In ARDS, bacterial pathogens damage host cells, activate innate immune responses, and create a pro-oxidant environment leading to cell death via one of the death programs. This Project will focus on a recently described death program, ferroptosis, realized via Fe-dependent activation of lipid peroxidation under conditions of deficiency of glutathione peroxidase 4 (GPX4), a seleno-enzyme uniquely capable of reducing phospholipid hydroperoxides. We identified 15-hydroperoxy-arachidonoyl-phosphatidylethanolamines (15-HOO-AA-PE) as specific lipid biomarkers of ferroptosis. We also discovered that complexes of 15-lipoxygenase (15LOX) with a scaffold protein, PEBP1, play the major role in generating 15-HOO-AA-PE signals. We divulged ferroptosis as a death program of the human pulmonary epithelium. Ferroptosis occurs in alternatively activated macrophages with low levels of NO?/iNOS, thus causing immuno-suppression. Unexpectedly, we discovered that a common Gram-negative pathogen, P. aeruginosa ? that does not contain polyunsaturated phospholipid oxidation substrates? expresses 15LOX (pLoxA) which oxidizes host polyunsaturated PE, generates 15-HOO-AA-PE and causes ferroptosis in epithelial cells and macrophages independently of the endogenous host 15-LOX. Ferroptosis-inducing pLoxA was detected in clinical P. aeruginosa isolates from ARDS patients. 15-HOO-AA- PE were identified in the lung samples from severely immuno-compromised patients with ARDS. Thus, we postulate the existence of a vicious cycle whereby inflammation/oxidative stress driven ferroptosis supported by endogenous 15-LOX acts as the major contributor to immunosuppression that sets the stage for the secondary P. aeruginosa infection of immune-impaired lung and further enhancement of ferroptosis by exogenous bacterial pLoxA. We propose to design and use selective small molecule pLoxA inhibitors, which will act as anti-ferroptotic agents thus representing new classes of pulmonary protectors.
Aim 1 will reveal and decipher pathogenic mechanisms through which reactions of phospholipid peroxidation catalyzed by isoforms of endogenous mammalian 15-LOX or exogenous bacterial pLoxA ? in conditions of GPX4/GSH deficiency ? lead to accumulation of hydroperoxy-phospholipids in murine lung epithelial cells (MLE) and alveolar macrophages and establish molecular identity and ferroptotic properties of these products. By using redox lipidomics we will identify and quantify 15-HOO-AA-PE biomarkers of ferroptosis in vivo using a two-hit model of immunosuppression. We will also employ iNOS KO animals exposed to P. aeruginosa to reveal the role of NO? as a regulator of pLoxA-driven AA-PE oxidation and ferroptotic death in the mouse lung vivo.
In Aim 2, we will design and develop selective inhibitors of pLoxA regulating P. aeruginosa-driven ferroptosis in epithelia and macrophages as a new class of small-molecule cytoprotective agents preventing breach of the barrier and immunosuppression.

Public Health Relevance

P. aeruginosa is a ubiquitous Gram-negative pathogen that causes severe respiratory tract infections with high mortality, particularly in immunocompromised individuals. Therefore, understanding pathogenesis of P. aeruginosa infections is essential for the development of novel therapeutic strategies. Death of pulmonary epithelial cells and immune cells are two important pathogenic factors. We will explore a new cell death program, ferroptosis, used by P. aeruginosa to kill the lung cells by hijacking and oxidizing the host phospholipids. We will design small molecule regulators of ferroptosis as new therapeutic protectors of the lung cells

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL114453-07
Application #
9922963
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Zhou, Guofei
Project Start
Project End
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
7
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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