardiac Registry (CR) 1–32 plasma protein was added to microwells. ELISA was used to measure the concen- (CR1–CR8 shown). Study approval and oversight were provided by the UBC tration of mouse and human IFN-α (Quantikine, R&D Systems), IL-1β, IFN-β Human Ethics Committee at the Office of Research Ethics. and IFN-γ (Invitrogen-BioSource) and MMP-12 (Kamiya Pharmaceuticals).

Cells. HeLa cells were cultured in 10% FBS (v/v) in DMEM. 1HAEo– cells MMP-12 cleavage assays. Recombinant IFN-α and IFN-β (1 × 102–1 × 105 IU ml−1) were cultured on fibronectin-coated tissue culture plates in DMEM and 10% and 100 µM quenched fluorescent synthetic peptide (ES010, R&D) were incu- FBS (v/v). HL1 cardiomyocytes were a gift from W. Claycomb. Wild-type and bated with 10 or 100 ng MMP-12, 37 °C in assay buffer at enzyme/substrate Nfkbia−/− NIH 3T3 cells were gifts from A. Hoffmann. ratios from 1:10 to 1:100. The ES010 peptide has the sequence Mca-K-P-L- G-L-Dpa-A-R-NH2 (Mca, 7-methoxycoumarin-4-yl)acetyl; Dpa, N-3-(2,4- Macrophage coculture with bronchial epithelial cells and cardiomyocytes. dinitrophenyl)-L-2,3 diaminopropionyl). Fluorometric substrate readings were THP1 macrophages were plated at 1 × 106 cells ml−1 in BD Falcon coculture obtained at 30-min intervals (excitation 420 nm, emission 320 nm), and the inserts (353090) and activated with 100 nM PMA in RPMI growth medium assay stopped at 3 h, when MMP-12 activity plateaued. IFN- cleavage was deter- containing 10% FBS. Following a 3-d incubation period, THP-1 cells from the mined by silver staining Tris-Tricine gels and western blot. To identify cleavage American Type Culture Collection (TIB 202) were washed three times and co- sites, Edman degradation and MALDI-TOF MS was performed as described30. cultured with bystander cells that had been plated the day before incubation with MMP-12 activity in plasma from mice treated with MMP-12 inhibitor RXP470.1 THP-1 cells. Cells were then co-cultured for up to 72 h in bystander cell medium or saline vehicle control was determined using the Sensolyte 520 MMP-12 (MEM for 1HAEO− cells or Claycomb medium for HL-1 cells). Bystander cells activity assay (Anaspec). were then collected for protein extraction using the Ne-Per nuclear cytoplasmic extraction kit (78833) from Pierce or fixed with paraformaldehyde and imaged Silencing of MMP-12 and IkBa. Vector lentiviral particles expressing using confocal immunofluorescence microscopy. short hairpin (sh) RNA sequences against MMP-12 (sc-41557-V) or a control scrambled shRNA (sc-108080) (Santa Cruz Biotechnology) were used to trans- Antibodies. Polyclonal rabbit anti–oligoadenylate synthase-1 (C terminus, duce HeLa cells at 37 °C for 3 h. Growth medium was then replaced and selec- AP6226a, 1/1,000 dilution for western blot) was from AbGent; secondary tion medium consisting of normal growth medium plus puromycin (10 µg/ml)

doi:10.1038/nm.3508 nature medicine was added 3 d later to select for stable transductants. Western blots to MMP-12 1HAEo– cells was also assessed. Total RNA was isolated using RNeasy (Qiagen), validated silencing in the clones. and 0.5 µg of RNA was converted to cDNA using Superscript reverse tran- The IκBα gene, NFKBIA, was silenced by transient transfection of RNA scriptase (Invitrogen). Quantitative PCR combined probe and primer assays interference oligonucleotides purchased from Life Technologies using (n = 3) (PSME3 #Hs00195072_m1; SPARCL1, #Hs00949881_m1; and NFKBIA, AllStars Negative Control siRNA oligonucleotide 1027280, Flexitube siRNA #Hs00153283_m1 (Applied Biosystems)) were performed