Serial Review: Redox Signaling in Immune Function and Cellular Responses in Lung Injury and Diseases Serial Review Editors: Victor Darley-Usmar, Lin Mantell
Genetic mechanisms of susceptibility to oxidative lung injury in miceā˜†

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Abstract

Genetic background is a known predisposing risk factor for many acute and chronic pulmonary disorders and responses to environmental oxidants. Variation in lung injury responses to oxidative stimuli such as ozone, particles, hyperoxia, and chemotherapeutic agents between genetically standardized inbred mouse strains has been demonstrated. In this review, we discuss quantitative trait loci (QTLs) which contain candidate genes that confer differential susceptibility to oxidative stimuli between strains in mouse models of airway toxicity and disease. We addressed multiple inflammatory, immunity, and antioxidant genes identified as candidate genetic determinants following these strategies, which include tumor necrosis factor (Tnf), toll-like receptor 4 (Tlr4), and the transcription factor NF-E2, related factor 2 (Nrf2). Mice with targeted deletion of these and related genes have provided initial proof of concept for their importance in the respective models. Interestingly, a few regions of the genome appear to have important roles in determining susceptibility to a number of stimuli which may suggest common genetic mechanisms in mice. Though more complete examination of functional association is required, results have potential implications for the role of these candidate genes in the pathogenesis of human pulmonary diseases including asthma, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), and emphysema.

Introduction

Molecular oxygen (O2) and its homeostasis are essential for the survival of all aerobic organisms. Incompletely reduced oxygen metabolites called reactive oxygen species (ROS) include hydrogen peroxide (H2O2), superoxide anion (O2āˆ’radical dot), and hydroxyl radical (OHradical dot), and they can be generated as metabolic by-products under normal physiological conditions. Endogenous cellular and extracellular antioxidant defense mechanisms exist to maintain equilibrium of the reductionā€“oxidation (redox) balance. However, excess ROS can overwhelm antioxidant capacity to perturb the redox balance, and cause tissue damage by oxidizing endogenous macromolecules (i.e., DNA, lipids, proteins) to generate peroxidation products (e.g., organic and inorganic ROS, DNA adducts, lipid peroxides), which eventually leads to oxidative stress. Reactive nitrogen species (RNS) are formed by reactions of superoxide with nitric oxide (NO) which is produced by the inducible form of nitric oxide synthase (iNOS, NOS2). Growing evidence has correlated increased oxidative stress with the pathogenesis of various human diseases including cancer, atherosclerosis, ischemiaā€“reperfusion injury, neurodegenerative disorders, and the aging process, though details of the molecular mechanisms and pathophysiology are not fully understood.

Redox balance is particularly important in the airways because they are the first points of contact with airborne oxidants such as environmental ozone, particles, and cigarette smoke, and thus are frequently exposed to higher oxidant burden than other tissues. Inhaled exogenous oxidants interact primarily with the epithelial lining fluid (ELF), which contains polyunsaturated fatty acids, numerous antioxidants, and protective agents including surfactants and mucus. Enhanced oxidative burden has been implicated in the pathogenesis of many acute and chronic pulmonary diseases including asthma, emphysema/chronic obstructive pulmonary disorders (COPD), cystic fibrosis, bronchopulmonary dysplasia (BPD), acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), and lung malignancies [1], [2], [3]. Depletion or alteration of antioxidants including glutathione (GSH) has been observed often in specimens from individuals affected by such diseases [4]. A variety of antioxidant therapies have been attempted in these patients, including supplementation with vitamins C and E or N-acetylcysteine (a GSH precursor), or intravenous treatment with polyethylene glycol-conjugated superoxide dismutase (SOD) and catalase [1], [5]. To date, however, the benefits of these therapies have been equivocal at best.

It is currently believed that multiple genetic and nongenetic (e.g., socioeconomic status, environmental exposures, gender, age, and diet) factors are involved in the etiology of most human diseases. Accumulating epidemiologic evidence has increasingly supported a role of genetic variation as an important intrinsic disease determinant. During the past two decades, extraordinary research endeavors have been initiated to identify genes associated with the pathogenesis of a number of human disorders. However, because most diseases have complex polygenic inheritance, and disease etiology involves interactions between genes (geneā€“gene interaction) identification of susceptibility genes has not progressed as rapidly as initially expected. Furthermore, it is also clear that complex diseases have important environmental components (geneā€“environment interaction), thus complicating our understanding of disease pathophysiology.

