Substrate-dependent effects of myeloperoxidase-derived oxidants on the metabolome and transcriptome of human airway epithelial cells Restricted; Files & ToC

Abstract

The airway epithelium is the primary barrier to the inhaled

environment and contributes to recruitment of immune cells in

response to insults like pathogens. Neutrophils, the initial

responders, release large amounts of myeloperoxidase (MPO), a

heme peroxidase enzyme that generates potent antimicrobial

oxidants. These include hypochlorous acid (HOCl), the active

ingredient in household bleach, as well as hypobromous acid

(HOBr) and hypothiocyanous acid (HOSCN). These oxidants differ

in reactivity, and in chronic obstructive pulmonary disease and

cystic fibrosis (CF), molecular markers consistent with HOCl and

HOBr, but not HOSCN, are associated with lung damage. Thus,

comprehending how each MPO-derived oxidant benefit or harm

airway epithelial cells in physiologically relevant milieu is crucial

to elucidate mechanisms of (mal)adaptation. To accomplish this,

we exposed human bronchoepithelial cells (BEAS-2B) to equal

dose-rates of enzymatically generated HOCl, HOBr, and HOSCN

by addition of different MPO substrates (chloride, bromide, and

thiocyanate, respectively). We profiled the intracellular

metabolome and transcriptome of BEAS-2B using high-resolution

mass spectrometry and RNA-sequencing. We found substratedependent

metabolic and transcriptional responses indicating the

divergent effects of MPO-derived oxidants. Thiocyanate,

promoting HOSCN production was tolerated by BEAS-2B for at

least 24 h in our model and caused pro-adaptive responses,

potentially through pentose phosphate pathway activation,

branched-chain amino acid catabolism, and antioxidant

transcriptional responses. However, chloride and bromide

(promoting HOCl and HOBr, respectively) oxidized methionine

and were associated with cytotoxicity. Methionine oxidation

products, methionine sulfoxide (MetO) and dehydromethionine

(dhMet), identified in our in vitro model were significantly

correlated with lung damage, neutrophils, and MPO activity in

airway fluid specimen from 5-year-olds with CF. This warrants

further exploration of MetO and dhMet as potential biomarkers of

early CF lung damage. Taken together, our results reveal

differences in effects of MPO-derived oxidants in complex milieu

which lead to distinct outcomes in epithelial cell viability and

function.

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About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, General Chemistry, Biochemistry
Keyword	Metabolomics Oxidants Lung disease Airway epithelium Mass spectrometry Transcriptomics Neutrophil
Committee Chair / Thesis Advisor	Joshua Chandler, Emory University
Committee Members	Nicholas Seyfried, Emory University Judith Fridovich-Keil, Emory University Rabindra Tirouvanziam, Emory University Christine Dunham, Emory University Michael Koval, Emory University

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