Redox regulation of lung inflammation: role of NADPH oxidase and NF-κB signalling

H. Yao, S.-R. Yang, A. Kode, S. Rajendrasozhan, S. Caito, D. Adenuga, R. Henry, I. Edirisinghe, I. Rahman


Regulation of reduction/oxidation (redox) state is critical for cell viability, activation, proliferation and organ function, and imbalance of oxidant/antioxidant balance is implicated in various chronic respiratory inflammatory diseases, such as asthma, pulmonary fibrosis and chronic obstructive pulmonary disease. CS (cigarette smoke) is a complex mixture of various noxious gases and condensed tar particles. These components elicit oxidative stress in lungs by continuous generation of ROS (reactive oxygen species) and various inflammatory mediators. In the present review, we have discussed the role of oxidative stress in triggering the inflammatory response in the lungs in response to CS by demonstrating the role of NADPH oxidase, redox-sensitive transcription factors, such as pro-inflammatory NF-κB (nuclear factor κB) and antioxidant Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), as well as HDAC (histone deacetylase) in pro-inflammatory cytokine release by disruption of HDAC–RelA/p65 NF-κB complex.

  • chronic obstructive pulmonary disease (COPD)
  • cigarette smoke
  • histone deacetylase (HDAC)
  • Nox
  • nuclear factor-erythroid 2 p45 subunit-related factor 2 (Nrf2)
  • oxidant


Redox signalling involves at least one reaction of reversible oxidation of a signalling molecule that is intended to modulate a signalling pathway. On the other hand, oxidative stress is often characterized as a decrease in the antioxidant/oxidant ratio, tilted towards a higher proportion of oxidants. The ultimate outcome of oxidative stress is cell-type-specific, which can not only elicit responses ranging from severe oxidative damage, loss of cell function and viability, to apoptosis and ultimately necrosis, but may also induce responses such as cell differentiation to cell cycle progression.

Owing to its large epithelial surface area and blood supply, the lungs are susceptible to oxidative injury by virtue of myriads of reactive forms of oxygen species and free radicals which can be generated by metabolic reaction (e.g. from mitochondrial electron transport during respiration or during activation of phagocytes) or exogenously, such as air pollutants or CS (cigarette smoke) [1,2]. Production of ROS (reactive oxygen species) has been directly linked to oxidation of proteins, DNA and lipids, which may cause direct lung injury or induce a variety of cellular responses through the generation of secondary metabolic reactive species. Furthermore, increased levels of ROS have been implicated in initiating inflammatory responses in the lungs through the activation of transcription factors such as NF-κB (nuclear factor κB) and AP-1 (activator protein-1), signal transduction, chromatin remodelling (histone acetylation and deacetylation) and gene expression of pro-inflammatory mediators [37]. Nevertheless, the lungs are endowed with an armamentarium of endogenous antioxidants to ward off such oxidant challenges [2]. The major non-enzymatic antioxidants of the lungs are glutathione, vitamins C and E, β-carotene and uric acid, and the enzymatic antioxidants are SODs (superoxide dismutases), catalase and peroxidases. The proteins such as peroxiredoxins, thioredoxins, glutaredoxins, haem oxygenases and reductases are also involved in cellular adaptation and protection against an oxidative assault. However, aberration of oxidant and antioxidant balance can lead to initiation and progression of a variety of respiratory inflammatory disease, such as asthma, pulmonary fibrosis and COPD (chronic obstructive pulmonary disease). In the present review, we have discussed the role of CS-mediated oxidative stress in triggering the inflammatory response in the lungs by assessing the role of NADPH oxidase, redox-sensitive transcription factors NF-κB and Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) and HDACs (histone deacetylases) in pro-inflammatory cytokine release.

