Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells.

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32 kb (R1) and ∼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Long-Acting ß2-Adrenoceptor Agonists Enhance Glucocorticoid Receptor (GR)-Mediated Transcription by Gene-Specific Mechanisms Rather Than Generic Effects via GR.

In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting ß2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.

Glucocorticoid and cytokine crosstalk: Feedback, feedforward, and co-regulatory interactions determine repression or resistance.

Inflammatory signals induce feedback and feedforward systems that provide temporal control. Although glucocorticoids can repress inflammatory gene expression, glucocorticoid receptor recruitment increases expression of negative feedback and feedforward regulators, including the phosphatase, DUSP1, the ubiquitin-modifying enzyme, TNFAIP3, or the mRNA-destabilizing protein, ZFP36. Moreover, glucocorticoid receptor cooperativity with factors, including nuclear factor-κB (NF-κB), may enhance regulator expression to promote repression. Conversely, MAPKs, which are inhibited by glucocorticoids, provide feedforward control to limit expression of the transcription factor IRF1, and the chemokine, CXCL10. We propose that modulation of feedback and feedforward control can determine repression or resistance of inflammatory gene expression toglucocorticoid.

Negative Feed-forward Control of Tumor Necrosis Factor (TNF) by Tristetraprolin (ZFP36) Is Limited by the Mitogen-activated Protein Kinase Phosphatase, Dual-specificity Phosphatase 1 (DUSP1): IMPLICATIONS FOR REGULATION BY GLUCOCORTICOIDS.

TNF is central to inflammation and may play a role in the pathogenesis of asthma. The 3'-untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (also known as zinc finger protein 36 (ZFP36)), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial and pulmonary epithelial A549 cells, we confirm that interleukin-1ß (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36, and TNF. Whereas IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36 exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2 h and profoundly repressed TNF mRNA at 6 h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, whereas DUSP1 overexpression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6 h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, silencing of ZFP36, nor silencing of both together prevented the repression of IL1B-induced TNF expression, thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.

DUSP1 Maintains IRF1 and Leads to Increased Expression of IRF1-dependent Genes: A MECHANISM PROMOTING GLUCOCORTICOID INSENSITIVITY.

Although the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, mediates dexamethasone-induced repression of MAPKs, 14 of 46 interleukin-1ß (IL1B)-induced mRNAs were significantly enhanced by DUSP1 overexpression in pulmonary A549 cells. These include the interferon regulatory factor, IRF1, and the chemokine, CXCL10. Of these, DUSP1-enhanced mRNAs, 10 including CXCL10, were IRF1-dependent. MAPK inhibitors and DUSP1 overexpression prolonged IRF1 expression by elevating transcription and increasing IRF1 mRNA and protein stability. Conversely, DUSP1 silencing increased IL1B-induced MAPK phosphorylation while significantly reducing IRF1 protein expression at 4 h. This confirms a regulatory network whereby DUSP1 switches off MAPKs to maintain IRF1 expression. There was no repression of IRF1 expression by dexamethasone in primary human bronchial epithelial cells, and in A549 cells IL1B-induced IRF1 protein was only modestly and transiently repressed. Although dexamethasone did not repress IL1B-induced IRF1 protein expression at 4-6 h, silencing of IL1B plus dexamethasone-induced DUSP1 significantly reduced IRF1 expression. IL1B-induced expression of CXCL10 was largely insensitive to dexamethasone, whereas other DUSP1-enhanced, IRF1-dependent mRNAs showed various degrees of repression. With IL1B plus dexamethasone, CXCL10 expression was also IRF1-dependent, and expression was reduced by DUSP1 silencing. Thus, IL1B plus dexamethasone-induced DUSP1 maintains expression of IRF1 and the IRF1-dependent gene, CXCL10. This is supported by chromatin immunoprecipitation showing IRF1 recruitment to be essentially unaffected by dexamethasone at the CXCL10 promoter or at the promoters of more highly repressed IRF1-dependent genes. Since IRF1-dependent genes, such as CXCL10, are central to host defense, these data may help explain the reduced effectiveness of glucocorticoids during asthma exacerbations.

Roles for the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, in feedback control of inflammatory gene expression and repression by dexamethasone.

