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Reducing protein oxidation reverses lung fibrosis


Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death1,2,3. Oxidative stress is believed to be critical in this disease pathogenesis4,5,6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)7. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.

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This work was funded by NIH R35HL135828, R01HL079331, R01HL060014 and R03HL095404 (Y.M.W.J.-H.), an ATS unrestricted grant and NIH R01HL122383 (V.A.), Parker B. Francis fellowships (V.A. and J.L.J.v.d.V.), NIH R01HL05646 (A.v.d.V.), T32HL076122 (D.H.M. and J.T.J.), NCRR 1S10RR019246 (D.J.T.), American Heart Association 16GRNT27660006, NIH R01DK103750 and 1UL1TR001430 (M.M.B.), NIH R01HL133013 and R03 AG051857 (R.M.), P30GM103532 (C.G.I.), FAMRI Young Clinical Scientist Award 113393 and 1K01HL125474-01 (J.L.G.) and U01HL108642 and RC2 HL101715 (N.K.). Analysis of biological specimens and data provided by the Lung Tissue Research Consortium (LTRC) was supported by R03HL095404 (Y.M.W.J.-H.). We would like to thank J. Whitsett (University of Cincinnati) for generously providing the CCSP-rtTA, TetOP-Cre mice, J. Gauldie for providing AdTGFB1, N. Bishop for help with imaging of GLRX and collagen, J. M. Siddesha for technical support and P. Vacek for statistical support.

Author information

V.A., K.G.L., S.M.H., D.H.M., J.T.J., E.E., J.D.N., D.T.C., J.L.J.v.d.V. and R.S. performed the animal experiments. S.A., S.B.C. and K.G.L. prepared and tested recombinant GLRX; V.A., S.M.H., E.C.R., R.A. and K.G.L. performed S-glutathionylation and glutaredoxin assays; K.G.L. and D.T.C. performed fibrosis analyses; K.J.B. reviewed histopathology; D.J.T. provided critical suggestions regarding imaging and image analysis. Y.-S.H. generated Glrx−/− mice and helped with data interpretation; R.A. and S.B.C. performed collagenolytic activity assays; M.D.L. and M.E.C. performed and analyzed CD studies; R.H. generated Casp8loxP/loxP mice and helped with data interpretation. R.M. and M.M.B. conducted the ageing study and participated in data analyses. A.v.d.V. and R.C.B. supported the studies with key experimental suggestions and helped interpret data. D.J.S., D.G.C., C.G.I., K.J.B., J.L.G., N.K. and K.K.B. evaluated and analyzed clinical data. Y.M.W.J.-H. and V.A. designed the research, interpreted the data and wrote the manuscript.

Competing interests

Y.M.W.J.-H. and V.A. hold patents entitled: ‘Treatments involving glutaredoxins and similar agents’, ‘Treatments of oxidative stress conditions’ (US patent no. 8,679,811 and 9,907,828; Y.M.W.J.-H. and V.A.) and ‘Detection of glutathionylated proteins’ (US patent no. 8,877,447; Y.M.W.J.-H.).

Correspondence to Yvonne M. W. Janssen-Heininger.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Dataset

    F, t and df values

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Fig. 1: Lower glutaredoxin-1 (GLRX) enzymatic activity and higher protein PSSG occur in lung tissues from subjects with IPF and correlate with disease severity.
Fig. 2: Bleomycin-induced lung fibrosis and PSSG are elevated in Glrx−/− mice and lowered following transgenic overexpression of Glrx in lung epithelial cells.
Fig. 3: Direct administration of recombinant Glrx to the lung protects against the progression of and reverses existing pulmonary fibrosis.
Fig. 4: Ageing-associated enhanced susceptibility to pulmonary fibrosis induced by bleomycin is associated with low Glrx activity and can be rescued following oropharyngeal administration of Glrx.