Role of NADPH oxidase in lung inflammation

NADPH oxidase is composed of five subunits, p40phox (phox for phagocyte NADPH oxidase), p47phox, p67phox, p22phox and gp91phox. The latter two are membrane-associated and together constitute the flavocytochrome b558, whereas the other components are located in the cytoplasm of resting cells. Upon activation, the cytoplasmic components translocate to the cell membrane where they bind to flavocytochrome b558, thus forming the active NADPH oxidase [8]. After formation of active NADPH oxidase, it catalyses the transfer of electrons from NADPH to molecular oxygen with subsequent production of O2 [9]. The classical NADPH oxidase was first described and characterized in phagocytes, such as neutrophils, and it was originally thought that the enzyme was restricted to leucocytes and used solely for host defences. However, subsequent studies indicate that similar NADPH oxidase is present in a wide variety of non-phagocytic cells and tissues (reviewed in [10,11]). These enzymes are distinct from the phagocyte oxidase and appear to play essential roles in functions other than host defence; however, structural features of many non-phagocyte oxidase proteins seem similar or even identical with those of their phagocyte counterparts. NADPH oxidase-derived ROS has been implicated in the activation of NF-κB and release of pro-inflammatory mediators (Figure 1).

Figure 1 CS-derived ROS activate kinase signalling, leading to histone acetylation

CS-mediated ROS generation via NADPH oxidase plays a pivotal role in activation of various kinases {MEKK [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase], PKC and NIK} and transcriptional factors (NF-κB and Nrf2). IKKβ can be activated by MEKK and/or PKC, leading to phosphorylation of RelA/p65, which would then interact with CBP, inducing acetylation and phosphorylation of histone. Acetylation and phosphorylation of histone result in uncoiling of DNA–histone complex, allowing increased accessibility for large protein complexes, such as RelA/p65, CBP and RNA polymerase II, hence increasing the pro-inflammatory genes expression. Activation of NIK by ROS will lead to IKKα-mediated phosphorylation of CBP, which would interact with RelA/p65. CS-mediated ROS generation disrupts the association of Nrf2 with Keap1, leading to diminished rates of proteolysis of Nrf2 and enhanced nuclear accumulation. Phosphorylation of Nrf2 by a series of kinases also affects its fate and distribution. Upon translocation into the nucleus, Nrf2 binds to AREs in the upstream sequence of target genes in association with other proteins such as small Maf proteins, c-Jun and co-activators, thereby mediating the transcriptional regulation of Phase II antioxidant genes.

NF-κB is an important intracellular signalling pathway for both innate and acquired immunity and involved in pro-inflammatory gene transcription. Previous studies showed that LPS (lipopolysaccharide) and Pseudomonas aeruginosa-induced lung NF-κB activation was impaired in p47phox−/− and gp91phox−/− mice [1214]. We have recently demonstrated that the activation of RelA/p65 subunit of NF-κB was decreased in lungs of p47phox−/− and gp91phox−/− mice compared with wild-type mice in response to CS exposure. Thus NADPH oxidase-derived ROS may have a role in the activation of NF-κB and MAPK (mitogen-activated protein kinase) pathways by CS. We have also demonstrated that CS exposure caused significant influx of neutrophils in BALF (bronchoalveolar lavage fluid), and macrophages in lung tissue of wild-type mice, which were augmented in p47phox−/− and gp91phox−/− mice, suggesting the role of NADPH oxidase subunits in chemotaxis (H. Yao, I. Edirisinghe, S.-R. Yang, S. Rajendrasozhan, A. Kode, S. Caito, D. Adenuga and I. Rahman, unpublished work). This connection of NADPH oxidase with chemotaxis may be linked with the ROS-dependent regulation of NF-κB and/or MAPK pathways, which in turn triggers the expression of genes transcribing various cytokines and chemotactic factors such as IL (interleukin)-6, IL-8 and MCP-1 (monocyte chemotactic protein-1). This is supported by the observations of lung levels of pro-inflammatory mediators, such as KC (keratinocyte chemoattractant), IL-6, MCP-1 and TNFα (tumour necrosis factor α), being significantly increased in both the knockout mice compared with wild-type mice in response to CS exposure. Although the basis of this finding is not clear, a possible explanation is the accumulation of ingested particles which cannot be destroyed in the phagosome, and decreased apoptosis in inflammatory cells, both of which are due to the defect in NADPH oxidase function [15,16]. This would lead to increased release of pro-inflammatory cytokines. The other possibility is the compensatory up-regulation of another isoform of NADPH oxidase, i.e. Nox4 and/or DUOXs, which would lead to increased ROS-mediated signalling leading to release of pro-inflammatory mediators. This observation is consistent with the findings in which LPS was used to induce lung inflammation [12,13]. However, it remains to be ascertained whether CS-mediated ROS generation, and therefore inflammation, is via activation of phagocytic or non-phagocytic cell NADPH oxidase or both. Investigation into this mechanism of CS action would underpin the actual cells affected/recruited by CS for its deleterious effects.

