International Immunopharmacology 19 (2014) 153–160

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Fluticasone furoate is more effective than mometasone furoate in restoring tobacco smoke inhibited SOCS-3 expression in airway epithelial cells Najmunnisa Nasreen a,b, Lixandra Gonzalves a, Sriram Peruvemba a,b, Kamal A. Mohammed a,b,⁎ a b

Division of Pulmonary Critical Care & Sleep Medicine, College of Medicine, University of Florida, United States NF/SGVHS, Malcom Randal VA Medical Center, Gainesville, FL, United States

a r t i c l e

i n f o

Article history: Received 21 July 2013 Received in revised form 20 December 2013 Accepted 30 December 2013 Available online 13 January 2014 Keywords: Bronchial airway epithelium COPD Tobacco smoke SOCS3 Fluticasone furoate Mometasone furoate

a b s t r a c t Fluticasone furoate (FF) and mometasone furoate (MF) are potent glucocorticoids recommended for the treatment of allergic rhinitis and other inflammatory diseases. However, whether these drugs render any antiinflammatory effects in Chronic Obstructive Pulmonary Disease (COPD) is unclear. Emerging data on suppressors of cytokine signaling-3 (SOCS-3) activation in the lungs during inflammation suggests that SOCS3 can be potential targets for regulating pulmonary inflammatory responses in COPD. In this study, we compared the effect of FF with MF on SOCS-3 expression in tobacco smoke (TS) exposed BAEpCs in vitro and in a mouse model of COPD in vivo. BAEpCs were exposed to TS or room air and later were treated with either FF (1 nmol–100 nmol) or MF (10–500 nmol) inhibitors in the presence and absence of Jak1 and Stat-3 inhibitors. C57BL/6 mice were exposed to TS for 6 months, and treated with either FF, MF for 2 and 4 weeks. FF induced 7 fold increases in SOCS-3 expression in BAEpCs whereas MF induced a three fold increase when compared to control. Jak1 and Stat-3 inhibitors significantly inhibited the FF and MF induced SOCS-3 expression in BAEpCs. In addition, FF and MF restored TS inhibited SOCS-3 expression in the airway epithelium of COPD mice. FF and MF treatments significantly reduced leukocyte infiltration in airways and inhibited lung inflammation. Our study elucidates a novel mechanism for the anti-inflammatory action of FF in COPD. The superior efficacy of FF may be in part due to the increased expression of SOCS-3 in BAEpCs. Published by Elsevier B.V.

1. Introduction Chronic obstructive pulmonary disease (COPD) is a progressive disease and currently it is the fourth leading cause of morbidity and mortality in United States [7]. About one quarter million Americans die each year due to COPD [19]. Airway hyper-responsiveness is a common feature of COPD, and cigarette smoking is the leading cause linked to the development of COPD [26]. In addition, long-term exposure of other lung irritants such as air-pollution, dust and chemical fumes are also known to contribute to COPD. COPD is characterized by destruction of lung and loss of alveolar cells secondary to airway inflammation [35]. The peripheral airways are the major site of airway obstruction in COPD [29]. Airway inflammation is associated with epithelial cell damage, enlarged sub-mucosal mucus-secreting glands, and an increase in the amount of smooth muscle and connective tissue in the airway wall. In recent years, corticosteroids such as FF and MF have become potential drugs to control the chronic inflammatory response characterized by

⁎ Corresponding author at: Division of Pulmonary, Critical Care & Sleep Medicine, Department of Medicine, University of Florida, P.O. Box 100225, Gainesville, FL, United States. Fax: + 1 352 392 7088. E-mail address: [email protected]fl.edu (K.A. Mohammed). 1567-5769/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.intimp.2013.12.029

asthma and chronic obstructive lung disease by modulation of AP-1 and F signaling pathway [6,15,28]. In addition members of signal transducer and activator of transcription (STAT) family members are involved in mediating the anti-inflammatory signals induced by these corticosteroids [3,13]. Attempts have been made to compare the efficacy of FF and MF by in vitro studies by measuring cellular activities and inflammatory markers [34,37]. In this study the efficacy of FF and MF was determined by evaluating the expression of SOCS-3, a master regulator of cytokine signaling network, and inflammatory signatures in BAEpCs and in a mouse model of COPD. Stat-3 as a transcription factor participates in the signaling pathways for many cytokines in various cells and organs that are regulated by the suppressor of cytokine signaling (SOCS) family, including SOCS-3. Recently, data on the activation and function of Stat-3 and SOCS-3 in the lung during acute inflammatory response are emerging, suggesting that these molecules can be potential targets for regulating pulmonary inflammatory responses. The Janus kinase (Jak) signaling pathway, which is activated in response to a variety of cytokines is regulated by SOCS-3, a feedback inhibitor of cytokine signaling [16]. SOCS proteins have been found to play a prominent role in T helper-2 cell mediated responses. In addition, forced expression of SOCS-3 down-regulates a variety of cytokine signaling pathways. SOCS-3 can be positively induced through Stat signaling [33]. Moreover, the expression of SOCS-3 can be induced by different mechanisms, particularly of extra-