on an ABI 7900HT Hs_NFKBIA_2 - SI00126812, Flexitube siRNA Hs_NFKBIA_3 - SI00126819. (Applied Biosystems) machine. Expression of the IFN-α genes in mouse fibrob- HeLa and 1HAEo– cells were transfected using Oligofectamine and gene silenc- lasts was detected using Taqman Gene Expression Assays from Life Technologies: ing was confirmed 3 days later by western blot for IκBα. Ifna2: Mm00833961_s1, Ifna9: Mm00833983_s1, Ifna12: Mm00616656_s1, Ifna13: Mm01731013_s1, Hprt: Mm01545399_m1. Luciferase NFKBIA promoter activity assay. Promoter activity assays were conducted in HeLa cells. MMP-12 silencing was performed using the Dual Statistical analyses. The normal distribution of the data was shown in all cases Luciferase Reporter kit (E1910) from Promega. Renilla plasmid was used as a and hence only parametric tests were required. s.d. and analysis of variance transfection control co-transfected with luciferase-expressing NFKBIA promoter were used to analyze data variability. The Student’s t-test was used to determine constructs31 and MMP-12 plasmid32. DNA was delivered to cells using Fugene 6 statistical significance between two treatments, and Tukey’s family compari- transfection reagent (Roche) in a 1:1:1 ratio of Renilla/IκBα luciferase/MMP-12. son was used for more than two treatments. A P value <0.05 was considered Cells were lysed 24 h later and assayed for luciferase and Renilla activity on a statistically significant. GENios (Tecan). Alternatively, the luciferase NFKBIA promoter construct and Renilla plasmids were transfected at a 1:1 ratio and recombinant MMP-12 pro- Proteomic sample preparation. Mouse embryonic fibroblasts (MEFs) were tein was added for 6 h and luciferase and Renilla activities measured. isolated from 129/SvEv Mmp12−/− mice. HeLa and Mmp12−/− MEF secretomes in the conditioned media were collected from eight T175 cell flasks (serum-free, Chromatin immunoprecipitation and ChIP-PCR. DNA chromatin immuno­ 24 h) and clarified (400g, 5 min; 2,250g, 30 min 4 °C) with protease inhibitor precipitation was made with the Quik ChIP kit from Imgenex from control HeLa tablets (Roche) added. Cell lysates of these cultures were prepared by nitrogen cells or after 1 h infection with coxsackievirus type B3. Sonication of the samples decompression of cells and separated from membranes by centrifugation as was conducted on a Vibra Cell VC50T 50 W sonicator (Sonics and Materials) described32. After concentration (Amicon, 30kDa cutoff) and buffer exchange with a 6-mm probe at 80% power, for ten cycles of 30 s sonication and 30 s on to 50 mM HEPES, pH 8.0 protein aliquots (500 µg in 250 µl) were incubated with ice to shear DNA into fragments of approximately 1 kb. Samples were incubated 5 µg AMPA-activated mouse MMP-12 in 50 mM HEPES, pH 7.6 100 mM NaCl with monoclonal rabbit anti–MMP-12 antibody (ab52897, AbCam) overnight at and 5 mM CaCl2 for 16 h, 37 °C. Control samples contained 1 mM APMA. 4 °C, and DNA was extracted after reverse crosslinking using the Qiagen blood and tissue minikit (69504) as described33. Immunoprecipitated DNA chromatin Terminal amine isotopic labeling of substrates (TAILS) mass spectrometry. was used for PCR screening through the NFKBIA promoter using primers shown MMP-12–treated lysates and secretome samples of MEFs and HeLa cells with in Supplementary Table 1. buffer controls were denatured in 2.5 M guanidinium hydrochloride, 250 mM HEPES, pH 8.0 at 65 °C for 15 min and then reduced by addition of 3 mM Tris Genomic library construction and sequencing. Using immunoprecipitated (2-carboxyethyl) phosphine hydrochloride