The transcription factor NF-κB, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory κB kinase (IKK) β-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-β Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity. Conversely, GRX1 knockdown sensitizes cells to oxidative inactivation of IKK-β and dampens TNF-α-induced IKK and NF-κB activation. Primary tracheal epithelial cells from Glrx1 -deficient mice display reduced NF-κB DNA binding, RelA nuclear translocation, and MIP-2 (macrophage inflammatory protein 2) and keratinocyte-derived chemokine production in response to LPS. Collectively, these findings demonstrate the physiological relevance of the S-glutathionylation–GRX redox module in controlling the magnitude of activation of the NF-κB pathway.

Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-κB. A commonly proposed mode of action for inhibition of NF-κBbyNO involves interference with NF-κB binding to DNA. Because activation of inhibitory κB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-κB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-κB activation. We demonstrate here that S -nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKβ, the catalytic subunit required for NF-κB activation, was a direct target for S-nitrosylation. A mutant version of IKKβ containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKβ, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor α to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-κB activation by cytokines. Collectively, our findings demonstrate that IKKβ is an important target for the redox regulation of NF-κB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.

NF-kappaB is an inducible transcription factor that plays a role in the expression of over one hundred genes involved in immunity, inflammation, proliferation, and in defense against apoptosis. NF-kappaB has been known to be redox regulated for some time and is a direct target for oxidation that can affect its ability to bind to DNA. Reactive oxygen species (ROS) have been identified as second messengers in cells, and play a role in receptor signaling and posttranslation modification of signaling molecules. These posttranslation modifications include oxidations of critical cysteines to sulfenic acids or mixed disulfides, which can affect the activity of proteins. Many kinases involved in direct or indirect activation of NF-kappaB are affected by oxidants and therefore, have the potential to alter NF-kappaB activity. This review will provide a summary of the NF-kappaB family, their activation and regulation, followed by a summary of cytoplasmic and nuclear kinases in this pathway whose activity is affected by oxidants. Additionally, recent investigations have revealed that the JNK signaling pathway, which is known to be redox regulated, and pro-apoptotic, is inhibited by NF-kappaB signaling. The crosstalk of NF-kappaB with other signaling pathways is therefore critical for cellular fate, notably survival or cell death under oxidative conditions, and will also be reviewed.

Rationale: Emphysema in chronic obstructive pulmonary disease (COPD) can be characterized by high-resolution chest computed tomography (HRCT); however, the repeated use of HRCT is limited because of concerns regarding radiation exposure and cost. Objectives: To evaluate biomarkers associated with emphysema and COPD-related clinical characteristics, and to assess the relationships of soluble receptor for advanced glycation endproducts (sRAGE), a candidate systemic biomarker identified in this study, with single-nucleotide polymorphisms (SNPs) in the gene coding for RAGE (AGER locus) and with clinical characteristics. Methods: Circulating levels of 111 biomarkers were analyzed for association with clinical characteristics in 410 patients with COPD enrolled in the TESRA study. sRAGE was also measured in the ECLIPSE cohort in 1,847 patients with COPD, 298 smokers and 204 nonsmokers. The association between 21 SNPs in the AGER locus with sRAGE levels and clinical characteristics was also investigated. Measurements and Main Results: sRAGE was identified as a biomarker of diffusing capacity of carbon monoxide and lung density in the TESRA cohort. In the ECLIPSE cohort, lower sRAGE levels were associated with increased emphysema, increased Global Initiative for Chronic Obstructive Lung Disease stage, and COPD disease status. The associations with emphysema in both cohorts remained significant after covariate adjustment (P < 0.0001). One SNP in the AGER locus, rs2070600, was associated with circulating sRAGE levels both in TESRA (P = 0.0014) and ECLIPSE (7.07 × 10⁻¹⁶), which exceeded genome-wide significance threshold. Another SNP (rs2071288) was also associated with sRAGE levels (P = 0.01) and diffusing capacity of carbon monoxide (P = 0.01) in the TESRA study. Conclusions: Lower circulating sRAGE levels are associated with emphysema severity and genetic polymorphisms in the AGER locus are associated with systemic sRAGE levels. Clinical trial registered with www.clinicaltrials.gov (NCT 00413205 and NCT 00292552).

Background: In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation. Methods: A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed. Results: We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation. Conclusion: Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.

Background. Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the innate immune system. Consid