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1ß (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.

Induction of regulator of G-protein signaling 2 expression by long-acting ß2-adrenoceptor agonists and glucocorticoids in human airway epithelial cells.

In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting ß2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.

TNF mediates the sustained activation of Nrf2 in human monocytes.

Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.

Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation.

Interleukin-23 (IL-23) is important for T helper type 17 (Th17) responses and strategies to regulate IL-23 in human dendritic cells (DC) are limited. This study describes a novel means to control IL-23 secretion by conditioning DC with a phosphatidyl inositol 3-kinase inhibitor Wortmannin (WM). Treatment of monocyte-derived DC with WM increased Toll-like receptor (TLR) -dependent IL-23 secretion 10-fold and IL-12p70 twofold, but IL-27 was unaffected. The effect of WM was restricted to TLR3/4 pathways, did not occur through TLR2, TLR7/8 or Dectin-1, and was characterized by increased p19, p35 and p40 transcription. These responses were not solely dependent on phosphatidyl inositol 3-kinase as the alternative inhibitor LY294002 did not modulate IL-23 production. The normal patterns of activation of mitogen-activated protein kinase pathways were unaffected by WM-conditioning but IL-23 secretion required p38, ERK and JNK pathways. Importantly, this effect was manifest in populations of blood DC. Conditioning freshly isolated myeloid DC with WM before TLR3 or TLR4 triggering resulted in high levels of IL-23 secretion and an absence of IL-12p70. These WM-conditioned myeloid DC were highly effective at priming Th17 responses from naive CD4(+) T cells. Our findings provide a novel means to generate IL-23-rich environments and Th17 responses and suggest as yet unidentified regulatory factors, identification of which will provide new approaches to control IL-23-dependent immunity in infectious disease, autoimmunity and malignancy.

Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells.

BACKGROUND: Glucocorticoids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and, as inhaled corticosteroids (ICS), are the cornerstone of treatment for asthma. However, reduced efficacy in severe disease or exacerbations indicates a need to improve ICS actions. METHODS: Glucocorticoid-driven transcriptomes were compared using PrimeView microarrays between primary human bronchial epithelial (HBE) cells and the model cell lines, pulmonary type II A549 and bronchial epithelial BEAS-2B cells. RESULTS: In BEAS-2B cells, budesonide induced (≥2-fold, P ≤ 0.05) or, in a more delayed fashion, repressed (≤0.5-fold, P ≤ 0.05) the expression of 63, 133, 240, and 257 or 15, 56, 236, and 344 mRNAs at 1, 2, 6, and 18 h, respectively. Within the early-induced mRNAs were multiple transcriptional activators and repressors, thereby providing mechanisms for the subsequent modulation of gene expression. Using the above criteria, 17 (BCL6, BIRC3, CEBPD, ERRFI1, FBXL16, FKBP5, GADD45B, IRS2, KLF9, PDK4, PER1, RGCC, RGS2, SEC14L2, SLC16A12, TFCP2L1, TSC22D3) induced and 8 (ARL4C, FLRT2, IER3, IL11, PLAUR, SEMA3A, SLC4A7, SOX9) repressed mRNAs were common between A549, BEAS-2B and HBE cells at 6 h. As absolute gene expression change showed greater commonality, lowering the cut-off (≥1.25 or ≤ 0.8-fold) within these groups produced 93 induced and 82 repressed genes in common. Since large changes in few mRNAs and/or small changes in many mRNAs may drive function, gene ontology (GO)/pathway analyses were performed using both stringency criteria. Budesonide-induced genes showed GO term enrichment for positive and negative regulation of transcription, signaling, proliferation, apoptosis, and movement, as well as FOXO and PI3K-Akt signaling pathways. Repressed genes were enriched for inflammatory signaling pathways (TNF, NF-κB) and GO terms for cytokine activity, chemotaxis and cell signaling. Reduced growth factor expression and effects on proliferation and apoptosis were highlighted. CONCLUSIONS: While glucocorticoids repress mRNAs associated with inflammation, prior induction of transcriptional activators and repressors may explain longer-term responses to these agents. Furthermore, positive and negative effects on signaling, proliferation, migration and apoptosis were revealed. Since many such gene expression changes occurred in human airways post-ICS inhalation, the effects observed in cell lines and primary HBE cells in vitro may be relevant to ICS in vivo.