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cellular signal-regulated kinase (Erk/1/2) and p38 mitogen-activated protein kinase (MAPK) [4,9]. The central role of SOCS-3 in modulation of inflammation is critical owing to its broad spectrum of singling events. SOCS-3 expression has been shown to have a beneficial role in attenuating inflammatory responses in various diseases [14,32]. However, the role of SOCS-3 in TS induced airway inflammation is not clear. We have established a mouse model of COPD that closely mimics human disease by exposing mice to TS [20]. In this mouse model of COPD we noticed increased airway resistance, decline in the lung tissue elastance (Htis), and increased airway remodeling all typical features associated with COPD. These physiological and morphological changes in lung architecture are accompanied with increased airway inflammation a typical feature of COPD. In a recent study we reported that TS exposure inhibits SOCS-3 expression in BAEpCs in vitro [22]. FF and MF are prescribed to inhibit the airway inflammation in acute inflammatory disorders. However, whether FF and MF induce the expression of SOCS-3 in BAEpC in COPD is not known. Therefore, in this study we determined the effect of FF and MF on the expression of SOCS-3 in BAEpC in vitro and in mouse model of COPD in vivo. We focused our studies to understand the role of Jak1/Stat-3 signaling pathway in TS mediated airway inflammation in COPD. 2. Materials and methods 2.1. Reagents Anti-SOCS-3 antibody (Santa Cruz Biotechnology, CA), Jak-1, Stat-3, Phospho Jak-1, phospho Stat-3 and SOCS-3 antibodies were purchased from Cell Signaling (Beverly, CA); fluticasone furoate received from GlaxoSmithKline, Durham, DL, UK. The inhibitors for Jak-1 and Stat-3 were purchased from Calbiochem (EMD Chemicals, Inc.). All the other reagents were purchased from Sigma-Aldrich unless otherwise indicated.

SOCS-3, Stat-3, phospho-Stat-3, Jak-1 and phospho-Jak-1 were detected using anti-rabbit SOCS-3, anti-rabbit Stat-3, anti-rabbit phospho-Stat-3, anti-rabbit Jak-1 and anti-rabbit phospho-Jak-1, respectively as reported earlier [22,23]. In brief, cells were lysed in RIPA (radioimmunoprecipitation assay) buffer 50 mM Tris–HCl, pH 8.0 with 150 mM sodium chloride, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. Protein concentration was measured as reported earlier [24] using Pierce's BCA™ Protein Assay Kit (PIERCE, Rockford, IL). Typically, 20 μg of total protein was resolved on 4%–20% Tris glycine SDS-PAGE gels (Bio-Rad Hercules, CA). Proteins were transferred to PVDF membranes, blocked, and incubated with respective antibodies at 1:1000 dilutions at 4 °C overnight. The blots were washed three times, and incubated with goat anti-rabbit secondary antibodies at 1:2000 dilutions for 1 h at room temperature. Proteins were detected by enhanced Immuno-Star™ HRP Chemiluminescent Kit (Bio-Rad, Hercules, CA). 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) BAEpCs were exposed to TS with and without FF or MF for indicated period of time and total RNA was extracted. SOCS-3, mRNA expression was evaluated by quantitative-PCR analysis using specific primers as reported earlier [22]. Total cellular RNA was isolated from BAEpCs after TS exposure using RNeasy kit (QIAGEN, Valencia CA) according to the manufacturer's recommendations as reported earlier [6]. 100 ng/μl, of RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase and oligo (dT). The SYBR Green JumpStart Taq Ready Mix™ was used to perform PCR as reported earlier [23]. PCR amplification consisted of 40 cycles (95 °C for 15 s, 60 °C for 1 min and 72 °C for 45 s) after the initial denaturation step (95 °C for 2 min). The gene expression level was based on the amount of the target message relative to the h18S RNA. 2.5. COPD mouse model

2.2. BAEpC culture and FF/MF drug exposure in vitro Primary cultures of human bronchial airway epithelial cells (BAEpCs) were obtained from Cell Applications (San Diego, CA), and were cultured in BEGM as reported earlier [21]. Culture dishes containing cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. When the cells reached confluence they were trypsinized and sub-cultured into vitrogen-coated dishes. Third-passage cultures were stored in liquid nitrogen. Cells of passages 3–6 were used in this project. cells were routinely checked with anti-human cytokeratin antibody and anti-vimentin antibody for purity. BAEpC in culture were keratin positive and vimentin negative. Depending upon the experiments, BAEpCs (0.5 × 106 cells/60 mm dish for RT-PCR analysis or 1 × 106 cells/100 mm dish for Western blot) were plated. The cell cultures were treated with different concentrations of either FF (1 nmol–100 nmol) or MF (10–500 nmol) and cells were harvested over time. Some parallel culture dishes were exposed to either 2-exposure units (2EU) or 4EU or TS or room air as reported earlier [22]. In this TS exposure protocol, one TS 1EU was defined as 15-minute TS exposure plus 45-minute incubation in 5% CO2 at 37° for recovery (a total of 1 h). Subsequently BAEpCs were treated with different concentrations of either FF or MF and SOCS-3 expression was determined. Cell lysate was prepared for Western blot analysis. Total RNA was isolated for quantitative RT-PCR analysis. In order to determine if the expression of SOCS-3 is dependent on or independent of Jak1/Stat signal transduction pathway in BAEpC, some of the cultures were pretreated with the Jak1/Stat-3 inhibitors prior to FF and MF treatments. 2.3. Western blot analysis BAEpCs cultured in 60 mm culture dishes (Corning, Tewksbury, MA) were exposed to varying concentrations of TS and the expressions of

