David H. McMillan

Platelets and Megakaryocytes Contain Functional Nuclear Factor-κB

Objective—To investigate the presence and role of NF-&kgr;B proteins in megakaryocytes and platelets. The nuclear factor-&kgr;B (NF-&kgr;B) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-&kgr;B family members, including the regulatory inhibitor-&kgr;B (I-&kgr;B) and I-&kgr; kinase (IKK) molecules. Methods and Results—Anucleate platelets exposed to NF-&kgr;B inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-&kgr;B inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-&kgr;B-&agr; phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-&bgr; or I-&kgr;B-&agr; protein to BAY inhibitor–treated platelets partially restored platelet spreading in I-&kgr;B-&agr; inhibited platelets, and addition of active IKK-&bgr; increased endogenous I-&kgr;B-&agr; phosphorylation levels. Conclusion—These novel findings support a crucial and nonclassical role for the NF-&kgr;B family in modulating platelet function and reveal that platelets are sensitive to NF-&kgr;B inhibitors. As NF-&kgr;B inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-&kgr;B is also identified as a new target to dampen unwanted platelet activation.

Lung-Targeted Overexpression of the NF-κB Member RelB Inhibits Cigarette Smoke–Induced Inflammation

Acute lung inflammation can be caused by a variety of respirable agents, including cigarette smoke. Long-term cigarette smoke exposure can cause chronic obstructive pulmonary disease (COPD), a serious illness that affects >10 million Americans. Cigarette smoke is a known inducer of inflammation and is responsible for approximately 90% of all COPD cases. RelB, a member of the NF-κB family, attenuates cigarette smoke-induced inflammatory mediator production in mouse lung fibroblasts in vitro. We hypothesized that overexpression of RelB in the airways of mice would dampen acute smoke-induced pulmonary inflammation. Mice received a recombinant adenovirus encoding RelB by intranasal aspiration to induce transient RelB overexpression in the lungs and were subsequently exposed to mainstream cigarette smoke. Markers of inflammation were analyzed after smoke exposure. Neutrophil infiltration, normally increased by smoke exposure, was significantly and potently decreased after RelB overexpression. Cigarette smoke-induced proinflammatory cytokine and chemokine production, cyclooxygenase-2 expression, and prostaglandin E(2) production were also significantly decreased in the context of RelB overexpression. The expression of intercellular adhesion molecule 1, an NF-κB-dependent protein, was decreased, indicating a potential mechanism through which RelB can regulate inflammatory cell migration. Therefore, increased expression and/or activation of RelB could be a novel therapeutic strategy against acute lung inflammation caused by respirable agents and possibly against chronic injury, such as COPD.

Ocular fibroblast diversity: implications for inflammation and ocular wound healing.

PURPOSE Various ocular and orbital tissues differ in their manifestations of inflammation, although the reasons for this are unclear. Such differences may be due to behaviors exhibited by resident cell types, including fibroblasts. Fibroblasts mediate immune function and produce inflammatory mediators. Chronic stimulation of ocular fibroblasts can lead to prolonged inflammation and, in turn, to impaired vision and blindness. Interleukin (IL)-1β, which is produced by various cells during inflammation, is a potent activator of fibroblasts and inducer of the expression of inflammatory mediators. The hypothesis for this study was that that human fibroblasts derived from distinct ocular tissues differ in their responses to IL-1β and that variations in the IL-1 signaling pathway account for these differences. METHODS Human fibroblasts were isolated from the lacrimal gland, cornea, and Tenons capsule and treated with IL-1β in vitro. Cytokine and prostaglandin (PG)E(2) production were measured by ELISA and EIA. Cyclooxygenase (Cox)-2 expression was detected by Western blot. Components of the IL-1 signaling pathway were detected by flow cytometry, ELISA, Western blot, and immunofluorescence. RESULTS Cytokine and PGE(2) production and Cox-2 expression were greatest in corneal fibroblasts. VEGF production was greatest in Tenons capsule fibroblasts. Variations in IL-1 receptor and receptor antagonist expression, IκBα degradation and p65 nuclear translocation, however, did not account for these differences, but overexpression of the NF-κB member RelB dampened Cox-2 expression in all three fibroblast types. CONCLUSIONS The results highlight the inherent differences between ocular fibroblast strains and provide crucial insight into novel, tissue-specific treatments for ocular inflammation and disease, such as RelB overexpression.

