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Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

Blaskovic, Sanja, Donati, Yves, Ruchonnet-Metrailler, Isabelle, Seredenina, Tamara, Krause, Karl-Heinz, Pache, Jean-Claude, Adler, Dan, Barazzone-Argiroffo, Constance, Jaquet, Vincent·2021·Antioxidants
DOI 10.3390/antiox10111833PMC PMC8615037
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Abstract

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

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Figure 1 (A) TGFβ1-induced upregulation of the expression of NOX4, the myofibroblast marker alpha-SMA and proteins of the ECM in MRC5 human lung fibroblasts. N = 4, * p < 0.05 using Mann−Whitney nonparametric test. (B). Fluorescent microscopy of DT in TGF-β1-treated MRC5. Addition of H2O2 in presence of HRP cells induces crosslinking of the fluorescein tyramide (TSA, green) with the extracellular matrix network. The cross-links are detected by the DT antibody (red) and colocalize with crosslinked TSA (yellow). (C). Similar experiment with human skin fibroblasts from a healthy donor and a patient carrying a p22phox mutation. Deletion of p22phox completely abolishes the formation of DT in the absence of exogenous H2O2. Blue, DAPI staining of cell nuclei. NOX4: NADPH oxidase isoform 4; ACTA 2: Actin Alpha2, Smooth Muscle; FN: Fibronectin; COL1A: Collagen1A; DAPI: 4′,6-diamidino-2-phenylindole; TSA: Alexa Fluor 488 tyramide Reagent; Di-TYR: dityrosine; TGFβ: Transforming growth factor β; Cont: control; HRP: horseradish peroxidase.
Figure 1 (A) TGFβ1-induced upregulation of the expression of NOX4, the myofibroblast marker alpha-SMA and proteins of the ECM in MRC5 human lung fibroblasts. N = 4, * p < 0.05 using Mann−Whitney nonparametric test. (B). Fluorescent microscopy of DT in TGF-β1-treated MRC5. Addition of H2O2 in presence of HRP cells induces crosslinking of the fluorescein tyramide (TSA, green) with the extracellular matrix network. The cross-links are detected by the DT antibody (red) and colocalize with crosslinked TSA (yellow). (C). Similar experiment with human skin fibroblasts from a healthy donor and a patient carrying a p22phox mutation. Deletion of p22phox completely abolishes the formation of DT in the absence of exogenous H2O2. Blue, DAPI staining of cell nuclei. NOX4: NADPH oxidase isoform 4; ACTA 2: Actin Alpha2, Smooth Muscle; FN: Fibronectin; COL1A: Collagen1A; DAPI: 4′,6-diamidino-2-phenylindole; TSA: Alexa Fluor 488 tyramide Reagent; Di-TYR: dityrosine; TGFβ: Transforming growth factor β; Cont: control; HRP: horseradish peroxidase.CC BY · PubMed Central
Figure 2 Representative images of DT (A) and NOX4 (C) staining in the indicated patients are shown for lungs. Quantification of staining for DT (B) and NOX4 (D). * p < 0.05 using Mann−Whitney nonparametric test. CTRL: control; IPF: idiopathic pulmonary fibrosis.
Figure 2 Representative images of DT (A) and NOX4 (C) staining in the indicated patients are shown for lungs. Quantification of staining for DT (B) and NOX4 (D). * p < 0.05 using Mann−Whitney nonparametric test. CTRL: control; IPF: idiopathic pulmonary fibrosis.CC BY · PubMed Central
Figure 3 Representative images for DT ad NOX4 staining of blood vessel smooth muscle cells in the control (A) and IPF samples (B) of human patients are shown. Inset shows a 10× higher magnification and depicts typical smooth muscle cell staining in these samples. DT and NOX4 staining are shown in brown.
Figure 3 Representative images for DT ad NOX4 staining of blood vessel smooth muscle cells in the control (A) and IPF samples (B) of human patients are shown. Inset shows a 10× higher magnification and depicts typical smooth muscle cell staining in these samples. DT and NOX4 staining are shown in brown.CC BY · PubMed Central
Figure 4 Representative images for DT and NOX4 staining of AECII cells (A) and bronchi (B) in the control (upper panel) and IPF/UIP samples (lower panel) of human patients are shown. Insets shows a 10× higher magnification and depicts AECII (A) and ciliary bronchial cells (B) in these samples. DT and NOX4 staining are shown in brown.
Figure 4 Representative images for DT and NOX4 staining of AECII cells (A) and bronchi (B) in the control (upper panel) and IPF/UIP samples (lower panel) of human patients are shown. Insets shows a 10× higher magnification and depicts AECII (A) and ciliary bronchial cells (B) in these samples. DT and NOX4 staining are shown in brown.CC BY · PubMed Central
Figure 5 Representative images for DT and NOX4 staining of blood vessel endothelial cells in the control (upper panels) and the IPF/UIP samples (lower panels) from human patients are shown. Inset shows a 10X higher magnification and depicts typical intimal endothelial region of these samples. DT and NOX4 staining are shown in brown. The red arrows indicate the NOX4 endothelial staining.
Figure 5 Representative images for DT and NOX4 staining of blood vessel endothelial cells in the control (upper panels) and the IPF/UIP samples (lower panels) from human patients are shown. Inset shows a 10X higher magnification and depicts typical intimal endothelial region of these samples. DT and NOX4 staining are shown in brown. The red arrows indicate the NOX4 endothelial staining.CC BY · PubMed Central
Figure 6 Histograms documenting the number of samples positive for DT and NOX4 for each cellular subtype. All 4 IPF patients were positive for DT in SMC and AECII while none of the controls showed DT staining (A). All cell types (SMC, AECII and EC were stained for NOX4 in IPF patients, while only EC were stained in the control (B). Macrophages were stained with both antibodies in both control and IPF (most likely not specific). SMC: smooth muscle cells, EC: endothelial cells, AECII: alveolar epithelial cells type 2, MAC macrophages.
Figure 6 Histograms documenting the number of samples positive for DT and NOX4 for each cellular subtype. All 4 IPF patients were positive for DT in SMC and AECII while none of the controls showed DT staining (A). All cell types (SMC, AECII and EC were stained for NOX4 in IPF patients, while only EC were stained in the control (B). Macrophages were stained with both antibodies in both control and IPF (most likely not specific). SMC: smooth muscle cells, EC: endothelial cells, AECII: alveolar epithelial cells type 2, MAC macrophages.CC BY · PubMed Central

