Matthew C. Catley

Differential IκB Kinase Activation and IκBα Degradation by Interleukin-1β and Tumor Necrosis Factor-α in Human U937 Monocytic Cells EVIDENCE FOR ADDITIONAL REGULATORY STEPS IN κB-DEPENDENT TRANSCRIPTION

The IκB kinases (IKKs) lie downstream of the NF-κB-inducing kinase (NIK) and activate NF-κB by phosphorylation of IκBα. This leads to IκBα degradation and release of NF-κB. In U937 monocytic cells, interleukin (IL)-1β (1 ng/ml) and tumor necrosis factor (TNF)-α; 10 ng/ml) induced κB-dependent transcription equally. However, IKK activity was strongly induced by TNF-α but not by IL-1β. This was consistent with IκBα phosphorylation and degradation, yet TNF-α-induced NF-κB DNA binding was only 30–40% greater than for IL-1β. This was not explained by degradation of IκBβ, IκBε, or p105 nor nuclear translocation of NF-κB·IκBα complexes or degradation-independent release of NF-κB. Dominant negative (NIK) repressed TNF-α and IL-1β-induced κB-dependent transcription by ∼60% and ∼35%, respectively. These data reveal an imprecise relationship between IKK activation, IκBα degradation, and NF-κB DNA binding, suggesting the existence of additional mechanisms that regulate NF-κB activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited κB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-κB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IκBα cascade may not produce equivalent reductions in NF-κB-dependent gene expression.

Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages.

Many of the healthcare consequences of cigarette smoking could be due to its ability to compromise the immune system, and in respiratory diseases like chronic obstructive pulmonary disease (COPD), a constant low level of infection could be responsible for some of the symptoms/pathology. The aim was to assess the impact of cigarette smoke (CS) on the release of innate effector cytokines in THP‐1 cells and human lung macrophages, and to determine the molecular mechanism behind the altered response. Cells were exposed to CS with and without endotoxin stimulus, cytokines, glutathione, mitogen‐activated protein kinase (MAPK) phosphorylation, IκB kinase‐2 (IKK‐2) activity, nuclear factor kappa B (NF‐κB), and activator protein‐1 (AP‐1) pathway activation was measured. Attempts were made to mimic or block the effect of CS by using nicotine, nitric oxide donors/inhibitors, prostanoid inhibitors, and anti‐oxidants. Results showed that CS initially delayed the production of “innate” cytokines (e.g., IL‐1β and IL‐6) and reduced glutathione levels. This was associated with a reduction in NF‐κB pathway activation, which suggested a causative link. CS also increased the phosphorylation of MAPKs and the production of IL‐8 but interestingly only in stimulated cells. Exogenous glutathione treatment reversed both these effects of CS, which suggests that this molecule may play a central role. In conclusion, this data provides a novel mechanistic explanation for why smokers have increased prevalence/severity of respiratory infections. In addition, the suppression of the innate response is accompanied by an increase in the neutrophil chemoattractant, IL‐8, which may suggest a link to the pathogenesis of smoking‐related inflammatory disease. J. Cell. Physiol. 214:27–37, 2008.

Repression of Inflammatory Gene Expression in Human Pulmonary Epithelial Cells by Small-Molecule IκB Kinase Inhibitors