However, continued progress in molecular and computational technology has led to the development of novel genetic approaches to discover disease genes for multifactorial polygenic diseases. Traditionally, two research approaches have been widely utilized to search for gene(s) that determine disease susceptibility [6]. The first, positional cloning (genetic linkage analysis, or meiotic mapping), is based on association of disease phenotype(s) with polymorphic markers [e.g., dinucleotide microsatellites, single nucleotide polymorphisms (SNPs)] located throughout the genome to identify quantitative trait loci (QTL) that contain gene(s) that are polymorphic and contribute to the differential phenotype (susceptibility). The genome-wide screen is especially useful in genetically well-controlled models, particularly inbred strains of mice, but dozens of disease QTLs have also been found in humans and other species using this approach. However, a QTL may span up to 10ā€“20 centimorgans (cM) and include hundreds of genes. Therefore, it is important to reduce the size of the QTL for identification of candidate genes, and inbred mice have proved to be an invaluable tool in this process. A number of approaches, including development of congenic mouse lines, comparative and haplotype mapping, and gene expression analysis, have been developed in the mouse to define candidate genes determined from genome scans (Fig. 1). The current review covers several inbred mouse models analyzed by this strategy, and readers can refer to specific articles (e.g., Ref. [7]) for additional information on these research approaches.

The second research strategy is candidate gene association which evaluates whether specific gene(s) polymorphisms of interest associate with a specific disease phenotype. Candidate genes can be selected for association based on biological evidence or plausibility, or may be identified from protein or mRNA expression analysis. Numerous recent investigations have utilized the candidate gene approach in humans and inbred mice. However, as genes are chosen a priori, this strategy may exclude other genes (loci) that are critical in the disease. Therefore, a combination of both methods has been a strong tool for defining genetic mechanisms of disease. In both strategies, the predictor (candidate susceptibility) gene is further validated through standard means including genetic sequence analysis for single nucleotide SNPs and functional analyses using genetically engineered mice (e.g., knockout or transgenic mice) or selective gene silencing techniques (e.g., siRNA).

Although family-based genome scan studies have been attempted in human subjects [8], [9], current technology and statistical methods have generally been insufficient to permit fine mapping of the location of multiple genes that influence a complex disorder in genetically heterogenous species like humans, particularly when environmental exposure may vary from one population to the next. Due to this limitation, successful positional cloning studies in mouse models of pulmonary disease have proved useful for identifying candidate genes. Because of the extensive linkage homology that exists between mouse and man, it is likely that genes identified using mouse models will have human homologues that have predictive value in understanding human disease.

Recent in silico haplotype mapping approaches utilize computational algorithms to identify the chromosomal loci responsible for strain-specific differences in multiple phenotypes. Haplotype mapping takes advantage of genetic diversity in thousands of common genetic markers (i.e., SNPs) across commonly used multiple inbred mouse strains [10], [11]. The approaches complement the traditional genotyping strategies which usually use two strains, and thus lack the genetic diversity across the genome. This new technology could prove to greatly enhance the ability to identify disease genes. In silico techniques also allow comparative analysis of the mouse and human genomes for orthologous disease genes to give a powerful tool for evolutional and functional studies between species [12], [13].

Genetic background predisposes to many pulmonary diseases, including COPD, ALI/ARDS, fibrosis, sarcoidosis, nonspecific interstitial pneumonitis, and cancer [14], [15], [16], [17]. Considering the importance of this risk factor for disease pathogenesis, association studies for candidate gene polymorphisms have expanded rapidly, and a large body of recent research has reported positive associations between candidate gene polymorphic variants with incidence and outcome of asthma, ARDS, COPD (emphysema), or lung cancer [18], [19], [20], [21], [22], [23].

In this review, we address genetic research that has identified pulmonary susceptibility genes conferring differential response to various oxidants including ozone, particles, hyperoxia, and fibrogens (radiation, bleomycin) in murine models. We also discuss functional analyses applied to candidate genes to verify their roles as genetic determinants. We primarily describe oxidative injury models in mice; a summary for a broader spectrum of mouse genetic research including neoplastic lung diseases may be found in a review by Bauer et al. [24].

Section snippets

Association of environmental ozone with pulmonary diseases

Ozone is a highly reactive oxidant in air pollution. Inhaled ozone reacts with unsaturated fatty acids on airā€“tissue interface (i.e., ELF and pulmonary cell bilayers) to form lipid ozonation products including Criegee ozonides, aldehydes, and hydroxyhydroperoxides [25]. These oxidized metabolites can trigger signal transduction pathways leading to airway toxicity accompanied by excess generation of ROS. Exposure to ozone causes cytokine and chemokine production and inflammation, epithelial

Summary

Genome-wide scans in mice have identified multiple chromosomal QTLs that confer differential pulmonary susceptibility to oxidative stimuli. Haplotype mapping, gene expression, and comparative sequence analysis of the QTLs have elucidated candidate susceptibility genes in some of these models. Chromosomal locations of significant and suggestive QTLs and their candidate genes determined by genetic linkage analyses are summarized in Table 1. Functional studies performed using gene knockout,

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    This article is part of a series of reviews on ā€œRedox signaling in Immune Function and Cellular Responses in Lung Injury and Diseasesā€. The full list of papers may be found on the home page of the journal.

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