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Research works printed but not linked: 31

  1. Wolters, 2014, Pathogenesis of Idiopathic Pulmonary Fibrosis, Annu. Rev. Pathol. Mech. Dis., 9, 157, 10.1146/annurev-pathol-012513-104706 · DOI
  2. Shumar, J., Chandel, A., and King, C. (2021). Antifibrotic Therapies and Progressive Fibrosing Interstitial Lung Disease (PF-ILD): Building on INBUILD. J. Clin. Med., 10. · DOI
  3. Betensley, A., Sharif, R., and Karamichos, D. (2016). A Systematic Review of the Role of Dysfunctional Wound Healing in the Patho-genesis and Treatment of Idiopathic Pulmonary Fibrosis. J. Clin. Med., 6. · DOI
  4. Veith, 2019, Redox Imbalance in Idiopathic Pulmonary Fibrosis: A Role for Oxidant Cross-Talk Between NADPH Oxidase Enzymes and Mitochondria, Antioxid. Redox Signal., 31, 1092, 10.1089/ars.2019.7742 · DOI
  5. Carnesecchi, 2011, A key role for NOX4 in epithelial cell death during development of lung fibrosis, Antioxid. Redox Signal., 15, 607, 10.1089/ars.2010.3829 · DOI · Paper page
  6. Bedard, 2007, The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology, Physiol. Rev., 87, 245, 10.1152/physrev.00044.2005 · DOI · Paper page
  7. Sies, 2020, Reactive oxygen species (ROS) as pleiotropic physiological signalling agents, Nat. Rev. Mol. Cell Biol., 21, 363, 10.1038/s41580-020-0230-3 · DOI · Paper page
  8. Geiszt, 2019, The Relationship of NADPH Oxidases and Heme Peroxidases: Fallin’ in and Out, Front. Immunol., 10, 394, 10.3389/fimmu.2019.00394 · DOI
  9. Aeschbach, 1976, Formation of dityrosine cross-links in proteins by oxidation of tyrosine residues, Biochim. Biophys. Acta Protein Struct., 439, 292, 10.1016/0005-2795(76)90064-7 · DOI
  10. Wong, 2004, The Oxidative Burst at Fertilization Is Dependent upon Activation of the Dual Oxidase Udx1, Dev. Cell, 7, 801, 10.1016/j.devcel.2004.10.014 · DOI
  11. Hensley, 1998, Electrochemical analysis of protein nitrotyrosine and dityrosine in the Alzheimer brain indicates region-specific accumulation, J. Neurosci., 18, 8126, 10.1523/JNEUROSCI.18-20-08126.1998 · DOI
  12. Biasetti, 2016, The involvement of dityrosine crosslinking in alpha-synuclein assembly and deposition in Lewy Bodies in Parkinson’s disease, Sci. Rep., 6, 39171, 10.1038/srep39171 · DOI
  13. Leeuwenburgh, 1997, Mass Spectrometric Quantification of Markers for Protein Oxidation by Tyrosyl Radical, Copper, and Hydroxyl Radical in Low Density Lipoprotein Isolated from Human Atherosclerotic Plaques, J. Biol. Chem., 272, 3520, 10.1074/jbc.272.6.3520 · DOI
  14. Locy, 2020, Oxidative cross-linking of fibronectin confers protease resistance and inhibits cellular migration, Sci. Signal., 13, eaau2803, 10.1126/scisignal.aau2803 · DOI
  15. Brault, 2014, Optimized Generation of Functional Neutrophils and Macrophages from Patient-Specific Induced Pluripotent Stem Cells:Ex VivoModels of X0-Linked, AR220- and AR470- Chronic Granulomatous Diseases, BioRes. Open Access, 3, 311, 10.1089/biores.2014.0045 · DOI
  16. Prior, 2016, CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity, Redox Biol., 9, 287, 10.1016/j.redox.2016.08.013 · DOI
  17. Vandesompele, 2002, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple in-ternal control genes, Genome Biol., 3, RESEARCH0034, 10.1186/gb-2002-3-7-research0034 · DOI · Paper page
  18. Weinberg, R.A., and Comb, M.J. (2015). Chapter 12: Immunohistochemistry (IHC) in CST Guide: Pathways & Protocols, Cell Signaling Technology, Inc.. [1st ed.].