The airway epithelium is critical in the pathogenesis of chronic inflammatory diseases, such as asthma and chronic obstructive pulmonary disease, and, by expressing numerous inflammatory genes, plays a prominent role in disease exacerbations. Since inflammatory gene expression often involves the transcription factor nuclear factor (NF)-κB, this signaling pathway represents a site for anti-inflammatory intervention. As the airway epithelium is targeted by inhaled therapeutic agents, for example corticosteroids, human A549 pulmonary cells and primary human bronchial epithelial (HBE) cells were selected to evaluate inhibitor of κB kinase (IKK) inhibitors. In A549 cells, interleukin (IL)-1β and tumor necrosis factor (TNF) α increased phosphorylation of IκBα, and this was followed by loss of IκBα, induction of NF-κB DNA binding, and the induction of NF-κB-dependent transcription. These events were repressed by the IKK-selective inhibitors, PS-1145 [N-(6-chloro-9H-β-carbolin-8-ly) nicotinamide] and ML120B [N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide]. Inhibition of NF-κB-dependent transcription was concentration-dependent and correlated with loss of intercellular adhesion molecule (ICAM)-1 expression. Similarly, IL-1β- and TNFα-induced expression of IL-6, IL-8, granulocyte macrophage-colony-stimulating factor (GM-CSF), regulated and activation normal T cell expressed and secreted (RANTES), growth-related oncogene α, and monocyte chemotactic protein-1 (MCP-1) was also significantly repressed. Likewise, PS-1145 and ML120B profoundly reduced NF-κB-dependent transcription induced by IL-1β and TNFα in primary HBE cells. Parallel effects on ICAM-1 expression and a significant repression of IL-8 release were observed. In contrast, the corticosteroid, dexamethasone, was without effect on NF-κB-dependent transcription or the expression of ICAM-1. The above data provide strong support for an anti-inflammatory effect of IKK2 inhibitors acting on the pulmonary epithelium and suggest that such compounds may prove beneficial in situations where traditional corticosteroid therapies prove inadequate.

P2X7 Receptor and Caspase 1 Activation Are Central to Airway Inflammation Observed after Exposure to Tobacco Smoke

Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently there is no effective medication to stop the relentless progression of this disease. It has recently been shown that an activator of the P2X7/inflammasome pathway, ATP, and the resultant products (IL-1β/IL-18) are increased in COPD patients. The aim of this study was to determine whether activation of the P2X7/caspase 1 pathway has a functional role in CS-induced airway inflammation. Mice were exposed to CS twice a day to induce COPD-like inflammation and the role of the P2X7 receptor was investigated. We have demonstrated that CS-induced neutrophilia in a pre-clinical model is temporally associated with markers of inflammasome activation, (increased caspase 1 activity and release of IL-1β/IL-18) in the lungs. A selective P2X7 receptor antagonist and mice genetically modified so that the P2X7 receptors were non-functional attenuated caspase 1 activation, IL-1β release and airway neutrophilia. Furthermore, we demonstrated that the role of this pathway was not restricted to early stages of disease development by showing increased caspase 1 activation in lungs from a more chronic exposure to CS and from patients with COPD. This translational data suggests the P2X7/Inflammasome pathway plays an ongoing role in disease pathogenesis. These results advocate the critical role of the P2X7/caspase 1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD.

IL-1β-dependent activation of NF-κB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase

Prostaglandin (PG) E2 release is induced in pulmonary A549 cells by the NF‐κB‐activating stimuli interleukin‐1β (IL‐1β) and phorbol 12‐myristate 13‐acetate (PMA). Adenoviral over‐expression of IκBαΔN, a dominant NF‐κB inhibitor, prevents NF‐κB‐dependent transcription and was used to qualify the role of NF‐κB in the release of PGE2. IκBαΔN repressed IL‐1β‐induced, but not PMA‐induced, cycloxygenase‐2 (COX‐2) and microsomal prostaglandin E synthase (mPGES) expression. These data conclusively demonstrate a substantial role for NF‐κB in the co‐ordinate induction of COX‐2, mPGES and in the corresponding release of PGE2 by IL‐1β. However, other pathways are primarily responsible for PGE2 release induced by PMA.

Validation of the Anti-Inflammatory Properties of Small-Molecule IκB Kinase (IKK)-2 Inhibitors by Comparison with Adenoviral-Mediated Delivery of Dominant-Negative IKK1 and IKK2 in Human Airways Smooth Muscle

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation. However, because patients with COPD and certain patients with asthma show little or no therapeutic benefit from existing corticosteroid therapies, there is an urgent need for novel anti-inflammatory strategies. The transcription factor nuclear factor-κB (NF-κB) is central to inflammation and is necessary for the expression of numerous inflammatory genes. Proinflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, activate the IκB kinase complex (IKK) to promote the degradation of inhibitory IκB proteins and activate NF-κB. This pathway and, in particular, the main IκB kinase, IKK2, are now considered prime targets for novel anti-inflammatory drugs. Therefore, we have used adenoviral overexpression to demonstrate NF-κB and IKK2 dependence of key inflammatory genes, including intercellular adhesion molecule (ICAM)-1, cyclooxygenase-2, IL-6, IL-8, granulocyte macrophage-colony-stimulating factor (GM-CSF), regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), growth-regulated oncogene-α (GROα), neutrophil-activating protein-2 (NAP-2), and epithelial neutrophil activating peptide 78 (ENA-78) in primary human airways smooth muscle cells. Because this cell type is central to the pathogenesis of airway inflammatory diseases, these data predict a beneficial effect of IKK2 inhibition. These validated outputs were therefore used to evaluate the novel IKK inhibitors N-(6-chloro-9H-β-carbolin-8-yl) nicotinamide (PS-1145) and N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide (ML120B) on IL-1β and TNFα-induced expression, and this was compared with the corticosteroid dexamethasone. As observed above, ICAM-1, IL-6, IL-8, GM-CSF, RANTES, MCP-1, GROα, NAP-2, and ENA-78 expression was reduced by the IKK inhibitors. Furthermore, this inhibition was either as effective, or for ICAM-1, MCP-1, GROα, and NAP-2, more effective, than a maximally effective concentration of dexamethasone. We therefore suggest that IKK inhibitors may be of considerable benefit in inflammatory airways diseases, particularly in COPD or severe asthma, in which corticosteroids are ineffective.

IκB kinase-2 independent and dependent inflammation in airway disease models: relevance of IKK- 2 inhibition to the clinic

Nuclear factor κB (NF-κB) is a transcription factor believed to be central in the expression of numerous inflammatory genes and the pathogenesis of many respiratory diseases. We have previously demonstrated increased NF-κB pathway activation in a steroid-sensitive animal model of lipopolysaccharide (LPS)-driven airway inflammation. It is noteworthy that this phenomenon was not observed in a steroid-insensitive model of elastase-induced inflammation in the rat. The aim of this study was to gather further evidence to suggest that these similar profiles of neutrophilic inflammation can be NF-κB-dependent or -independent by determining the impact of an IκB kinase-2 (IKK-2) inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). In the LPS model, TPCA-1 blocked the increase in NF-κB DNA binding, a marker of NF-κB pathway activation. This inhibition was associated with a reduction in inflammatory mediator release [tumor necrosis factor α (TNFα)/interleukin-1β (IL-1β)/matrix metalloproteinase-9 (MMP-9)] and lung inflammatory cell burden (neutrophilia/eosinophilia). These data were paralleled with a steroid and in human cell based assays. In the elastase-driven inflammation model, in which our group has previously failed to measure an increase in NF-κB DNA binding, neither TPCA-1 nor the steroid, affected mediator release (IL-1β/MMP-9) or cellular burden (neutrophilia/lymphomononuclear cells). This is the first study to examine the effect of an IKK-2 inhibitor in well validated models that mimic aspects of the inflammatory lesion evident in diseases such as COPD. In conclusion, we have demonstrated that animal models with similar profiles of airway inflammation can be IKK-2 inhibitor/steroid-sensitive or -insensitive. If both profiles of inflammation exist in the clinic, then this finding is extremely exciting and may lead to greater understanding of disease pathology and the discovery of novel anti-inflammatory targets.

Novel role for the Liver X nuclear receptor in the suppression of lung inflammatory responses

The liver X receptors (LXRα/β) are part of the nuclear receptor family and are believed to regulate cholesterol and lipid homeostasis. It has also been suggested that LXR agonists possess anti-inflammatory properties. The aim of this work was to determine the effect of LXR agonists on the innate immune response in human primary lung macrophages and a pre-clinical rodent model of lung inflammation. Before profiling the impact of the agonist, we established that both the human macrophages and the rodent lungs expressed LXRα/β. We then used two structurally distinct LXR agonists to demonstrate that activation of this transcription factor reduces cytokine production in THP-1 cells and lung macrophages. Then, using the expression profile of ATP binding cassettes A1 (ABCA-1; a gene directly linked to LXR activation) as a biomarker for lung exposure of the compound, we demonstrated an LXR-dependent reduction in lung neutrophilia rodents in vivo. This inhibition was not associated with a suppression of c-Fos/c-Jun mRNA expression or NF-κB/AP-1 DNA binding, suggesting that any anti-inflammatory activity of LXR agonists is not via inhibition of NF-κB/AP-1 transcriptional activity. These data do not completely rule out an impact of these agonists on these two prominent transcription factors. In summary, this study is the first to demonstrate anti-inflammatory actions of LXRs in the lung. Chronic innate inflammatory responses observed in some airway diseases is thought to be central to disease pathogenesis. Therefore, data suggest that LXR ligands have utility in the treatment of lung diseases that involves chronic inflammation mediated by macrophages and neutrophils.

Identification in human airways smooth muscle cells of the prostanoid receptor and signalling pathway through which PGE2 inhibits the release of GM-CSF

The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE2 on interleukin (IL)‐1β‐induced granulocyte/macrophage colony‐stimulating factor (GM‐CSF) release has been classified. IL‐1β evoked the release of GM‐CSF from HASM cells, which was suppressed by PGE2, 16,16‐dimethyl PGE2 (nonselective), misoprostol (EP2/EP3‐selective), ONO‐AE1‐259 and butaprost (both EP2‐selective) with pIC50 values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP‐receptor agonists that have selectivity for the EP1‐ (17‐phenyl‐ω‐trinor PGE2) and EP3‐receptor (sulprostone) subtypes as well as cicaprost (IP‐selective), PGD2, PGF2α and U‐46619 (TP‐selective) were poorly active or inactive at concentrations up to 10 μM. AH 6809, a drug that can be used to selectively block EP2‐receptors in HASM cells, antagonised the inhibitory effect of PGE2, 16,16‐dimethyl PGE2 and ONO‐AE1‐259 with apparent pA2 values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP4‐receptor antagonists, AH 23848B and L‐161,982, failed to displace to the right the concentration–response curves that described the inhibition of GM‐CSF release evoked by PGE2 and ONO‐AE1‐259. Inhibition of GM‐CSF release by PGE2 and 8‐Br‐cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP‐dependent protein kinase (PKA) but not by H‐89, a purported small molecule inhibitor of PKA. We conclude that prostanoid receptors of the EP2‐subtype mediate the inhibitory effect of PGE2 on GM‐CSF release from HASM cells by recruiting a PKA‐dependent pathway. In addition, the data illustrate that caution should be exercised when using H‐89 in studies designed to assess the role of PKA in biological processes.

The Role of IκB Kinase 2, but Not Activation of NF-κB, in the Release of CXCR3 Ligands from IFN-γ-Stimulated Human Bronchial Epithelial Cells

The severity of chronic obstructive pulmonary disease correlates with increased numbers of cytotoxic CD8+ T lymphocytes in the lung parenchyma. CD8+ T lymphocytes release IFN-γ which stimulates airway epithelial cells to produce CXCR3 chemokines leading to further recruitment of CD8+ T lymphocytes. To evaluate the signaling pathways involved in regulation of CXCR3 ligands, the human bronchial epithelial cell line BEAS-2B was stimulated with IFN-γ and the release of the CXCR3 ligands was measured by ELISA. The release of CXCL9, CXCL10, and CXCL11 was inhibited by an IκB kinase 2 (IKK2) selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) (EC50 values were 0.50 ± 0.03, 0.17 ± 0.06, and 0.45 ± 0.10 μM, respectively (n = 6)) and an IKK1/2 selective inhibitor 2-amino-6-(2′cyclopropylemethoxy-6′-hydroxy-phenyl)-4-piperidin-3-yl-pyridine-3-carbonitrile (EC50 values 0.74 ± 0.40, 0.27 ± 0.06, and 0.88 ± 0.29 μM, respectively (n = 6)). The glucocorticosteroid dexamethasone had no effect on CXCR3 ligand release. The release of CXCL10 was most sensitive to inhibition by IKK2 and a role for IKK2 in CXCL10 release was confirmed by overexpression of dominant-negative adenoviral constructs to IKK2 (68.2 ± 8.3% n = 5), but not of IKK1. Neither phosphorylation of IκBα, translocation of p65 to the nucleus, or activation of a NF-κB-dependent reporter (Ad-NF-κB-luc) were detected following stimulation of BEAS-2B cells with IFN-γ. These data suggest that IKK2 is also involved in the IFN-γ-stimulated release of the CXCR3 ligands through a novel mechanism that is independent NF-κB.