for 45 min, 65 °C (ref. 32). Proteins DNA chromatin, genomic libraries were constructed from uninfected control were alkylated in 5 mM iodoacetamide for 30 min at 65 °C in the dark. Before HeLa cells and cells infected for 1 h with coxsackievirus type B3. The libraries trypsinization, the different protein samples (conditions) were labeled for 4-plex were sequenced (two technical replicates) on the Illumina-Solexa genome experiments with iTRAQ-isobaric isotopic labeling as follows: 114, secretome analysis platform, and 1 Gb data were analyzed as described33. treated with MMP-12; 115, secretome treated with buffer; 116, cell lysate treated with MMP-12; 117, cell lysate treated with buffer34,35. For 2-plex analyses, 2.5 mg ChIP-Seq data analysis. Sequence tags were obtained and mapped to the human of isobaric CLIP-TRAQ 113 (MMP-12 treatment) or 114 (buffer treatment) were 33 36 Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature genome using the Solexa Analysis Pipeline . The output of the Solexa Analysis used . Excess label was quenched with 100 mM ammonium bicarbonate. The Pipeline was converted to wiggle (.wig) files for viewing in the UCSC Genome different isotopic-labeled samples were combined and precipitated with ice-cold Browser (http://genome.ucsc.edu/). acetone/ methanol 8:1 (v/v) for 3 h, −80 °C. After resuspension to 1 ml 100 mM HEPES, pH 8.0, the samples were trypsin digested and the unblocked unlabeled npg Electrophoretic mobility shift assay. Nuclear and cytoplasmic extracts were internal and C-terminal tryptic peptides were coupled to fivefold excess isolated from control and HeLa cells infected with coxsackievirus type B3 for 1 h polyaldehyde-derivatized high-molecular-weight polymer under reductive using the Ne-Per nuclear cytoplasmic extraction kit included with the Pierce conditions21,32 and then removed by ultrafiltration (Centricon, 10 kDa cutoff). Chemiluminescent electromobility shift assay kit. Target oligonucleotides (oligos 3 The remaining N-terminal peptide solution was fractionated by strong cation and 11) and a negative control oligonucleotide (oligo N) (sequences listed in exchange HPLC35. Fractions were concentrated to 100 µl volume and desalted Supplementary Fig. 7b) with 5′ biotin labels were from Sigma. Sense oligo­ using C18 OMIX tips (Agilent Technologies) and analyzed by LC-MS/MS on a nucleotides were annealed to their antisense partner at 95 °C for 5 min, followed QSTAR XL Hybrid ESI mass-spectrometer (Applied Biosystems)35. by 70 cycles for 1 min at 95 °C with a 1 °C step down in temperature per cycle. Test oligonucleotides (50 fmol) and the positive control EDNA oligonucleotide MS/MS peptide assignments and iTRAQ quantification of substrates. (20 fmol) (provided by the manufacturer) were added to full-length MMP-12 Acquired MS/MS scans were searched against International Protein Index or catalytic domain only of MMP-12 or buffer control and incubated at room protein databases (v.3.69) by Mascot version 2.2.2 (Matrix Science) and X! temperature for 30 min. Each 20-µl reaction was loaded onto a 6% polyacryla- Tandem (2007.07.01 release). Searches were performed with the following mide gel in 0.5× Tris-borate EDTA and electrophoresed at 100 V for 1 h (when parameters: Semi-ArgC cleavage specificity with up to two missed cleavages; the dye front was 75% down the gel). Gels were blotted at 400 mA for 1 h onto cysteine carbamidomethylation and peptide lysine iTRAQ or CLIP-TRAQ as positively charged nylon membrane (Boehringer Mannheim) in 0.5× Tris-borate fixed modifications; N-terminal iTRAQ or CLIP-TRAQ36, N-terminal acetyla- EDTA. Detection was on a Chemigenius (Syngene). tion and methionine oxidation as variable modifications; peptide tolerance and MS/MS tolerance of 0.4 Da; ESI-QUAD-TOF scoring scheme. Search Reverse transcriptase quantitative PCR. Reverse-transcriptase polymerase results were modeled and validated by the Trans Proteomic Pipeline (TPP)37,38 chain reaction (RT-PCR) was used to detect transcript levels of MMP12, PSME3, (TPPv.4.3, rev 0, Build 200902191420) using PeptideProphet for peptide/protein SPARCL1 and NFKBIA versus housekeeping HPRT in control HeLa, 1HAEo– identification and Libra for quantification of reporter ion intensities39. Final data cells and primary human bronchial epithelial cells (for which patient informed sets include only peptides with a PeptideProphet probability error rate ≤5%. consent was obtained as approved by the UBC Human Ethics Committee and To determine high confidence MMP-12 substrates we used custom open-source the Office of Research Ethics). RNA from MMP-12 shRNA–silenced HeLa and software (CLIPPER)40 to statistically distinguish MMP-12 generated neo–N

nature medicine doi:10.1038/nm.3508 termini (iTRAQ ratios >7.3 or <0.13 are high confidence (P < 0.05) MMP-12 34. auf dem Keller, U., Prudova, A., Gioia, M., Butler, G.S. & Overall, C.M. A statistics- substrates) from N termini of background proteolysis products and natural based platform for quantitative N-terminome analysis and identification of protease cleavage products. Mol. Cell. Proteomics 9, 912–927 (2010). N termini of proteins (iTRAQ ratios centered on 1.0). 35. Prudova, A., auf dem Keller, U., Butler, G.S. & Overall, C.M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative 29. Cheung, C. et al. Ablation of matrix metalloproteinase-9 increases severity of viral proteomics. Mol. Cell. Proteomics 9, 894–911 (2010). myocarditis in mice. Circulation 117, 1574–1582 (2008). 36. Fahlman, R.P., Chen, W. & Overall, C.M. Absolute proteomic quantification of the 30. Starr, A.E. & Overall, C.M. Chapter 13. Characterizing proteolytic processing of activity state of proteases and proteolytic cleavages using proteolytic signature chemokines by mass spectrometry, biochemistry, neo-epitope antibodies and peptides and isobaric tags. J. Proteomics 100, 79–91 (2014). functional assays. Methods Enzymol. 461, 281–307 (2009). 37. Pedrioli, P.G. Trans-proteomic pipeline: a pipeline for proteomic analysis. Methods 31. Buroker, N.E., Barboza, J. & Huang, J.Y. The IκBα gene is a peroxisome proliferator– Mol. Biol. 604, 213–238 (2010). activated receptor cardiac target gene. FEBS J. 276, 3247–3255 (2009). 38. Deutsch, E.W. et al. A guided tour of the Trans-Proteomic Pipeline. Proteomics 10, 32. Kleifeld, O. et al. Identifying and quantifying proteolytic events and the natural 1150–1159 (2010). N terminome by terminal amine isotopic labeling of substrates. Nat. Protoc. 6, 39. Keller, A., Nesvizhskii, A.I., Kolker, E. & Aebersold, R. Empirical statistical model 1578–1611 (2011). to estimate the accuracy of peptide identifications made by MS/MS and database 33. Robertson, G. et al. Genome-wide profiles of STAT1 DNA association using chromatin search. Anal. Chem. 74, 5383–5392 (2002). immunoprecipitation and massively parallel sequencing. Nat. Methods 4, 651–657 40. auf dem Keller, U. & Overall, C.M. An add-on to the Trans-Proteomic Pipeline for (2007). the Automated Analysis of TAILS data. Biol. Chem. 393, 1477–1483 (2012). Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature npg

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