  19. Diebold, 2019, Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR, Methods Mol. Biol., 1982, 191, 10.1007/978-1-4939-9424-3_12 · DOI
  20. Augsburger, 2019, Pharmacological characterization of the seven human NOX isoforms and their inhibitors, Redox Biol., 26, 101272, 10.1016/j.redox.2019.101272 · DOI
  21. Rajaram, 2019, Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution, Redox Biol., 26, 101234, 10.1016/j.redox.2019.101234 · DOI
  22. Kato, 2000, Immunochemical Detection of Protein Dityrosine in Atherosclerotic Lesion of Apo-E-Deficient Mice Using a Novel Monoclonal Antibody, Biochem. Biophys. Res. Commun., 275, 11, 10.1006/bbrc.2000.3265 · DOI
  23. Larios, 2001, Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts, J. Biol. Chem., 276, 17437, 10.1074/jbc.M100426200 · DOI
  24. Woods, 2003, Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques, Biochem. J., 370, 729, 10.1042/bj20021710 · DOI
  25. Maher, 2019, Antifibrotic therapy for idiopathic pulmonary fibrosis: Time to treat, Respir. Res., 20, 1, 10.1186/s12931-019-1161-4 · DOI
  26. Mora, 2017, Emerging therapies for idiopathic pulmonary fibrosis, a progressive age-related disease, Nat. Rev. Drug Discov., 16, 755, 10.1038/nrd.2017.170 · DOI
  27. Modarressi, 2016, NADPH oxidase 4 deficiency leads to impaired wound repair and reduced dityrosine-crosslinking, but does not affect myofibroblast formation, Free. Radic. Biol. Med., 96, 374, 10.1016/j.freeradbiomed.2016.04.194 · DOI
  28. Luxen, 2009, Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells, J. Cell Sci., 122, 1238, 10.1242/jcs.044123 · DOI
  29. Forteza, 2005, Regulated hydrogen peroxide production by Duox in human airway epithelial cells, Am. J. Respir. Cell Mol. Biol., 32, 462, 10.1165/rcmb.2004-0302OC · DOI
  30. Sarr, 2018, Antimicrobial actions of dual oxidases and lactoperoxidase, J. Microbiol., 56, 373, 10.1007/s12275-018-7545-1 · DOI · Paper page
  31. Louzada, 2021, NADPH oxidase DUOX1 sustains TGF-beta1 signalling and promotes lung fibrosis, Eur. Respir. J., 57, 1901949, 10.1183/13993003.01949-2019 · DOI

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Metadata

Authors
Blaskovic, S., Donati, Y., Ruchonnet-Metrailler, I., Seredenina, T., Krause, K.-H., Pache, J.-C., Adler, D., Barazzone-Argiroffo, C., & Jaquet, V.
Affiliations
  • Department of Pediatrics, Gynecology and Obstetrics, Children’s Hospital, 1211 Geneva, Switzerland
  • Department of Pathology and Immunology, Medical School, University of Geneva, 1211 Geneva, Switzerland
  • Service of Clinical Pathology, Department of Pathology and Immunology, Medical School, University of Geneva, 1211 Geneva, Switzerland
  • Division of Pulmonary Diseases, Department of Medicine, Geneva University Hospitals, 1211 Geneva, Switzerland
ORCID
Venue
Antioxidants
Publisher
MDPI AG
Published
2021-11-18
Volume
10
Issue
11
Pages
1833
ISSN
2076-3921
DOI
10.3390/antiox10111833
PMC
PMC8615037
Cited by
11
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Swiss national science foundation (310030-159500/1)

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CoChat. (2026). Citation check: Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis. https://app.cochat.ai/papers/10.3390/antiox10111833
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Blaskovic, S., Donati, Y., Ruchonnet-Metrailler, I., Seredenina, T., Krause, K.-H., Pache, J.-C., Adler, D., Barazzone-Argiroffo, C., & Jaquet, V. (2021). Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis. Antioxidants. https://doi.org/10.3390/antiox10111833