Karolyn G. Lahue

Epithelial NF-κB Orchestrates House Dust Mite–Induced Airway Inflammation, Hyperresponsiveness, and Fibrotic Remodeling

NF-κB activation within the epithelium has been implicated in the pathogenesis of asthma, yet the exact role of epithelial NF-κB in allergen-induced inflammation and airway remodeling remains unclear. In the current study, we used an intranasal house dust mite (HDM) extract exposure regimen time course in BALB/c mice to evaluate inflammation, NF-κB activation, airway hyperresponsiveness (AHR), and airway remodeling. We used CC10-IκBαSR transgenic mice to evaluate the functional importance of epithelial NF-κB in response to HDM. After a single exposure of HDM, mRNA expression of proinflammatory mediators was significantly elevated in lung tissue of wild-type (WT) mice, in association with increases in nuclear RelA and RelB, components of the classical and alternative NF-κB pathway, respectively, in the bronchiolar epithelium. In contrast, CC10-IκBαSR mice displayed marked decreases in nuclear RelA and RelB and mRNA expression of proinflammatory mediators compared with WT mice. After 15 challenges with HDM, WT mice exhibited increases in inflammation, AHR, mucus metaplasia, and peribronchiolar fibrosis. CC10-IκBαSR transgenic mice displayed marked decreases in neutrophilic infiltration, tissue damping, and elastance parameters, in association will less peribronchiolar fibrosis and decreases in nuclear RelB in lung tissue. However, central airway resistance and mucus metaplasia remained elevated in CC10-IκBαSR transgenic mice, in association with the continued presence of lymphocytes, and partial decreases in eosinophils and IL-13. The current study demonstrates that following airway exposure with an asthma-relevant allergen, activation of classical and alternative NF-κB pathways occurs within the airway epithelium and may coordinately contribute to allergic inflammation, AHR, and fibrotic airway remodeling.

Endoplasmic reticulum stress mediates house dust mite-induced airway epithelial apoptosis and fibrosis

BackgroundThe endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness.MethodsHouse Dust Mite (HDM) was used as an allergen for in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively.ResultsChallenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 α (ATF6α) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6α and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6α and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis.ConclusionCollectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6α and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function.

Oxidative Processing of Latent Fas in the Endoplasmic Reticulum Controls the Strength of Apoptosis

ABSTRACT We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241–252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.

Cooperation between classical and alternative NF-κB pathways regulates proinflammatory responses in epithelial cells.

The transcription factor NF-κB has been causally linked to inflammatory lung diseases. Recent studies have unraveled the complexity of NF-κB activation by identifying two parallel activation pathways: the classical NF-κB pathway, which is controlled by IκB kinase complex-β (IKKβ) and RelA/p50, and the alternative pathway, which is controlled by IKKα and RelB/p52. The alternative pathway regulates adaptive immune responses and lymphoid development, yet its role in the regulation of innate immune responses remains largely unknown. In this study, we determined the relevance of the alternative NF-κB pathway in proinflammatory responses in lung epithelial cells. The exposure of C10 murine alveolar lung epithelial cells to diverse stimuli, or primary murine tracheal epithelial cells to LPS, resulted in the activation of both NF-κB pathways, based on the nuclear translocation of RelA, p50, RelB, and p52. Increases in the nuclear content of RelA occurred rapidly, but transiently, whereas increases in nuclear RelB content were protracted. The small interfering (si) RNA-mediated knockdown of IKKα, RelA, or RelB resulted in decreases of multiple LPS-induced proinflammatory cytokines. Surprisingly, the siRNA ablation of IKKα or RelB led to marked increases in the production of IL-6 in response to LPS. The simultaneous expression of constitutively active (CA)-IKKα and CA-IKKβ caused synergistic increases in proinflammatory mediators. Lastly, the disruption of the IKK signalsome inhibited the activation of both NF-κB pathways. These results demonstrate that the coordinated activation of both NF-κB pathways regulates the magnitude and nature of proinflammatory responses in lung epithelial cells.

Glutaredoxin-1 Attenuates S-Glutathionylation of the Death Receptor Fas and Decreases Resolution of Pseudomonas aeruginosa Pneumonia

RATIONALE The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection. OBJECTIVES Fas ligand (FasL)-induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection. METHODS Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1(-/-)), Glrx1(-/-) mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa. MEASUREMENTS AND MAIN RESULTS Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1(-/-) cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1(-/-) mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals. CONCLUSIONS These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.

Emerging mechanisms of glutathione-dependent chemistry in biology and disease

Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S‐glutathionylation. The formation of an S‐glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S‐glutathionylation. In pathophysiological settings, S‐glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S‐glutathionylation in situ, which will enable further discovery into the role of S‐glutathionylation in biology and disease. J. Cell. Biochem. 114: 1962–1968, 2013.

Absence of c-Jun NH2-terminal kinase 1 protects against house dust mite-induced pulmonary remodeling but not airway hyperresponsiveness and inflammation

Chronic allergic asthma leads to airway remodeling and subepithelial fibrosis via mechanisms not fully understood. Airway remodeling is amplified by profibrotic mediators, such as transforming growth factor-β1 (TGF-β1), which plays a cardinal role in various models of fibrosis. We recently have identified a critical role for c-Jun-NH2-terminal-kinase (JNK) 1 in augmenting the profibrotic effects of TGF-β1, linked to epithelial-to-mesenchymal transition of airway epithelial cells. To examine the role of JNK1 in house dust mite (HDM)-induced airway remodeling, we induced allergic airway inflammation in wild-type (WT) and JNK1-/- mice by intranasal administration of HDM extract. WT and JNK1-/- mice were sensitized with intranasal aspirations of HDM extract for 15 days over 3 wk. HDM caused similar increases in airway hyperresponsiveness, mucus metaplasia, and airway inflammation in WT and JNK1-/- mice. In addition, the profibrotic cytokine TGF-β1 and phosphorylation of Smad3 were equally increased in WT and JNK1-/- mice. In contrast, increases in collagen content in lung tissue induced by HDM were significantly attenuated in JNK1-/- mice compared with WT controls. Furthermore HDM-induced increases of α-smooth muscle actin (α-SMA) protein and mRNA expression as well as the mesenchymal markers high-mobility group AT-hook 2 and collagen1A1 in WT mice were attenuated in JNK1-/- mice. The let-7 family of microRNAs has previously been linked to fibrosis. HDM exposure in WT mice and primary lung epithelial cells resulted in striking decreases in let-7g miRNA that were not observed in mice or primary lung epithelial cells lacking JNK1-/- mice. Overexpression of let-7g in lung epithelial cells reversed the HDM-induced increases in α-SMA. Collectively, these findings demonstrate an important requirement for JNK1 in promoting HDM-induced fibrotic airway remodeling.

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

Protein disulfide isomerase–endoplasmic reticulum resident protein 57 regulates allergen-induced airways inflammation, fibrosis, and hyperresponsiveness

BACKGROUND Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum-localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown. OBJECTIVES Here we sought to examine the contribution of airway epithelium-specific ERp57 in the pathogenesis of allergic asthma. METHODS We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57. RESULTS Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite-challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite-induced endoplasmic reticulum stress and airways fibrosis. CONCLUSIONS Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype.