Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Abstract

Epithelia express oxidative anti-microbial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H 2 O 2 ) and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H 2 O 2 for use by the LPO. To investigate regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells, challenged with Pseudomonas aeruginosa flagellin or IFNγ. Flagellin upregulated Duox2 mRNA 20-fold, but only upregulated LPO mRNA 2.5-fold. IFNγ increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFNγ. Both stimuli increased H 2 O 2 synthesis and LPO-dependent killing of Pseudomonas aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H 2 O 2 production, while Duox2 siRNA markedly reduced basal H 2 O 2 production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2 mediated H 2 O 2 production appear to be coordinated with increases in LPO mRNA and, without increased LPO, H 2 O 2 levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H 2 O 2 synthesis despite the presence of greater amounts of Duox1.

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

“…In our setting, the H 2 O 2 /LPO system is unlikely to have a role based on the following observations. First, LPO is not secreted from in vitro cultured AECs [44] and all the studies that previously demonstrated an LPO-dependent antimicrobial function required the addition of LPO in the experiments [38,39,43]. However, here we observe an antiviral effect of DUOX2 without the ectopic addition of LPO.…”

“…To date, studies based on the use of antioxidant enzymes support the necessity of H 2 O 2 in the LPOdependent antibacterial defense [38]. Further studies suggest that DUOX2 is a source of H 2 O 2 production in the airways [39,40]. However, none of these studies have clearly demonstrated a functional connection between DUOX2-mediated H 2 O 2 production and LPO-dependent airway antibacterial defenses.…”

H2O2 Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

“…We previously showed that IFN-g-stimulated NHBE cells cultured at the ALI produced high levels of H 2 O 2 , at up to 5-10 mM in apical washes used for assay (10). Thus, much higher concentrations exist in the ASL before washes were used to collect samples (dilution (23), corresponding to a surface volume of approximately 6 ml, because of the meniscus at the edge of the culture surface (24).…”

“…Duox1 and Duox2 are regulated by cytokines (10-12) and bacterial products (10,13,14) to increase H 2 O 2 synthesis to high levels (10). Stimulation with interferon-g increased H 2 O 2 synthesis levels 30-fold when measured in air-liquid interface (ALI) cultures of normal human airway epithelial cells (10), leading to 10 mM H 2 O 2 in washes used for assays.…”

Dual Oxidase–1 Is Required for Airway Epithelial Cell Migration and Bronchiolar Reepithelialization after Injury

“…Epithelial H 2 O 2 production, presumably by DUOX2, is thought to aid in direct oxidative microbial killing in association with secreted lactoperoxidase (3,4), and the prominent induction of DUOX2 in response to microbial and viral stimuli further supports its role in host defense (5,6). Studies of Drosophila and zebrafish have established the importance of their single DUOX gene in the maintenance of gut immunity (7)(8)(9), and analogous host-defense properties have been demonstrated for DUOX2 in the mammalian gastrointestinal tract (10,11).…”