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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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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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