Thiol Redox Chemistry: Role of Protein Cysteine Oxidation and Altered Redox Homeostasis in Allergic Inflammation and Asthma

Asthma is a pulmonary disorder, with an estimated 300 million people affected worldwide. While it is thought that endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydrogen peroxide and nitric oxide, are important mediators of natural physiological processes, inflammatory cells recruited to the asthmatic airways have an exceptional capacity for producing a variety of highly reactive ROS and RNS believed to contribute to tissue damage and chronic airways inflammation. Antioxidant defense systems form a tightly regulated network that maintains the redox environment of the intra‐ as well as extracellular environment. Evidence for an oxidant‐antioxidant imbalance in asthmatic airways is demonstrated in a number of studies, revealing decreased total antioxidant capacity as well as lower levels of individual antioxidants. Thiols in the form of GSH and sulfhydryl groups of proteins are among the most susceptible oxidant‐sensitive targets, and hence, studies investigating protein thiol redox modifications in biology and disease have emerged. This perspective offers an overview of the combined efforts aimed at the elucidation of mechanisms whereby cysteine oxidations contribute to chronic inflammation and asthma, as well as insights into potential cysteine thiol‐based therapeutic strategies. J. Cell. Biochem. 116: 884–892, 2015.

Attenuation of inflammatory mediator production by the NF-κB member RelB is mediated by microRNA-146a in lung fibroblasts

Lung inflammation can result from exposure to multiple types of inflammatory stimuli. Fibroblasts, key structural cells in the lung that are integral to inflammation and wound healing, produce inflammatory mediators after exposure to stimuli such as IL-1β. We and others have shown that the NF-κB member RelB has anti-inflammatory properties in mice. Little is known, however, about the anti-inflammatory role of RelB in human cells and how it functions. MicroRNAs (miRNAs), a novel class of small, noncoding RNAs, can mediate inflammatory signaling pathways, including NF-κB, through regulation of target gene expression. Our goal was to analyze the anti-inflammatory properties of RelB in human lung fibroblasts. We hypothesized that RelB regulates inflammatory mediator production in lung fibroblasts in part through a mechanism involving miRNAs. To accomplish this, we transfected human lung fibroblasts with a plasmid encoding RelB and small interfering (si)RNA targeting RelB mRNA to overexpress and downregulate RelB, respectively. IL-1β, a powerful proinflammatory stimulus, was used to induce NF-κB-driven inflammatory responses. RelB overexpression reduced IL-1β-induced cyclooxygenase (Cox)-2, PGE₂, and cytokine production, and RelB downregulation increased Cox-2 expression and PGE₂ production. Furthermore, RelB overexpression increased IL-1β-induced expression of miRNA-146a, an NF-κB-dependent miRNA with anti-inflammatory properties, whereas RelB downregulation reduced miRNA-146a. miR-146a overexpression ablated the effects of RelB downregulation on IL-1β-induced Cox-2, PGE₂, and IL-6 production, suggesting that RelB mediates IL-1β-induced inflammatory mediator production in lung fibroblasts through miRNA-146a. RelB and miRNA-146a may therefore be new therapeutic targets in the treatment of lung inflammation caused by various agents and conditions.

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.

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp-/- mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Reducing protein oxidation reverses lung fibrosis

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death1–3. Oxidative stress is believed to be critical in this disease pathogenesis4–6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)7. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.Targeting a post-translational modification of Fas by recombinant Glrx reverses established lung fibrosis in a mouse model of age-related idiopathic pulmonary fibrosis.

IL-1/inhibitory κB kinase ε–induced glycolysis augment epithelial effector function and promote allergic airways disease

Background: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. Objectives: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. Methods: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. Results: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL‐1–dependent manner. Furthermore, administration of IL‐1&bgr; into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL‐1&bgr; or IL‐1&agr; resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL‐1&bgr;– or IL‐1&agr;–mediated proinflammatory responses and the stimulatory effects of IL‐1&bgr; on house dust mite (HDM)–induced release of thymic stromal lymphopoietin and GM‐CSF from tracheal epithelial cells. Inhibitor of &kgr;B kinase &egr; was downstream of HDM or IL‐1&bgr; and required for HDM‐induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM‐induced increases in lactate levels and attenuated HDM‐induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL‐1&bgr; levels, and lactate content correlated negatively with lung function. Conclusions: Collectively, these findings demonstrate that IL‐1&bgr;/inhibitory &kgr;B kinase &egr; signaling plays an important role in HDM‐induced glycolysis and pathogenesis of allergic airways disease. Graphical abstract: Figure. No caption available.