An inhaled dose of budesonide induces genes involved in transcription and signaling in the human airways: enhancement of anti- and proinflammatory effector genes.

Although inhaled glucocorticoids, or corticosteroids (ICS), are generally effective in asthma, understanding their anti-inflammatory actions in vivo remains incomplete. To characterize glucocorticoid-induced modulation of gene expression in the human airways, we performed a randomized placebo-controlled crossover study in healthy male volunteers. Six hours after placebo or budesonide inhalation, whole blood, bronchial brushings, and endobronchial biopsies were collected. Microarray analysis of biopsy RNA, using stringent (≥2-fold, 5% false discovery rate) or less stringent (≥1.25-fold, P ≤ 0.05) criteria, identified 46 and 588 budesonide-induced genes, respectively. Approximately two third of these genes are transcriptional regulators (KLF9, PER1, TSC22D3, ZBTB16), receptors (CD163, CNR1, CXCR4, LIFR, TLR2), or signaling genes (DUSP1, NFKBIA, RGS1, RGS2, ZFP36). Listed genes were qPCR verified. Expression of anti-inflammatory and other potentially beneficial genes is therefore confirmed and consistent with gene ontology (GO) terms for negative regulation of transcription and gene expression. However, GO terms for transcription, signaling, metabolism, proliferation, inflammatory responses, and cell movement were also associated with the budesonide-induced genes. The most enriched functional cluster indicates positive regulation of proliferation, locomotion, movement, and migration. Moreover, comparison with the budesonide-induced expression profile in primary human airway epithelial cells shows considerable cell type specificity. In conclusion, increased expression of multiple genes, including the transcriptional repressor, ZBTB16, that reduce inflammatory signaling and gene expression, occurs in the airways and blood and may contribute to the therapeutic efficacy of ICS. This provides a previously lacking insight into the in vivo effects of ICS and should promote strategies to improve glucocorticoid efficacy in inflammatory diseases.

Cytokine-induced loss of glucocorticoid function: effect of kinase inhibitors, long-acting ß(2)-adrenoceptor [corrected] agonist and glucocorticoid receptor ligands.

Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2 × glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1ß inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally, formoterol-enhanced 2×GRE reporter activity was also proportional to agonist efficacy and functionally reversed repression by TNF. As similar effects were apparent on glucocorticoid-induced gene expression, the most effective strategy to overcome glucocorticoid resistance in this model was addition of formoterol to high efficacy NR3C1 agonists.

Role of mitogen-activated protein kinase and PI3K pathways in the regulation of IL-12-family cytokines in dendritic cells and the generation of T H-responses.

Mitogen-activated protein kinases (MAPK) are targets for the immune-modulation of dendritic cells (DC). However, our knowledge of their role in the regulation of IL-12-family cytokines is limited. This study investigated the roles of p38, JNK, p44/42 and PI3K pathways in IL-12/23/27 production by human DC, and their impact on naïve T(H)-responses. We first identified TOP and UBC as robust DC housekeeping genes. Peak transcription of p35 and p40 occurred by 12h, p19 and p28 by 8h and EBI3 by 12-24h. Using selective antagonists, we showed that p38 was a positive regulator of IL-12, 23 and 27, JNK positively regulated IL-12 and IL-27, and inhibition of MEK1/2 had no marked effect. In contrast, the PI3K pathway markedly attenuated IL-23 responses and, to a lesser extent, IL-12, but not IL-27. To identify the role of these soluble factors, we co-stimulated naïve CD4+ T-cells in the presence of DC supernatant. The presence of mature DC supernatant induced not only strong IFNγ responses, but also IL-10 and IL-17A. Inhibition of p38 ablated T(H1), and IL-10 and IL-17A responses, whilst modestly enhancing IL-5 secretion. In contrast, inhibition of MEK1/2 abolished IL-17A production, whilst leaving other responses unaffected, whereas inhibition of JNK or PI3K had no discernable effect. In summary, we describe the expression of IL-12-family cytokines from DC and propose a modified model for their regulation. This study further clarifies the potential for therapeutic modulation through these mediators.