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

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Abstract

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12−/− but not wild-type mice to express IκBα and thus export IFN-α 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-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3–infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α 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.

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Figure 1: Mmp12−/− mice demonstrate increased viral load, morbidity, mortality and lower IFN-α levels in viral infections.
Figure 2: IFN-α is synthesized but not secreted from Mmp12−/− cells as a result of IκBα deficiency.
Figure 3: Nuclear localization of MMP-12 in human and mouse cells.
Figure 4: Nuclear MMP-12 binds the human NFKBIA promoter during viral infection, activating transcription.
Figure 5: MMP-12 exon binding represses expression of PSME3 and SPARCL1 genes, and MMP-12 cleaves PSME3 and SPARCL1 protein.
Figure 6: Targeted drug inhibition of extracellular MMP-12 during virus infection enhances plasma levels of IFN-α, significantly reducing viral load and morbidity.

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Acknowledgements

We thank C. Smits at St. Paul's Hospital for technical assistance and expert advice regarding the humane care of animal models used in this study. We thank T. Buroker at Seattle Children's Hospital for IκBα plasmid promoter constructs and A. Hoffmann at the University of California–San Diego for Nfkbia−/− cells and advice. HL1 cardiomyocytes were a gift from W. Claycomb (Louisiana State University). This work was supported by Canadian Institutes of Health Research grants on MMPs during viral infection (no. 08-0369 (B.M.M., D.J.M.)) and on MMPs in inflammation (nos. MOP-37937 and MOP-111055 (C.M.O.)) and an Infrastructure Grant from the Michael Smith Research Foundation (University of British Columbia Centre for Blood Research) and by the British Columbia Proteomics Network (C.M.O.); salary support for D.J.M. is provided by a Canada Research Chair in Viral Pathogenesis and is supported by research fellowships from the US Myocarditis Foundation and the Heart and Stroke Foundation of Canada; H.L. is funded by the Heart and Stroke Foundation of British Columbia and Yukon; salary support for C.M.O. is provided by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology B.M.M. is funded by the Heart and Stroke Foundation of British Columbia and Yukon, Genome Canada/British Columbia and the Networks of Centres of Excellence–CECR Centre of Excellence for Prevention of Organ Failure.

Author information

D.J.M. conceived of, performed and planned most experiments, analyzed the data and wrote the paper. C.L.B. performed TAILS, analyzed proteomic data, performed biochemical cleavage assays and expressed and purified MMP-12; T.J.M. planned and conducted the respiratory syncytial virus in vivo experiments in Figure 1 and supplementary information; S.J.W. provided guidance on MMP biology and the direction of the project; A.D. performed western blotting, mass spectrometry/biochemical analyses of IFN-α cleavage assays and biochemical validation of the MMP-12 inhibitor RXP470.1 on MMP-s in vitro and in vivo; G.S.B. expressed and purified MMP-2, MMP-8 and MMP-14, edited the paper and did IFN-α digestion assays; L.M.B. performed macrophage coculture experiments, ran western blots, 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 and ran western blots; A.G.R. analyzed ChIP-PCR and ChIP-Seq data and identified MMP-12 target genes; C.T.C. performed the first Mmp12−/− in vivo experiment; J.N. performed ELISAs, confocal microscopy and experiments on human biopsy samples; L.A. performed surgeries and RXP470.1 in vivo treatment experiments; Z.L. performed western blots, ELISAs, in situ hybridization for virus and immunohistochemistry and analyzed data; K.H. performed ChIP-PCR; M.J.N. performed in vivo respiratory syncytial virus experiments in Figure 1 and supplementary information ; W.D. performed in vivo respiratory syncytial virus experiments in Figure 1 and supplementary information; T.B. performed ELISAs 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 edited the manuscript; H.L. provided crucial advice and guidance regarding the cell culture and cell biology experiments and the mouse experiments; D.J.G. performed surgeries and RXP470.1 in vivo treatment experiments; V.D. designed and synthesized RXP470.1; B.M.M. conceived of the in vivo experiments, analyzed data, conducted histopathological analysis and supervised the project; C.M.O. conceived of in vivo and in vitro experiments, designed proteomics analyses, wrote and edited the paper, analyzed data and supervised and provided support for the project.

Correspondence to Bruce M McManus or Christopher M Overall.

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Marchant, D., Bellac, C., Moraes, T. et al. A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity. Nat Med 20, 493–502 (2014) doi:10.1038/nm.3508

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