Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases

Abstract

-Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8 -10 wk) treated Ϯ ethanol in drinking water (20% wt/vol, 12 wk) Ϯ orally gavaged GSH in methylcellulose vehicle (300 mg·kg Ϫ1 ·day Ϫ1 , during week 12). MH-S cells, a mouse AM cell line, were treated Ϯ ethanol (0.08%, 3 days) Ϯ GSH (500 M, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice Ϯ inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) Ϯ orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2=,7=-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia. glutathione; alveolar macrophage; nicotinamide adenine dinucleotide phosphate oxidases; oxidative stress; phagocytic function CHRONIC ALCOHOL ABUSE INCREASES susceptibility to acute respiratory distress syndrome (ARDS) (54), a severe form of lung injury with a 26% mortality rate (23), and increases risk of developing respiratory infections, such as pneumonia (49). ARDS is characterized by the development of pulmonary inflammation and edema in response to aspiration, sepsis, or trauma, resulting in systemic proinflammatory cascade activation (68). Compared with a 22% incidence of ARDS in nonalcoholic patients at risk for lung injury, alcoholic patients have a 43% incidence of ARDS (52). Alcohol predisposes to ARDS development through multiple mechanisms, including oxidative stress (11,33,35,37,52). Furthermore, subjects with a history of alcohol use disorders have a higher prevalence of community-acquired pneumonia (20), resulting in worse clinical outcomes than nonalcoholics. Chronic alcohol ingestion increases oxidative stress, which primes the lung for respiratory infections and injury (34).Chronic alcohol consumption depleted levels of the critical antioxidant glutathione (GSH) in the bronchoalveolar lavage (BAL) fluid of human subjects (53) and in the alveolar macro...

Peroxisome Proliferator–Activated Receptor γ Regulates Chronic Alcohol-Induced Alveolar Macrophage Dysfunction

“…The data presented herein extend our previous findings that ethanol increases AM oxidative stress and phagocytic dysfunction (10,19) by demonstrating that PPARg plays an important role in both the pathogenesis and potential treatment of alcohol-induced derangements in both hAMs and mAMs. These findings indicate that targeting PPARg represents a novel therapeutic strategy for attenuating chronic alcohol-induced AM derangements.…”

“…Protein levels of PPARg, Noxs, and TGF-b 1 (fluorescent immunomicroscopy) and TLR4 (confocal immunomicroscopy) were assessed. Cellular ROS production, using 29,79-dichlorofluorescein-diacetate fluorescence (Invitrogen, Carlsbad, CA), and H 2 O 2 release, using Amplex Red analysis (Invitrogen), was determined (10,19). In vitro phagocytic capacity was measured using pHrodo Staphylococcus aureus BioParticles conjugate (Invitrogen) (34).…”

“…Lung Klebsiella pneumoniae Clearance In Vivo Male C57BL/6J mice with or without ethanol (20% wt/vol, 12 wk) were anesthetized, and 100 ml of 2 3 10 6 CFU/ml K. pneumoniae (online supplement) was delivered intratracheally (10). After 4 hours, selected mice were gavaged (25, 26) with 10 mg/kg RSG in 100 µl methylcellulose vehicle or vehicle alone and then killed after 24 hours.…”

“…AMs phagocytose and clear infectious particles from the lower airways (8), and chronic ethanol consumption impairs AM bacterial phagocytosis (9), increasing the risk for lung infection and injury (9,10). Although the mechanisms are incompletely defined, several studies have provided important mechanistic insights for the deleterious effects of alcohol on AM phagocytosis.…”

“…In addition, chronic alcohol ingestion stimulates AM oxidative stress, and attenuating this alcohol-induced oxidative stress reduces the deleterious effects of alcohol on the AMs (10). Reduced nicotinamide adenine dinucleotide oxidase (Nox) enzymes, including Nox1, Nox2, and Nox4, are critical sources of reactive oxygen species (ROS) in the AMs (16,17), and Nox2 is essential for respiratory burst involved in killing microbes after phagocytosis (16).…”