MMPs (matrix metalloproteinases) such as gelatinase B (MMP-9), gelatinase A (MMP-2) and macrophage metalloelastase (MMP-12) play a predominant role in the lung inflammation. Consistent with the previous finding [17], MMP-9, MMP-2 and MMP-12 activities and expressions were increased in lung tissue in response to CS exposure in wild-type mice. Interestingly, genetic ablation of p47phox and gp91phox enhanced the MMP-9 and MMP-2 activities in response to CS exposure, raising the possibility that ROS derived from NADPH oxidase inhibited MMPs (H. Yao, I. Edirisinghe, S.-R. Yang, S. Rajendrasozhan, A. Kode, S. Caito, D. Adenuga and I. Rahman, unpublished work). Indeed, ROS can potently and efficiently inactivate MMP-7 by cross-linking adjacent tryptophan/glycine residues within the catalytic domain of the enzyme [18,19]. Moreover, reactive intermediates by phagocyte NADPH oxidase could inactivate MMP-12 in vitro and in vivo [20]. Thus it appears that on one hand CS provokes generation of ROS via NADPH oxidase and inflammatory reactions by the cells and on the other generation of ROS inhibits certain MMPs, hence regulating extracellular matrix remodelling. It therefore becomes more pertinent to ask the question whether ROS generated by NADPH oxidase from phagocytic and non-phagocytic cells have different roles in the ultimate outcome of CS effects. It would be an interesting proposition to study whether or not activation of NADPH oxidase from the two different cell types has differential effects. In addition to their ability to destroy extracellular matrix, MMPs can affect inflammation by directly or indirectly regulating the activity of inflammatory mediators such as chemokines [2123]. Therefore the increased levels of MMPs may explain the heightened inflammatory response in lungs of mice where NADPH oxidase was ablated than those in wild-type mice in response to CS exposure. Overall, it suggests that NADPH oxidase-derived ROS regulate lung inflammation but genetic ablation of NADPH oxidase leads to augmentation of CS-induced lung inflammation.

Role of Nrf2 in redox signalling

Nrf2 is a redox-sensitive basic leucine zipper transcription factor, which is essential for the ARE (antioxidant responsive element)-mediated induction of Phase II detoxifying genes. It plays a pivotal role in cellular defence against electrophiles and ROS. Keap1 (Kelch-like enoyl-CoA hydratase-associated protein 1) negatively regulates Nrf2 activity by targeting it to proteasomal degradation. In response to oxidative stress, Nrf2 dissociates from Keap1 and then binds to AREs in the upstream sequence of target genes in association with several other proteins such as small Maf proteins, c-Jun and CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 and p160 family co-activators, thereby mediating the transcriptional regulation of Phase II antioxidant genes.

Studies have shown that acute CS exposure increased but chronic exposure decreased the levels of Phase II drug-metabolizing enzymes in both rat lungs and nasal epithelium, despite Nrf2 protein levels remaining stable [24]. The authors believe that Nrf2 becomes less sensitive to the oxidants from smoke with repeated exposures, although no mechanism for this decreased sensitivity has been demonstrated. Therefore it is important to determine whether or not CS components directly affect Nrf2 protein expression, which appears to be unlikely due to no change in Nrf2 protein levels in response to CS in lung epithelical cells in vitro. The other possibility is that CS components orchestrate post-translational modification of Nrf2 protein and related pathways, leading to alteration of nuclear translocation or its affinity for ARE–DNA binding. The functional impact of decreased Phase II metabolism is significant; as the redox capabilities of the cell are decreased, the initiation and/or propagation of inflammatory response increases. Nrf2−/− mice have been shown to be more susceptible to airway inflammation caused by bleomycin [25] and ovalbumin challenge [26]. Studies have also shown increased susceptibility of Nrf2−/− mice to CS-induced emphysema compared with wild-type mice [27,28], suggesting the protective role of Nrf2. Recent studies have shown that activating Nrf2 with the compound CDDO (2-cyano-3,12-dioxo-oleana-1,9-dien-28-oic acid)-imidazolide can reduce LPS-induced inflammation and mortality, providing a novel therapeutic strategy for diseases where oxidative stress is implicated [29]. On the other hand, studies in our laboratory revealed that treatment of human alveolar/airway epithelial cells and macrophages with CSE (CS extract) had no effect on nuclear translocation of Nrf2 due to the post-translational modifications of Nrf2 and Keap1 caused by aldehydes present in CSE. Immunohistochemistry data on human peripheral lung tissue also showed sequestration of Nrf2 predominantly in the cytoplasm of airway epithelium, alveolar type II cells and macrophages in smokers and patients with COPD compared with non-smokers, suggesting that CS causes sequestration of Nrf2 in the cytoplasm by its post-translational modifications.

In addition, decreased Nrf2 activity caused by CS may have an effect on the transcription factors that interact with Nrf2. A protein–protein interaction has been demonstrated in vitro between Nrf2 and PPARγ (peroxisome-proliferator-activated receptor γ) in the rat thromboxane synthase gene [30] and NF-κB. PPARγ has been shown to be anti-inflammatory, so oxidative stresses such as CS could diminish a PPARγ-mediated anti-inflammatory response through decreased Nrf2 activity or post-translational modification of PPARγ by CS-derived reactive aldehydes [31]. Further studies are required to discern the role of Nrf2 in CS-induced lung inflammation particularly by its interaction/association with NF-κB and PPARγ. Thus it appears that multiple pathways may be operational in a cell to attain a redox balance during oxidative stress; which of these pathways is actually altered by CS would be an interesting study for the future.

Role of NF-κB and IKK [IκB (inhibitory κB) kinase] in lung inflammation

The IKK complex, responsible for the cytokine-induced activation of the latent NF-κB, consists of the two highly related kinase subunits IKKα and IKKβ, as well as a third structural subunit, IKKγ. All three subunits are necessary for NF-κB-dependent gene expression. IKKα's main role is in the phosphorylation and acetylation of histone H3; upon activation, IKKα is able to phosphorylate H3 at Ser10. This modification is followed by the subsequent acetylation of H3 at Lys14 by CBP. It has been shown that this acetylation is facilitated by the interaction between IKKα and CBP in addition to the phosphorylation of Ser10 [32]. It has also been suggested that NIK (NF-κB-inducing kinase) is a key upstream regulator of IKKα and that IKKα activation occurs due to the nuclear translocation of NIK, which is then able to activate nuclear IKKα, resulting in IKKα phosphorylating histone H3 [33]. As a part of the canonical NF-κB pathway, the action of IKKβ has been well characterized. It is known that IKKβ's role is to phosphorylate IκBα, leading to the subsequent ubiquitination and degradation of this inhibitor. This leads to the disassociation of RelA/p65, which is then able to be phosphorylated by a number of kinases such as PKA (protein kinase A) and MSK1 (mitogen- and stress-activated kinase) (both at Ser276), and PKC (protein kinase C) ζ (Ser311), each of which leads to an increase in RelA/p65's ability to induce cytokine release [34]. This is because phosphorylation of the p65 subunit facilitates binding of CBP and p300, which are able to acetylate p65 at Lys218, Lys221 and Lys310. Acetylation at Lys218/Lys221 increases DNA binding and decreases binding to IκBα, while acetylation at Lys310 is believed to promote binding of RelA/p65 to another factor, all of which promotes RelA/p65-mediated transcription [35]. Previously we have shown that CS exposure induced the neutrophilic influx, which was associated with increased levels of various NF-κB-dependent pro-inflammatory mediators, inducing the increased level of IKKα and acetylation of histone H3 in vitro in macrophages and airway epithelial cells as well as in lungs of Sprague–Dawley rats and C57BL/6J mice [4,6,7]. Further investigation on the role of IKKα in acetylation of both histone protein and RelA/p65 in regulation of CS-induced lung inflammation is required so as to demonstrate the mechanism sustained pro-inflammatory effects of CS in lungs.

