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Peroxiredoxin V Contributes to Antioxidant Defense of Lung Epithelial Cells

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Abstract

The aim of this study was to determine the functional significance of peroxiredoxin V (PRXV) in defense against oxidative stress and changes of its expression in human lung inflammation. We used in vitro cell cultures and retrospective analyses of human sputum samples to perform the study. We found that stable clones of lung epithelial cell lines A549 and U1810 with reduced expression of PRXV were prone to oxidative damage. Upregulation of PRXV decreased induction of DNA double-strand breaks and protein oxidation by cigarette smoke extract and hydrogen peroxide. Transfection with PRXV-carrying plasmid protected Calu-3 confluent epithelial cell sheets from alterations in barrier permeability induced by oxidative stress. In human sputum proinflammatory cytokines, myeloperoxidase, and PRXV were increased during viral-induced inflammation. We conclude that PRXV is an important antioxidant protein of lung epithelial cells. Its expression in the human lung increases in inflammation.

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Acknowledgments

Research described in this article was supported by Philip Morris USA Inc. and by Philip Morris International, and by INTAS Genomics grant 05-1000004-7755. Clinical samples were collected under funding from NIAID (AI50496 and AI057506). P. Avila is currently funded by the Ernest S. Bazley Grant to Northwestern University. The authors thank Dr. Homer Boushey, Ms. Theresa Ward, and Ms. Jane Liu for their assistance in conducting the clinical common cold study, and Ms. Junqing Shen for technical work with rhinovirus infection of epithelial cell cultures.

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Authors and Affiliations

  1. Feinberg School of Medicine, Northwestern University, Chicago, Illinois, 60611, USA

    Pedro C. Avila

  2. Institute of Cytology, RAS, St. Petersburg, Russia

    Andrei V. Kropotov & Nikolay V. Tomilin

  3. Department of Cell Biology, St. Petersburg State University, St. Petersburg, Russia

    Anna Krasnodembskay

  4. Children’s Hospital Oakland Research Institute, Serikov Lab, 5700 Martin Luther King Jr. Way, Oakland, CA, 94609, USA

    Raisa Krutilina & Vladimir B. Serikov

Authors
  1. Pedro C. Avila
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  2. Andrei V. Kropotov
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  3. Raisa Krutilina
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  4. Anna Krasnodembskay
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  5. Nikolay V. Tomilin
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  6. Vladimir B. Serikov
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Correspondence to Vladimir B. Serikov.

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408_2007_9066_MOESM4_ESM.tif

[DTT is known to affect measurements of some cytokines. Study was performed to determine whether the presence of this reducing agent could influence measurements of PRXV levels by Western blot. DTT and sputum did not affect measurements of PRXV. Representative Western blot analyses for PRXV. Ten nanograms of recombinant PRXV were incubated in PBS, PBS with 10 mM DTT (A), or different sputum samples (B) with known very low or zero concentration of endogenous PRXV for 30 min at 37°C]

408_2007_9066_MOESM5_ESM.tif

[Level of myeloperoxidase (MPO) is an index of the presence of neutrophils, indicative of the level of inflammation. Correlation between MPO levels and PRXV levels was investigated to determine whether levels of PRXV upregulation are related to inflammatory reaction. PRXV levels correlated strongly with MPO levels in induced-sputum samples]

408_2007_9066_MOESM6_ESM.tif

[Experiments were performed in vitro to determine whether inoculation of epithelial cell cultures with RV16 virus changes expression of PRXV without the presence of immune cells. Expression of PRXV was not changed by RV16 infection in vitro in epithelial cells. These data indicate that upregulation of PRXV requires the presence of activated immune cells, which may trigger secretion of PRXV by paracrine mechanisms or become an alternative source of PRXV. A Confocal microscopy images of nasal epithelial cultures stained for PRXV with FITC-labeled secondary antibody. A1 Control staining without primary antibody, no nonspecific green fluorescence is present, costaining with propidium iodine (red fluorescence). Upper left panel – PI red fluorescence; upper right panel – FITC green fluorescence; lower panel – combined image. A2 Control noninfected culture. A3 Culture infected with RV16. Original magnification 40×. Diagrams at the bottom demonstrate distribution of green and red fluorescence signals along the median horizontal axis. B Western blot analyses of tissue lysates for PRXV. Actin was used as control]

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Avila, P.C., Kropotov, A.V., Krutilina, R. et al. Peroxiredoxin V Contributes to Antioxidant Defense of Lung Epithelial Cells. Lung 186, 103–114 (2008). https://doi.org/10.1007/s00408-007-9066-2

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