Extracellular Matrix Oxidised by the Granulocyte Oxidants Hypochlorous and Hypobromous Acid Reduces Lung Fibroblast Adhesion and Proliferation In Vitro.

Chronic airway inflammation and oxidative stress play crucial roles in the pathogenesis of chronic inflammatory lung diseases, with airway inflammation being a key driving mechanism of oxidative stress in the lungs. Inflammatory responses in the lungs activate neutrophils and/or eosinophils, leading to the generation of hypohalous acids (HOX). These HOX oxidants can damage the extracellular matrix (ECM) structure and may influence cell-ECM interactions. The ECM of the lung provides structural, mechanical, and biochemical support for cells and determines the airway structure. One of the critical cells in chronic respiratory disease is the fibroblast. Thus, we hypothesised that primary human lung fibroblasts (PHLF) exposed to an oxidised cell-derived ECM will result in functional changes to the PHLF. Here, we show that PHLF adhesion, proliferation, and inflammatory cytokine secretion is affected by exposure to HOX-induced oxidisation of the cell-derived ECM. Furthermore, we investigated the impact on fibroblast function from the presence of haloamines in the ECM. Haloamines are chemical by-products of HOX and, like the HOX, haloamines can also modify the ECM. In conclusion, this study revealed that oxidising the cell-derived ECM might contribute to functional changes in PHLF, a key mechanism behind the pathogenesis of inflammatory lung diseases.

Tropomyosin 2.1 collaborates with fibronectin to promote TGF-ß1-induced contraction of human lung fibroblasts.

Many lung diseases are characterized by fibrosis, leading to impaired tissue patency and reduced lung function. Development of fibrotic tissue depends on two-way interaction between the cells and the extra-cellular matrix (ECM). Concentration-dependent increased stiffening of the ECM is sensed by the cells, which in turn increases intracellular contraction and pulling on the matrix causing matrix reorganization and further stiffening. It is generally accepted that the inflammatory cytokine growth factor ß1 (TGF-ß1) is a major driver of lung fibrosis through the stimulation of ECM production. However, TGF-ß1 also regulates the expression of members of the tropomyosin (Tm) family of actin associating proteins that mediate ECM reorganization through intracellular-generated forces. Thus, TGF-ß1 may mediate the bi-directional signaling between cells and the ECM that promotes tissue fibrosis. Using combinations of cytokine stimulation, mRNA, protein profiling and cellular contractility assays with human lung fibroblasts, we show that concomitant induction of key Tm isoforms and ECM by TGF-ß1, significantly accelerates fibrotic phenotypes. Knocking down Tpm2.1 reduces fibroblast-mediated collagen gel contraction. Collectively, the data suggest combined ECM secretion and actin cytoskeleton contractility primes the tissue for enhanced fibrosis. Our study suggests that Tms are at the nexus of inflammation and tissue stiffening. Small molecules targeting specific Tm isoforms have recently been designed; thus targeting Tpm2.1 may represent a novel therapeutic target in lung fibrosis.

Cataloguing the phosphorylation sites of tristetraprolin (TTP): Functional implications for inflammatory diseases.

Tristetraprolin (TTP) is a destabilizing mRNA binding protein known to regulate gene expression of a wide variety of targets, including those that control inflammation. TTP expression, regulation and function is controlled by phosphorylation. While the importance of key serine (S) sites (S52 and S178 in mice and S186 in humans) has been recognized, other sites on the hyperphosphorylated TTP protein have more recently emerged as playing an important role in regulating cellular signalling and downstream functions of TTP. In order to propel investigation of TTP and fully exploit its potential as a drug target in inflammatory disease, this review will catalogue TTP phosphorylation sites in both the murine and human TTP protein, the known and unknown roles and functions of these sites, the kinases and phosphatases that act upon TTP and overview methodological approaches to increase our knowledge of this important protein regulated by phosphorylation.

Regulation of ATP binding cassette transporter A1 (ABCA1) expression: cholesterol-dependent and - independent signaling pathways with relevance to inflammatory lung disease.

The role of the ATP binding cassette transporter A1 (ABCA1) in maintaining cellular lipid homeostasis in cardiovascular disease is well established. More recently, the important beneficial role played by ABCA1 in modulating pathogenic disease mechanisms, such as inflammation, in a broad range of chronic conditions has been realised. These studies position ABCA1 as a potential therapeutic target in a diverse range of diseases where inflammation is an underlying cause. Chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD) are driven by inflammation, and as such, there is now a growing recognition that we need a greater understanding of the signaling pathways responsible for regulation of ABCA1 expression in this clinical context. While the signaling pathways responsible for cholesterol-mediated ABCA1 expression have been clearly delineated through decades of studies in the atherosclerosis field, and thus far appear to be translatable to the respiratory field, less is known about the cholesterol-independent signaling pathways that can modulate ABCA1 expression in inflammatory lung disease. This review will identify the various signaling pathways and ligands that are associated with the regulation of ABCA1 expression and may be exploited in future as therapeutic targets in the setting of chronic inflammatory lung diseases.

The effect of statins and the synthetic LXR agonist T0901317 on expression of ABCA1 transporter protein in human lung epithelial cell lines in vitro.

BACKGROUND: The pathogenesis of chronic obstructive pulmonary disease (COPD) is associated with dyslipidemia, an established co-morbidity. Statins treat hypercholesterolemia, but more recently have been trailed in the setting of COPD for their potential anti-inflammatory benefits. The outcomes of prospective trials however have been inconsistent. Thus, we hypothesize that the variation in results may have been due to statin-induced downregulation of ATP-binding cassette transporter A1 (ABCA1), thereby reducing cholesterol export. This study aims to elucidate whether statin treatment in a cellular model of COPD leads to a decrease in ABCA1 protein expression. METHODS: To mimic the inflammatory environment of COPD, two commonly used lung epithelial cell lines (BEAS-2B and A549) were treated with tumor necrosis factor (TNF), and co-treated with cholesterol/25-hydroxycholesterol (25-OH) to mimic dyslipidemia. ABCA1 protein was detected by Western Blotting. RESULTS: We unexpectedly showed that statins did not affect ABCA1 expression. However, the LXR agonist T0901317 significantly increased ABCA1 expression in both cell lines, while TNF, cholesterol or 25-OH induced ABCA1 protein upregulation in BEAS-2B cells, indicating cell line differences in response. There was also evidence of synergistic impacts of combined treatments on ABCA1 upregulation in BEAS-2B cells. CONCLUSION: Statins did not have an impact on ABCA1 expression in lung epithelial cell lines, disproving our original hypothesis. However, we showed for the first time, the effect of the inflammatory cytokine TNF, cholesterol/25-OH, statins and the LXR agonist T0901317 on expression of ABCA1 transporter protein in human lung epithelial cell lines in vitro. We hope that these in vitro studies may prove beneficial for addressing dyslipidemia in COPD in the future.

Protein phosphatase 2A (PP2A): a key phosphatase in the progression of chronic obstructive pulmonary disease (COPD) to lung cancer.

Lung cancer (LC) has the highest relative risk of development as a comorbidity of chronic obstructive pulmonary disease (COPD). The molecular mechanisms that mediate chronic inflammation and lung function impairment in COPD have been identified in LC. This suggests the two diseases are more linked than once thought. Emerging data in relation to a key phosphatase, protein phosphatase 2A (PP2A), and its regulatory role in inflammatory and tumour suppression in both disease settings suggests that it may be critical in the progression of COPD to LC. In this review, we uncover the importance of the functional and active PP2A holoenzyme in the context of both diseases. We describe PP2A inactivation via direct and indirect means and explore the actions of two key PP2A endogenous inhibitors, cancerous inhibitor of PP2A (CIP2A) and inhibitor 2 of PP2A (SET), and the role they play in COPD and LC. We explain how dysregulation of PP2A in COPD creates a favourable inflammatory micro-environment and promotes the initiation and progression of tumour pathogenesis. Finally, we highlight PP2A as a druggable target in the treatment of COPD and LC and demonstrate the potential of PP2A re-activation as a strategy to halt COPD disease progression to LC. Although further studies are required to elucidate if PP2A activity in COPD is a causal link for LC progression, studies focused on the potential of PP2A reactivating agents to reduce the risk of LC formation in COPD patients will be pivotal in improving clinical outcomes for both COPD and LC patients in the future.

Role of Dual-Specificity Phosphatase 1 in Glucocorticoid-Driven Anti-inflammatory Responses.

Glucocorticoids (GCs) potently inhibit pro-inflammatory responses and are widely used for the treatment of inflammatory diseases, such as allergies, autoimmune disorders, and asthma. Dual-specificity phosphatase 1 (DUSP1), also known as mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), exerts its effects by dephosphorylation of MAPKs, i.e., extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Endogenous DUSP1 expression is tightly regulated at multiple levels, involving both transcriptional and post-transcriptional mechanisms. DUSP1 has emerged as a central mediator in the resolution of inflammation, and upregulation of DUSP1 by GCs has been suggested to be a key mechanism of GC actions. In this review, we discuss the impact of DUSP1 on the efficacy of GC-mediated suppression of inflammation and address the underlying mechanisms.

New therapeutic targets for the prevention of infectious acute exacerbations of COPD: role of epithelial adhesion molecules and inflammatory pathways.

Chronic respiratory diseases are among the leading causes of mortality worldwide, with the major contributor, chronic obstructive pulmonary disease (COPD) accounting for approximately 3 million deaths annually. Frequent acute exacerbations (AEs) of COPD (AECOPD) drive clinical and functional decline in COPD and are associated with accelerated loss of lung function, increased mortality, decreased health-related quality of life and significant economic costs. Infections with a small subgroup of pathogens precipitate the majority of AEs and consequently constitute a significant comorbidity in COPD. However, current pharmacological interventions are ineffective in preventing infectious exacerbations and their treatment is compromised by the rapid development of antibiotic resistance. Thus, alternative preventative therapies need to be considered. Pathogen adherence to the pulmonary epithelium through host receptors is the prerequisite step for invasion and subsequent infection of surrounding structures. Thus, disruption of bacterial-host cell interactions with receptor antagonists or modulation of the ensuing inflammatory profile present attractive avenues for therapeutic development. This review explores key mediators of pathogen-host interactions that may offer new therapeutic targets with the potential to prevent viral/bacterial-mediated AECOPD. There are several conceptual and methodological hurdles hampering the development of new therapies that require further research and resolution.

Prostaglandin E2, but not cAMP nor ß2-agonists, induce tristetraprolin (TTP) in human airway smooth muscle cells.

Tristetraprolin (TTP) is an anti-inflammatory molecule known to post-transcriptionally regulate cytokine production and is, therefore, an attractive drug target for chronic respiratory diseases driven by inflammation, such as asthma and chronic obstructive pulmonary disease. Our recent in vitro studies in primary human airway smooth (ASM) cells have confirmed the essential anti-inflammatory role played by TTP as a critical partner in a cytokine regulatory network. However, several unanswered questions remain. While prior in vitro studies have suggested that TTP is regulated in a cAMP-mediated manner, raising the possibility that this may be one of the ways in which ß2-agonists achieve beneficial effects beyond bronchodilation, the impact of ß2-agonists on ASM cells is unknown. Furthermore, the effect of prostaglandin E2 (PGE2) on TTP expression in ASM cells has not been reported. We address this herein and reveal, for the first time, that TTP is not regulated by cAMP-activating agents nor following treatment with long-acting ß2-agonists. However, PGE2 does induce TTP mRNA expression and protein upregulation in ASM cells. Although the underlying mechanism of action remains undefined, we can confirm that PGE2-induced TTP upregulation is not mediated via cAMP, or EP2/EP4 receptor activation, and occurred in a manner independent of the p38 MAPK-mediated pathway. Taken together, these data confirm that ß2-agonists do not upregulate TTP in human ASM cells and indicate that another way in which PGE2 may achieve beneficial effects in asthma and COPD may be via upregulation of the master controller of inflammation-TTP.

EP2 and EP4 receptor antagonists: Impact on cytokine production and ß2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for ß2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) ß2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores ß2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Enhancing tristetraprolin activity reduces the severity of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a progressive disease that causes significant mortality and morbidity worldwide and is primarily caused by the inhalation of cigarette smoke (CS). Lack of effective treatments for COPD means there is an urgent need to identify new therapeutic strategies for the underlying mechanisms of pathogenesis. Tristetraprolin (TTP) encoded by the Zfp36 gene is an anti-inflammatory protein that induces mRNA decay, especially of transcripts encoding inflammatory cytokines, including those implicated in COPD. METHODS: Here, we identify a novel protective role for TTP in CS-induced experimental COPD using Zfp36aa/aa mice, a genetically modified mouse strain in which endogenous TTP cannot be phosphorylated, rendering it constitutively active as an mRNA-destabilising factor. TTP wild-type (Zfp36 +/+) and Zfp36aa/aa active C57BL/6J mice were exposed to CS for four days or eight weeks, and the impact on acute inflammatory responses or chronic features of COPD, respectively, was assessed. RESULTS: After four days of CS exposure, Zfp36aa/aa mice had reduced numbers of airway neutrophils and lymphocytes and mRNA expression levels of cytokines compared to wild-type controls. After eight weeks, Zfp36aa/aa mice had reduced pulmonary inflammation, airway remodelling and emphysema-like alveolar enlargement, and lung function was improved. We then used pharmacological treatments in vivo (protein phosphatase 2A activator, AAL(S), and the proteasome inhibitor, bortezomib) to promote the activation and stabilisation of TTP and show that hallmark features of CS-induced experimental COPD were ameliorated. CONCLUSION: Collectively, our study provides the first evidence for the therapeutic potential of inducing TTP as a treatment for COPD.

Repurposing of statins via inhalation to treat lung inflammatory conditions.

Despite many therapeutic advancements over the past decade, the continued rise in chronic inflammatory lung diseases incidence has driven the need to identify and develop new therapeutic strategies, with superior efficacy to treat these diseases. Statins are one class of drug that could potentially be repurposed as an alternative treatment for chronic lung diseases. They are currently used to treat hypercholesterolemia by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, that catalyses the rate limiting step in the mevalonate biosynthesis pathway, a key intermediate in cholesterol metabolism. Recent research has identified statins to have other protective pleiotropic properties including anti-inflammatory, anti-oxidant, muco-inhibitory effects that may be beneficial for the treatment of chronic inflammatory lung diseases. However, clinical studies have yielded conflicting results. This review will summarise some of the current evidences for statins pleiotropic effects that could be applied for the treatment of chronic inflammatory lung diseases, their mechanisms of actions, and the potential to repurpose statins as an inhaled therapy, including a detailed discussion on their different physical-chemical properties and how these characteristics could ultimately affect treatment efficacies. The repurposing of statins from conventional anti-cholesterol oral therapy to inhaled anti-inflammatory formulation is promising, as it provides direct delivery to the airways, reduced risk of side effects, increased bioavailability and tailored physical-chemical properties for enhanced efficacy.

Selectively targeting prostanoid E (EP) receptor-mediated cell signalling pathways: Implications for lung health and disease.

Arachidonic acid is metabolized by cyclooxygenases (COX-1 and COX-2) into various prostanoids which exert different functions in mammalian physiology. One of these prostanoids, prostaglandin E2 (PGE2), interacts with four different G protein-coupled receptors, named EP1, EP2, EP3 and EP4, to initiate different downstream signalling pathways. Prostanoid receptors are diversely expressed throughout different tissues all over the body and PGE2 is responsible for a large variety of beneficial and disadvantageous effects. We have recently achieved a greater understanding of the biology of prostanoid E receptors and the potential for specific drug targeting with the advent of potent and selective EP receptor agonists and antagonists. This has important implications for lung health and disease as PGE2-mediated EP receptor activation impacts upon migration of airway smooth muscle cells, airway microvascular leak, tone regulation of pulmonary blood vessels, mast cell degranulation, bronchodilatation, cough, angiogenesis and airway inflammation, to name a few. In this review, we overview the EP receptor family and the related signalling pathways, summarize a variety of EP1-4 receptor agonists and antagonists, provide an overview of pharmacological tools used to implicate EP receptor function in the context of respiratory health and disease and finally highlight some of the more selective pharmacological reagents that have recently been developed. The availability of selective pharmacological agonists and antagonists for the distinct EP receptors, as well as the development of specific prostanoid receptor knock-out mice, offer hitherto unattainable opportunities for achieving an in depth understanding of the role and function of PGE2 in respiratory disease and the exciting potential of targeting EP receptors more broadly.

Role and regulation of MKP-1 in airway inflammation.

Mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a protein with anti-inflammatory properties and the archetypal member of the dual-specificity phosphatases (DUSPs) family that have emerged over the past decade as playing an instrumental role in the regulation of airway inflammation. Not only does MKP-1 serve a critical role as a negative feedback effector, controlling the extent and duration of pro-inflammatory MAPK signalling in airway cells, upregulation of this endogenous phosphatase has also emerged as being one of the key cellular mechanism responsible for the beneficial actions of clinically-used respiratory medicines, including ß2-agonists, phosphodiesterase inhibitors and corticosteroids. Herein, we review the role and regulation of MKP-1 in the context of airway inflammation. We initially outline the structure and biochemistry of MKP-1 and summarise the multi-layered molecular mechanisms responsible for MKP-1 production more generally. We then focus in on some of the key in vitro studies in cell types relevant to airway disease that explain how MKP-1 can be regulated in airway inflammation at the transcriptional, post-translation and post-translational level. And finally, we address some of the potential challenges with MKP-1 upregulation that need to be explored further to fully exploit the potential of MKP-1 to repress airway inflammation in chronic respiratory disease.

para-Sulfonatocalix[4]arene and polyamidoamine dendrimer nanocomplexes as delivery vehicles for a novel platinum anticancer agent.

Novel para-sulfonatocalix[4]arene (sCX[4]) and polyamidoamine (PAMAM) dendrimer nanocomplexes were evaluated as delivery vehicles for the platinum anticancer agent [(1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)] chloride (PHENSS). Different ratios of sCX[4] to PHENSS were tested for their compatibility, with a ratio of 6:1 sCX[4]:PHENSS having the best solubility. The loading of sCX[4], and sCX[4]-bound PHENSS, onto three different generations of PAMAM dendrimers (G3.0-5.0) was examined using UV-visible spectrophotometry. The quantity of sCX[4] bound was found to increase exponentially with dendrimer size: G3, 15 sCX[4] molecules per dendrimer; G4, 37; and G5, 78. Similarly, the loading of sCX[4]-bound PHENSS also increased with increasing dendrimer size: G3, 7 PHENSS molecules per dendrimer; G4, 14; and G5, 28.5. The loading of sCX[4]-bound PHENSS molecules is significantly lower when compared with that of sCX[4], which indicates that less than half of the binding sites were occupied (45, 44, and 44%, respectively). By 1H NMR and UV-vis analysis, the nanocomplex was found to be stable in NaCl solutions at concentrations up to 150mM. While PHENSS is more active in vitro than cisplatin against the human breast cancer cell line, MCF-7, delivery of PHENSS using the sCX[4]-dendrimer nanocomplexes, regardless of dendrimer generation, had little effect on PHENSS cytotoxicity. The results of this study may have application in the delivery of a variety of small molecule metal-based drugs for which chemical conjugation to a nanoparticle is undesired or not feasible.

Doxofylline does not increase formoterol-induced cAMP nor MKP-1 expression in ASM cells resulting in lack of anti-inflammatory effect.

The xanthine doxofylline has been examined in clinical trials and shown to have efficacy and greater tolerability than theophylline in asthma and chronic obstructive pulmonary disease. The 'novofylline' doxofylline has demonstrated bronchodilatory and anti-inflammatory actions in in vivo and ex vivo experimental models of respiratory disease. However, there are limited studies in vitro. We address this herein and examine whether doxofylline has anti-inflammatory impact on primary cultures of airway smooth muscle (ASM) cells. We conduct a series of investigations comparing and contrasting doxofylline with the archetypal xanthine, theophylline, and the specific phosphodiesterase (PDE) 4 inhibitor, cilomilast. We confirm that the xanthine drugs do not have action as PDE inhibitors in ASM cells. Unlike cilomilast, doxofylline (and theophylline) do not increase cAMP production in ASM cells induced by long-acting ß2-agonist formoterol. Similar to theophylline, and consistent with the lack of cAMP potentiation, doxofylline does not augment formoterol-induced upregulation of the anti-inflammatory protein mitogen-activated protein kinase phosphatase 1 (MKP-1). However, when we examine the effect of doxofylline on secretion of the interleukin 8 from ASM cells stimulated by tumour necrosis factor (an in vitro surrogate measure of inflammation), there was no repression of inflammation. This is in contrast to the anti-inflammatory impact exerted by theophylline and cilomilast in confirmatory experiments. In summary, our study is the first to examine the effect of doxofylline on ASM cells in vitro and highlights some distinct differences between two key members of xanthine drug family, doxofylline and theophylline.

Examining the role of ABC lipid transporters in pulmonary lipid homeostasis and inflammation.

Respiratory diseases including asthma and chronic obstructive pulmonary disease (COPD) are characterised by excessive and persistent inflammation. Current treatments are often inadequate for symptom and disease control, and hence new therapies are warranted. Recent emerging research has implicated dyslipidaemia in pulmonary inflammation. Three ATP-binding cassette (ABC) transporters are found in the mammalian lung - ABCA1, ABCG1 and ABCA3 - that are involved in movement of cholesterol and phospholipids from lung cells. The aim of this review is to corroborate the current evidence for the role of ABC lipid transporters in pulmonary lipid homeostasis and inflammation. Here, we summarise results from murine knockout studies, human diseases associated with ABC transporter mutations, and in vitro studies. Disruption to ABC transporter activity results in lipid accumulation and elevated levels of inflammatory cytokines in lung tissue. Furthermore, these ABC-knockout mice exhibit signs of respiratory distress. ABC lipid transporters appear to have a crucial and protective role in the lung. However, our knowledge of the underlying molecular mechanisms for these benefits requires further attention. Understanding the relationship between cholesterol and inflammation in the lung, and the role that ABC transporters play in this may illuminate new pathways to target for the treatment of inflammatory lung diseases.

Roflumilast N-Oxide in Combination with Formoterol Enhances the Antiinflammatory Effect of Dexamethasone in Airway Smooth Muscle Cells.

Roflumilast is an orally active phosphodiesterase 4 inhibitor approved for use in chronic obstructive pulmonary disease. Roflumilast N-oxide (RNO) is the active metabolite of roflumilast and has a demonstrated antiinflammatory impact in vivo and in vitro. To date, the effect of RNO on the synthetic function of airway smooth muscle (ASM) cells is unknown. We address this herein and investigate the effect of RNO on ß2-adrenoceptor-mediated, cAMP-dependent responses in ASM cells in vitro, and whether RNO enhances steroid-induced repression of inflammation. RNO (0.001-1,000 nM) alone had no effect on AMP production from ASM cells, and significant potentiation of the long-acting ß2-agonist formoterol-induced cAMP could only be achieved at the highest concentration of RNO tested (1,000 nM). At this concentration, RNO exerted a small, but not significantly different, potentiation of formoterol-induced expression of antiinflammatory mitogen-activated protein kinase phosphatase 1. Consequently, tumor necrosis factor-induced IL-8 secretion was unaffected by RNO in combination with formoterol. However, because there was the potential for phosphodiesterase 4 inhibitors and long-acting ß2-agonists to interact with corticosteroids to achieve superior antiinflammatory efficacy, we examined whether RNO, alone or in combination with formoterol, enhanced the antiinflammatory effect of dexamethasone by measuring the impact on IL-8 secretion. Although RNO alone did not significantly enhance the cytokine repression achieved with steroids, RNO in combination with formoterol significantly enhanced the antiinflammatory effect of dexamethasone in ASM cells. This was linked to increased mitogen-activated protein kinase phosphatase 1 expression in ASM cells, suggesting that a molecular mechanism is responsible for augmented antiinflammatory actions of combination therapeutic approaches that include RNO.

The phosphorylated form of FTY720 activates PP2A, represses inflammation and is devoid of S1P agonism in A549 lung epithelial cells.

Protein phosphatase 2A (PP2A) activity can be enhanced pharmacologically by PP2A-activating drugs (PADs). The sphingosine analog FTY720 is the best known PAD and we have shown that FTY720 represses production of pro-inflammatory cytokines responsible for respiratory disease pathogenesis. Whether its phosphorylated form, FTY720-P, also enhances PP2A activity independently of the sphingosine 1-phosphate (S1P) pathway was unknown. Herein, we show that FTY720-P enhances TNF-induced PP2A phosphatase activity and significantly represses TNF-induced interleukin 6 (IL-6) and IL-8 mRNA expression and protein secretion from A549 lung epithelial cells. Comparing FTY720 and FTY720-P with S1P, we show that unlike S1P, the sphingosine analogs do not induce cytokine production on their own. In fact, FTY720 and FTY720-P significantly repress S1P-induced IL-6 and IL-8 production. We then examined their impact on expression of cyclooxygenase 2 (COX-2) and resultant prostaglandin E2 (PGE2) production. S1P did not increase production of this pro-inflammatory enzyme because COX-2 mRNA gene expression is NF-κB-dependent, and unlike TNF, S1P did not activate NF-κB. However, TNF-induced COX-2 mRNA expression and PGE2 secretion is repressed by FTY720 and FTY720-P. Hence, FTY720-P enhances PP2A activity and that PADs can repress production of pro-inflammatory cytokines and enzymes in A549 lung epithelial cells in a manner devoid of S1P agonism.

TLR2 ligation induces corticosteroid insensitivity in A549 lung epithelial cells: Anti-inflammatory impact of PP2A activators.

Corticosteroids are effective anti-inflammatory therapies widely utilized in chronic respiratory diseases. But these medicines can lose their efficacy during respiratory infection resulting in disease exacerbation. Further in vitro research is required to understand how infection worsens lung function control in order to advance therapeutic options to treat infectious exacerbation in the future. In this study, we utilize a cellular model of bacterial exacerbation where we pretreat A549 lung epithelial cells with the synthetic bacterial lipoprotein Pam3CSK4 (a TLR2 ligand) to mimic bacterial infection and tumor necrosis factor α (TNFα) to simulate inflammation. Under these conditions, Pam3CSK4 induces corticosteroid insensitivity; demonstrated by substantially reduced ability of the corticosteroid dexamethasone to repress TNFα-induced interleukin 6 secretion. We then explored the molecular mechanism responsible and found that corticosteroid insensitivity induced by bacterial mimics was not due to altered translocation of the glucocorticoid receptor into the nucleus, nor an impact on the NF-κB pathway. Moreover, Pam3CSK4 did not affect corticosteroid-induced upregulation of anti-inflammatory MAPK deactivating phosphatase-MKP-1. However, Pam3CSK4 can induce oxidative stress and we show that a proportion of the MKP-1 produced in response to corticosteroid in the context of TLR2 ligation was rendered inactive by oxidation. Thus to combat inflammation in the context of bacterial exacerbation we sought to discover effective strategies that bypassed this road-block. We show for the first time that known (FTY720) and novel (theophylline) activators of the phosphatase PP2A can serve as non-steroidal anti-inflammatory alternatives and/or corticosteroid-sparing approaches in respiratory inflammation where corticosteroid insensitivity exists.