HDACs play an important role in maintaining the balance of histone acetylation/deacetylation and/or transcriptional activity of pro-inflammatory genes in cells by the removal of acetyl groups from lysine (K) residues at N-terminal tails of core histone proteins [1]. HDAC2 is known to be required for the anti-inflammatory effects of glucocorticoids as reduced levels of HDAC2 have been shown to occur in patients with COPD with subsequent corticosteroid resistance [36]. CSE-mediated reduction in HDAC2 was associated with increased RelA/p65, and indicated that RelA/p65 interacts with HDAC2 and RelA/p65 becomes available or retained in the nucleus for pro-inflammatory gene transcription when HDAC2 is decreased [7]. Thus understanding the mechanism of regulation of HDAC2 by kinase signalling will provide a venue for therapeutic modulation of steroid resistance in CS-induced lung inflammation.


Oxidants play an important role in triggering the inflammatory response by activation of NF-κB, down-modulation of Nrf2, and/or disruption of deacetylase–RelA/p65 complex. However, genetic ablation of components of NADPH oxidase assembly enhances susceptibility to lung inflammation, implicating a protective role of endogenous ROS in controlling CS-induced inflammation. Since different cells respond differently to a given oxidant stimulus and also since multiple signalling pathways may be involved, which again may be cell-type-specific, it is an imperative that these subtle differences be dissected in order to have a better understanding of the in situ scenario during a pathological condition involving oxidative stress and redox alterations. Overall, the interplay of endogenous ROS and transcriptional factors such as NF-κB, PPARγ and Nrf2, and co-repressor HDAC2, in response to CS in regulation of lung inflammation needs further investigation. This knowledge would lead to better understanding of the pathology due to CS and would also lead to development of a specific therapeutic agent.


  • Inflammation: A Focus Topic at Life Sciences 2007, held at SECC Glasgow, U.K., 9–12 July 2007. Edited by J. Allen (University College Dublin, Ireland), G. Brown (Cambridge, U.K.), G. Cirino (Napoli, Italy), A. Floto (Cambridge, U.K.), I. Megson (UHI Millennium Institute, U.K.), C. Page (King's College London, U.K.), M. Perretti (Queen Mary, University of London, U.K.) and S. Ward (Bath, U.K.).

Abbreviations: ARE, antioxidant responsive element; CREB, cAMP-response-element-binding protein; CBP, CREB-binding protein; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; CSE, CS extract; HDAC, histone deacetylase; IκB, inhibitory κB; IKK, IκB kinase; IL, interleukin; Keap1, Kelch-like enoyl-CoA hydratase-associated protein 1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MEKK, MAPK/extracellular-signal-regulated kinase kinase kinase; MCP-1, monocyte chemotactic protein-1; MMP, matrix metalloproteinase; NF-κB, nuclear factor κB; NIK, NF-κB-inducing kinase; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2; PKC, protein kinase C; PPARγ, peroxisome-proliferator-activated receptor γ; ROS, reactive oxygen species


View Abstract