Aldose reductase inhibition suppresses airway inflammation

https://doi.org/10.1016/j.cbi.2011.02.014Get rights and content

Abstract

Airway inflammation induced by reactive oxygen species (ROS)-mediated activation of redox-sensitive transcription factors is the hallmark of asthma, a prevalent chronic respiratory disease. In various cellular and animal models, we have recently demonstrated that, in response to multiple stimuli, aldose reductase (AKR1B1) regulates the inflammatory signals via NF-kappa B activation. Since NF-κB activation is implicated in asthma pathogenesis, we investigated whether AKR1B1 inhibition could prevent ovalbumin (Ova)- and ragweed pollen extract (RWE)-induced airway inflammation and hyper-responsiveness in mice models and tumor necrosis factor-alpha (TNF-α)-, lipopolysachharide (LPS)- and RWE-induced cytotoxic and inflammatory signals in primary human small airway epithelial cells (SAEC). Sensitization and challenge with Ova or RWE caused airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid, airway hyperresponsiveness, elevated IgE levels and release of Th2 cytokines in the airway and treatment with AKR1B1 inhibitors markedly reduced these pathological changes in mice. In SAEC, treatment with TNF-α, LPS or RWE induced apoptosis, reactive oxygen species generation, synthesis of inflammatory markers IL-6, IL-8, and PGE2 and activation of NF-κB and AP-1. Pharmacological inhibition prevented these changes suggesting that AKR1B1 mediates ROS induced inflammation in small airway epithelial cells. Our results indicate that AKR1B1 inhibitors may offer a novel therapeutic approach to treat inflammatory airway diseases such as asthma.

Introduction

Airway inflammation is one of the main characteristic features of asthma, which affects approximately 300 million people worldwide [1]. In the US, approximately 8% of the population suffers from asthma and according to an estimate the annual expenditure on asthma treatment was approximately 37 billion dollars in 2007 [2], [3]. In spite of progress made in the therapy and management of the disease, there has been increase in the prevalence of asthma in last few decades which suggests the lack of clear understanding of the pathophysiology of asthma [4]. The ever increasing evidences suggest that decrease in the antioxidant capacity of the airway and resultant oxidative stress due to either external stimuli such as environmental pollutants, allergens, gases, particulate matters, pets, exercise, cold or intrinsic factors such as inappropriate immune response, genetic predisposition or compromised host-pathogen interaction could be significant player in asthma pathogenesis [5], [6], [7], [8], [9], [10]. The decrease in antioxidant capacity of the lung results from an enhanced production of reactive oxygen species (ROS) upon exposure to the extrinsic or intrinsic stimulants results in the generation of inflammatory mediators that drive the pathophysiology of asthma [11], [12]. The altered redox status of the airway cells due to overwhelming level of ROS activates the cascade of molecular signals involving an array of intermediate protein kinases eventually leading to activation of transcription factors that transcribe a number of inflammatory genes including cytokines, chemokines and other mediators such as prostaglandins, leukotrienes, nitric oxide, adhesion molecules, and proteases [13]. The inflammatory mediators, when released in the local microenvironment, attract an entire array of immune cells such as eosinophils, macrophages, neutrophils and other leukocytes, which further secrete inflammatory cytokines and chemokines starting a cycle of events resulting in tissue damage, cytotoxicity, and remodeling which contribute to the progression of asthma [14].

There has been increased correlation between the markers of oxidative stress including malondialdehyde, thiobarbituric acid reactive products, and oxidized glutathione (GSSG) in the urine, plasma, sputum, and BAL fluid of patients with severity of asthma [15]. The increased ROS levels lead to lipid peroxidation resulting in the formation of lipid-derived aldehydes such 4-hydroxynonenal (HNE), which could readily combine with the cellular reduced glutathione (GSH) to form GS-lipid aldehydes conjugates thereby trapping cellular glutathione and increasing the oxidative stress even further [16]. In addition, we have shown that both lipid derived aldehydes such as HNE as well as their glutathione conjugates (GS-HNE) are the excellent substrates (Km = 10–30 μM) for aldose reductase, an enzyme linked to diabetic complications for its role in the reduction of glucose to sorbitol via polyol pathway [17], [18]. We have demonstrated that the reduced product of GS-lipid aldehydes i.e. GS-lipid alcohol (such as GS-DHN) is an important inducer of signaling cascade that activates the signaling kinases including PKC, PLC, MAPKs in different cellular models eventually activating transcription factors leading to transcription of various genes involved in inflammation and pathologies [19], [20] and inhibition of AKR1B1 prevents ROS-induced inflammatory changes in cellular and animal models probably by blocking the formation of GS-lipid alcohol species and breaks the cycle of events that results in the pathological development [18], [19], [20]. Based on these evidences, we hypothesized that since ROS and ROS-mediated lipid peroxidation products are involved in asthma pathogenesis, blocking the ROS-mediated activation of inflammatory signals by AKR1B1 inhibition could prevent airway inflammation.

Here we have investigated whether stimulants such as RWE, bacterial endotoxin, LPS or TNF-α-induced inflammatory changes in human primary small airway epithelial cells could be prevented by pharmacological inhibition or siRNA ablation of AKR1B1. Further, we have also used animal models of Ova- or RWE-induced asthma in mice to further confirm our hypothesis. Our results demonstrate that inhibition of AKR1B1 in airway cells prevented various oxidants – induced cytotoxicity, ROS levels, inflammatory mediators, activation of signaling intermediates and airway resistance, cytokines and chemokines synthesis in mouse model of Ova- or RWE-induced airway inflammation. These results suggest important role of AKR1B1 in asthma pathogenesis and that the use of AKR1B1 inhibitors could be a potential therapeutic approach for airway inflammation in asthma.

Section snippets

Reagents

The media and reagent pack for cell culture such as small airway epithelial basal medium (SABM), and small airway epithelial growth media (SAGM™) bulletkit and Reagent pack containing Trypsin and EDTA in the ratio of 0.025%:0.01%, Trypsin neutralizing solution and HEPES buffered saline solution were purchased from Lonza Walkersville Inc. (Walkersville, MD). Two structurally different AKR1B1 inhibitors, Sorbinil and Zopolrestat, were obtained as gifts from Pfizer (New York, NY). Dimethyl

TNF-α-, LPS- and RWE-induced ROS levels prevented by AKR1B1 inhibition in SAEC

First we examined if SAEC showed increased generation of ROS in response to the treatment with TNF-α, LPS or RWE. As shown in Fig. 1, approximately 2-fold increase in the level of ROS was observed after stimulation with TNF-α, LPS or RWE compared to control cells not stimulated with any of these agents and treatment with AKR1B1 inhibitor prevented the increase in ROS levels in SAEC.

TNF-α-, LPS- and RWE-induced cellular apoptosis prevented by AKR1B1 inhibition

Since ROS-induction is well known to induce cytotoxic effects resulting in the altered cell fate, we determined

Discussion

Epidemiological studies suggest multiple interacting risk factors for asthma such as inhaled pollutants, environmental tobacco smoke, particulate matter, oxides of nitrogen, ozone, and repeated respiratory virus exposures [26], [27], [28]. These factors, directly or indirectly, induce and/or augment ROS generation in the airways [27]. Excessive immune response to a particular antigen resulting in the chemo-attraction of inflammatory cells in the lung also results in the increased ROS generation

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

This study was supported in parts by American Asthma Foundation Grant AAF 08-0219 and NIH grant DK 36118 to William Bowes Scholar SKS and NIH Grant GM-71036 to KVR.

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