Anna Miller-Larsson

Altered lung function relates to inflammation in an acute LPS mouse model.

Preclinical in vivo models of lipopolysaccharide (LPS) -induced acute lung injury are commonly used to recapitulate pathophysiological features of chronic obstructive pulmonary disease and acute exacerbations. The LPS-induced lung inflammation is well described; however, whether the inflammatory response relates temporally to specific alterations in lung function has not been elucidated. We have investigated the effects of acute LPS inhalation in mice up to 96 h post LPS. Quantitation of inflammatory cells and inflammatory mediators in bronchoalveolar lavage fluid and non-invasive and invasive lung function measurements were performed at corresponding time points. The inhibitory effect of the glucocorticoid, budesonide, on LPS-induced lung inflammation and lung function was determined. LPS inhalation induced distinct histopathological changes, and infiltration of inflammatory cells to the lungs peaked at 48 h. At this time point, significantly increased inflammatory mediators and significantly altered lung capacity and mechanics parameters were observed. Budesonide given per os prevented the LPS-induced lung inflammation and lung dysfunction. These results demonstrate a temporal relationship between the peak of inflammatory cell influx and significant impairment of lung function, suggestive of a causative role of inflammation. These results allow better understanding of the functional consequences of lung inflammation in respiratory diseases.

Increased corticosteroid sensitivity by a long acting β2 agonist formoterol via β2 adrenoceptor independent protein phosphatase 2A activation.

Long-acting β2-adrenoceptor agonists (LABAs) are reported to enhance anti-inflammatory effects of corticosteroids in vitro and in vivo, although the molecular mechanisms have not yet been elucidated. We investigated the role of serine/threonine protein phosphatase 2A (PP2A) on regulation of corticosteroid sensitivity via inhibition of glucocorticoid receptor (GR) phosphorylation as the target of formoterol, an LABA. Corticosteroid sensitivity was determined as IC50 to dexamethasone (Dex) on TNFα-induced IL-8 release in a U937 monocytic cell line (Dex-IC50). Phosphorylation levels of GR-Ser226 and c-Jun N-terminal kinase (JNK) were determined by western-blotting. Phosphatase activity of immunopurified PP2A was measured by fluorescence-based assay. Exposure to IL-2/IL-4 for 48 h decreased Dex sensitivity with a concomitant increase of GR phosphorylation at Ser226 with JNK1 activation. Formoterol restored Dex sensitivity by inhibiting phosphorylation of GR-Ser226 and JNK1. PP2A inhibition by okadaic acid, a phosphatase inhibitor, abrogated formoterol-mediated effects. In addition, formoterol enhanced PP2A activity in intact or IL-2/IL-4 treated U937 cells and human peripheral blood mononuclear cells. In addition, PP2A activation by formoterol was not antagonized by ICI-118551, and formoterol could activate PP2A directly in cell free system. Taken together, formoterol increases corticosteroid sensitivity via activation of PP2A in receptor independent manner, explaining its benefits as add-on therapy for the treatment of corticosteroid-insensitive diseases, such as severe asthma.

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses

BackgroundCombination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.MethodsTo test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×106 plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.ResultsRV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.ConclusionsBudesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

The role of intracellular esterification in budesonide once-daily dosing and airway selectivity

BACKGROUND Since their introduction in the 1970s, inhaled corticosteroids (ICSs) have been used to control airway inflammation associated with asthma. Budesonide is one of the ICSs recommended as first-line therapy for mild to moderate persistent asthma. OBJECTIVE This article describes the esterification of budesonide and how it results in prolonged, location-specific retention of drug in the airways, allowing once-daily dosing. RESULTS Studies conducted over the past decade have shown that budesonide forms reversible fatty acid esters within the cells of airway tissue, resulting in the formation of an intracellular depot pool of inactive drug. As the intracellular concentration of free budesonide decreases, these budesonide esters are hydrolyzed back to their active state. This process increases budesonides retention in the airways, prolongs its duration of action, and lowers the risk of systemic effects. CONCLUSIONS By extending budesonides local anti-inflammatory effect and increasing its airway selectivity, the esterification process appears to contribute to the drugs efficacy, particularly during once-daily administration. Reducing the number of required daily inhalations may increase patient compliance with asthma therapy, although this remains to be evaluated.

