15LO1 dictates glutathione redox changes in asthmatic airway epithelium to worsen type 2 inflammation
Tadao Nagasaki, … , Valerian E. Kagan, Sally E. Wenzel
Tadao Nagasaki, … , Valerian E. Kagan, Sally E. Wenzel
Published November 11, 2021
Citation Information: J Clin Invest. 2022;132(1):e151685. https://doi.org/10.1172/JCI151685.
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Research Article Metabolism Pulmonology

15LO1 dictates glutathione redox changes in asthmatic airway epithelium to worsen type 2 inflammation

Abstract

Altered redox biology challenges all cells, with compensatory responses often determining a cell’s fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway–induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

Authors

Tadao Nagasaki, Alexander J. Schuyler, Jinming Zhao, Svetlana N. Samovich, Kazuhiro Yamada, Yanhan Deng, Scott P. Ginebaugh, Stephanie A. Christenson, Prescott G. Woodruff, John V. Fahy, John B. Trudeau, Detcho Stoyanovsky, Anuradha Ray, Yulia Y. Tyurina, Valerian E. Kagan, Sally E. Wenzel

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Figure 3

15LO1 expression and activity regulate intracellular redox balance by lowering GSH and GSH:GSSG.

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15LO1 expression and activity regulate intracellular redox balance by lo...
(A) Under IL-13–stimulated conditions, ALOX15 (15LO1) knockdown (KD) increases intracellular GSH and the GSH:GSSG ratio toward control conditions (n = 6). (B) Under IL-13 conditions, inhibition of 15LO1 by the specific 15LO1 inhibitor BLX2477 also restores intracellular GSH and GSH:GSSG (n = 6). (C) ALOX15 KD lowers extracellular GSSG and restores a normal GSH:GSSG ratio (n = 6). (D) Representative Western blots demonstrating that ALOX15 KD also decreases GPX4 protein expression (n = 4). (E) Representative Western blots show decreased expression of GPX4 under conditions of 15LO1 inhibition (n = 4). (F) Ex vivo 15LO1 protein expression correlates with intraepithelial cell GSH and GSH:GSSG (n = 15). (G) Indexing SLC7A11, as a critical regulator of intracellular GSH levels, to 15LO1 improves the relationship of 15LO1 to intracellular GSH (n = 12). Matched-pair analysis (A–C) of log-transformed data was used to compare conditions, while Pearson’s correlation was used for F and G.