Ine Kuipers

Ablation of Glutaredoxin-1 Attenuates Lipopolysaccharide-Induced Lung Inflammation and Alveolar Macrophage Activation

Protein S-glutathionylation (PSSG), a reversible posttranslational modification of reactive cysteines, recently emerged as a regulatory mechanism that affects diverse cell-signaling cascades. The extent of cellular PSSG is controlled by the oxidoreductase glutaredoxin-1 (Grx1), a cytosolic enzyme that specifically de-glutathionylates proteins. Here, we sought to evaluate the impact of the genetic ablation of Grx1 on PSSG and on LPS-induced lung inflammation. In response to LPS, Grx1 activity increased in lung tissue and bronchoalveolar lavage (BAL) fluid in WT (WT) mice compared with PBS control mice. Glrx1(-/-) mice consistently showed slight but statistically insignificant decreases in total numbers of inflammatory cells recovered by BAL. However, LPS-induced concentrations of IL-1β, TNF-α, IL-6, and Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) in BAL were significantly decreased in Glrx1(-/-) mice compared with WT mice. An in situ assessment of PSSG reactivity and a biochemical evaluation of PSSG content demonstrated increases in the lung tissue of Glrx1(-/-) animals in response to LPS, compared with WT mice or PBS control mice. We also demonstrated that PSSG reactivity was prominent in alveolar macrophages (AMs). Comparative BAL analyses from WT and Glrx1(-/-) mice revealed fewer and smaller AMs in Glrx1(-/-) mice, which showed a significantly decreased expression of NF-κB family members, impaired nuclear translocation of RelA, and lower levels of NF-κB-dependent cytokines after exposure to LPS, compared with WT cells. Taken together, these results indicate that Grx1 regulates the production of inflammatory mediators through control of S-glutathionylation-sensitive signaling pathways such as NF-κB, and that Grx1 expression is critical to the activation of AMs.

Cigarette Smoke Targets Glutaredoxin 1, Increasing S-glutathionylation and Epithelial Cell Death

It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death.

Increased glutaredoxin-1 and decreased protein S-glutathionylation in sputum of asthmatics

To the Editor: Total reduced glutathione (GSH), the main pulmonary antioxidant, is increased in asthma patients and, in some studies, increased amounts of oxidised glutathione (GSSG) were also found. In addition, enzymes that regulate the GSH redox cycle are altered in asthmatics (reviewed in [1]). GSH, with its redox cycle partners, serves to maintain the reduced state of protein thiols, which can be achieved by scavenging oxidants or by the covalent reversible binding of GSH to protein thiols. The latter occurs under physiological conditions, is induced upon mild oxidative stress and is known as S -glutathionylation (PSSG) [2]. PSSG protects targeted thiols from irreversible oxidations and can modulate protein function. Of significant relevance in asthma, SERCA (sarco/endoplasmic reticulum calcium ATPase) is activated by PSSG, increasing smooth muscle relaxation, and PSSG of the RyR (ryanodine receptor) calcium channel was associated with impaired coupling. With respect to inflammation, nuclear factor-κB and activator protein-1 are negatively affected by PSSG (reviewed in [2]). GSH can be removed from proteins by glutaredoxins (Grx), which restores the function of proteins targeted by PSSG [2]. The Grx1 isoform localises to the cytosol, and Grx2 to mitochondria and the nucleus. Grx1 can also reduce low molecular weight disulfides and, thus, proteins with functionally important disulfide bonds could also be affected by Grx1 alterations. In contrast to damaging oxidations and measurements of GSH, PSSG and Grx have rarely been studied in lung diseases, and never in conjunction. In chronic obstructive pulmonary disease (COPD), Grx1-positive alveolar macrophages were negatively correlated with forced expiratory volume in 1 s (FEV1) and sputum Grx1 levels were higher during exacerbations [3]. In a murine model of allergic airway disease, we found increased Grx1 expression and activity [4]. Given the importance of …

Altered Cigarette Smoke-Induced Lung Inflammation Due to Ablation of Grx1

Glutaredoxins (Grx) are redox enzymes that remove glutathione bound to protein thiols, know as S-glutathionylation (PSSG). PSSG is a reservoir of GSH and can affect the function of proteins. It inhibits the NF-κB pathway and LPS aspiration in Grx1 KO mice with decreased inflammatory cytokine levels. In this study we investigated whether absence of Grx1 similarly repressed cigarette smoke-induced inflammation in an exposure model in mice. Cigarette smoke exposure for four weeks decreased lung PSSG levels, but increased PSSG in lavaged cells and lavage fluid (BALF). Grx1 KO mice had increased levels of PSSG in lung tissue, BALF and BAL cells in response to smoke compared to wt mice. Importantly, levels of multiple inflammatory mediators in the BALF were decreased in Grx1 KO animals following cigarette smoke exposure compared to wt mice, as were levels of neutrophils, dendritic cells and lymphocytes. On the other hand, macrophage numbers were higher in Grx1 KO mice in response to smoke. Although cigarette smoke in vivo caused inverse effects in inflammatory and resident cells with respect to PSSG, primary macrophages and epithelial cells cultured from Grx1 KO mice both produced less KC compared to cells isolated from WT mice after smoke extract exposure. In this manuscript, we provide evidence that Grx1 has an important role in regulating cigarette smoke-induced lung inflammation which seems to diverge from its effects on total PSSG. Secondly, these data expose the differential effect of cigarette smoke on PSSG in inflammatory versus resident lung cells.

Smoke decreases reversible oxidations S-glutathionylation and S-nitrosylation in mice

Cigarette smoke causes irreversible oxidations in lungs, but its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Here the effect of cigarette smoke exposure in mice was investigated on the covalent binding of glutathione to protein thiols, known as S-glutathionylation (PSSG), which can be reversed by glutaredoxins (Grx). Also, protein S-nitrosylation (PSNO) which is the modification of protein thiols by NO and which is reversed by the enzyme alcohol dehydrogenase (ADH) 5 was examined. Both PSSG and PSNO levels in lung tissue were markedly decreased after 4 weeks of cigarette smoke exposure. This coincided with attenuated protein free thiol levels and increased protein carbonylation. The expression of NOX4, DHE sensitive oxidant production and iNOS levels were induced by smoke, whereas Grx1 mRNA expression and activity were attenuated. Free GSH levels, protein expression and activity of ADH5 were unaffected by smoke. Taken together, smoke exposure decreases reversible cysteine oxidations PSSG and PSNO and enhances protein carbonylation. These alterations are not associated with differences in some of the regulatory enzymes, but are likely the result of oxidative stress.