TGFβ-induced matrix production by bronchial fibroblasts in asthma: Budesonide and formoterol effects.

To investigate the mechanisms of enhanced airway deposition of subepithelial collagen in asthma and its sensitivity to drug therapy with combination of an inhaled glucocorticosteroid (GC) and a long-acting β(2)-agonist (LABA), a cell model system involving bronchial fibroblasts derived from biopsies from patients with stable mild-to-moderate asthma has been used. To mimic unstable conditions and severe asthma, fibroblasts were stimulated ex vivo with TGFβ1. Primary fibroblasts established from central bronchial biopsies from 8 asthmatic patients were incubated for 24 h with 0.4% serum or TGFβ1 (10 ng/ml) with/without the GC budesonide (BUD; 10 nM) and/or the LABA formoterol (FORM; 0.1 nM). Procollagen peptide I (PICP), metalloproteinase (MMP)-1 and tissue inhibitor of MMPs (TIMP-1) were determined in culture media using ELISA while the activity of MMP-2, -3, -9 by zymography. Metabolically labeled proteoglycans, biglycan and decorin, associated with collagen fibrillation/deposition, were separated using chromatography and SDS-PAGE. The levels of PICP and biglycan were increased 2-fold by TGFβ1 (p < 0.05). The BUD and FORM combination reduced the PICP increase by 58% (p < 0.01) and the biglycan by 36% (p < 0.05) while each drug alone had no effect. Decorin levels were reduced by TGFβ1 in fibroblasts of most patients; BUD alone and BUD and FORM completely counteracted this decrease. MMPs and TIMP-1 were not affected by TGFβ1 or the drugs. These results suggest that BUD and FORM combination therapy, without affecting metalloproteolytic balance, has a potential to counteract enhanced collagen production by bronchial fibroblasts in asthma and to normalize the production of small proteoglycans which may affect collagen fibrillation and deposition.

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 β2-Adrenoceptor 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-1β 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.

Differences in Endogenous Esterification and Retention in the Rat Trachea between Budesonide and Ciclesonide Active Metabolite

The airway retention of inhaled glucocorticosteroids (GCs) depends largely on their lipophilicity. Inhaled budesonide (BUD) becomes highly lipophilic reversibly by the formation of esters acting as a reservoir of active BUD. Ciclesonide (CIC) was also reported to form esters after hydrolysis to active metabolite (CIC-AM). We have investigated lipophilicity and airway retention of BUD, CIC/CIC-AM, fluticasone propionate (FP), and mometasone furoate (MF), and compared esterification of BUD and CIC-AM and its contribution to GC airway retention. Rat tracheas were preincubated with the esterification inhibitor cyclandelate or vehicle. A 3H-GC (∼10-7 M: BUD, CIC, CIC-AM, FP, MF) was added for 20 min. After incubation, one half of the trachea was used for analysis of GC uptake and the other to analyze GC release during 3 h in drug-free medium. GC species in trachea halves were analyzed by radiochromatography. At 20 min, the uptake of BUD was similar to that of CIC/CIC-AM; however, the BUD-ester pool was 9-fold greater (p < 0.01). BUD overall retention in trachea at 3 h was greater than that of other GCs (p < 0.01), and the BUD-ester pool was 3-fold greater than the CIC-AM-ester pool (p < 0.01). Cyclandelate decreased the initial BUD- and CIC-AM-ester pools (p < 0.01), and reduced the overall retention of BUD at 3 h (p < 0.01) but not of CIC-AM. Thus, BUD becomes esterified in the airways more promptly and to a greater extent than CIC-AM, and BUD esterification prolongs BUD airway retention. In contrast, airway retention of CIC-AM and CIC seems to be determined mainly by their lipophilicity, similar to FP and MF, which are not esterified.

Modulation of transcriptional responses by poly(I:C) and human rhinovirus: Effect of long-acting β2-adrenoceptor agonists

Exacerbations of asthma, a chronic inflammatory respiratory disease, are associated with viral upper respiratory tract infections involving human rhinovirus. Although glucocorticoids (corticosteroids) effectively control airways inflammation in many asthmatics, human rhinovirus-associated exacerbations show reduced glucocorticoid responsiveness. Using human bronchial epithelial BEAS-2B cells, we show that human rhinovirus reduced glucocorticoid-inducible activation of glucocorticoid response element (GRE) reporter systems in a time- and concentration-dependent manner. The synthetic double-stranded viral RNA mimetic, polyinosinic:polycytidylic acid (poly(I:C)), also reduced activation of GRE reporter systems in BEAS-2B and pulmonary A549 cells. In addition, poly(I:C) decreased transcription from cAMP response element (CRE)-, TATA-, simian virus 40- and nuclear factor-kappa B (NF-κB)-dependent reporter systems. The effects of poly(I:C) on GRE-reporter activation were countered by the long-acting β2-adrenoceptor agonists, formoterol and salmeterol. Likewise, increased expression of the gene cyclin-dependent kinase inhibitor 1C (CDKN1C; p57(KIP2)) by dexamethasone was reduced by poly(I:C), but was substantially enhanced by the addition of formoterol. Poly(I:C) induced the expression of interleukin-8 (IL8; CXCL8) and this was significantly decreased by dexamethasone, formoterol or their combination. This confirms that not all transcriptional responses were attenuated by poly(I:C) and that decreased glucocorticoid-dependent transcription can be counteracted by the addition of long-acting β2-adrenoceptor agonists. These data show how human rhinovirus may attenuate glucocorticoid-induced transcription to reduce anti-inflammatory activity. However, addition of long-acting β2-adrenoceptor agonist to the glucocorticoid functionally restored this response and shows how glucocorticoid plus long-acting β2-adrenoceptor agonist combinations may prove beneficial during virus-induced exacerbations of asthma.

Pharmacological modulation of the bradykinin-induced differentiation of human lung fibroblasts: Effects of budesonide and formoterol

Objective. Bradykinin (BK) induces differentiation of lung fibroblasts into myofibroblasts, which play an important role in extracellular matrix remodeling in the airways of asthmatic patients. It is unclear whether this process is affected by antiasthma therapies. Here, we evaluated whether a glucocorticoid, budesonide (BUD), and a long-acting β2-agonist, formoterol (FM), either alone or in combination, modified BK-induced lung fibroblast differentiation, and affected the BK-activated intracellular signaling pathways. Methods. Human fetal lung fibroblasts were incubated with BUD (0.001–0.1 μM) and/or FM (0.0001–0.1 μM) before exposure to BK (0.1 or 1 μM). Fibroblast differentiation into α-smooth-muscle-actin-positive (α-SMA+) myofibroblasts, BK2 receptor (B2R) expression, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation (p-ERK1/2), intracellular Ca2+ concentration ([Ca2+]i), and p65 nuclear factor kappa B translocation were evaluated. Results. BUD (0.1 μM) and FM (0.1 μM), either alone or in combination, completely inhibited BK-induced α-SMA protein expression and decreased the numbers of α-SMA+ fibroblasts, with a clear reduction in α-SMA stress fibers organization. BUD also completely inhibited the increase of B2R, whereas FM with or without BUD had no effect. BK-induced increases of [Ca2+]i and p-ERK1/2 were significantly reduced to similar levels by BUD and FM, either alone or in combination, whereas p65 translocation was completely inhibited by all treatments. Conclusion. Both BUD and FM, either alone or in combination, effectively inhibited the BK-induced differentiation of fibroblasts into α-SMA+ myofibroblasts and the intracellular signaling pathways involved in fibroblast activation. These results suggest that BUD and FM combination therapy has potential to inhibit fibroblast-dependent matrix remodeling in the airways of asthmatic patients.