We have established a COPD animal model by exposing mice to TS. The animal protocol was reviewed and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). Eight week old C57BL/6 mice were individually exposed to mainstream (smoke that comes out from unlighted-end of cigarette due to suction) and side stream smoke (smoke that spontaneously comes out from lighted-end of the cigarette). Mice were exposed to smoke of 6 cigarettes (2R4F reference cigarettes, Tobacco and Health Research Institute, University of Kentucky) using a nose only inhalation system (CH-Technologies, NJ) 5 days/week for 6 months. In order to study the effect of corticosteroids on airway inflammation and on SOCS-3 expression in COPD, mice after 6 month TS exposure were allowed either to inhale 30 μg of FF (GlaxoSmithKline, Stockley Park, UK) or 30 μg of MF (US Pharmacopeia, Rockville, MD) each separately for 2 and 4 weeks. Mice were euthanized and the lungs were perfused via pulmonary artery with heparinized normal saline. The lungs were inflated with 0.5% soft agar at 30 cm gravity, harvested and either flash frozen or preserved in 4% paraformaldehyde. SOCS-3 expression in the airway epithelium was detected by quantitative-PCR and immunohistochemistry as reported earlier [22]. 2.6. Histochemical immuno-staining of SOCS-3 in lung tissues The lung sections were immuno-stained with avidin–biotin conjugate and peroxidase as described earlier [25]. In brief, after euthanasia of mice, the harvested lungs were processed by treating with 4% paraformaldehyde, embedded in Paraffin blocks and 5 μm thick sections were cut. The sections were treated in 3 changes of xylene and ethanol. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in PBS. The sections were rinsed 3 times in PBS and were blocked with 1% normal horse serum for 30 min to

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reduce nonspecific binding. They were incubated for 30 min in the presence of rabbit anti-SOCS-3 antibody (at 1:500 dilution in buffer). The negative controls were incubated in the presence of normal rabbit IgG (isotype) antibody. The sections were again rinsed with PBS 3 times, then incubated for 40 min with avidin–biotin-conjugated goat anti-rabbit IgG (VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA). The lung sections were washed in PBS and incubated for 5 min in peroxidase substrate (DAB substrate for peroxidase; Vector Laboratories), rinsed in PBS, and counterstained with Mayer's hematoxylin (Sigma, St Louis). The cells were dehydrated in graded alcohol solutions and xylene, and then mounted in Permount (Fisher, Fair Lawn, NJ). The images were analyzed by light microscopy (Nikon inverted microscope). 2.7. Broncho alveolar lavage Mice were euthanized by inhalation of 5% isoflurane following NF/SG VHA IACUC approved procedures. After euthanasia, tracheal cannula was inserted, bronchoalveolar lavage (BAL) fluid was harvested from all groups of mice by introducing 1 ml of phosphate buffered saline and gentle manual aspiration three times. BAL fluids were centrifuged and cells were harvested. Total cell counts were determined using a hemocytometer. In order to determine the differential leukocyte counts, cytospin slides were prepared. Differential cell counts on 1000 cells were performed on cytocentrifuged preparations using standard morphologic criteria after May–Grünwald–Giemsa staining as reported earlier [5]. The data is expressed as percent population by rounding to the nearest whole number.

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(Fig. 1A). The expression of SOCS-3 was also determined in BAEpCs treated with MF (10, 25, 50, 100, 250 and 500 nM) after 4 h and 24 h. MF induced SOCS-3 expression in BAEpCs in a concentration and time dependent manner. The lower concentration (10 nM) MF did not show any significant increase in SOCS-3 expression. At 50 nM concentration MF induced three fold increases in SOCS-3 expression when compared to control cells (Fig. 1B). By contrast, at higher concentrations (100 nM, 250 nM, and 500 nM), MF turned to be less effective on SOCS-3 induction in BAEpCs. Perhaps both FF and MF at higher concentrations may be cytotoxic to BAEpCs. The concentration dependent SOCS-3 expression was also confirmed by Western blot analysis. The translational expression of SOCS-3 at 10 nM and 25 nM concentrations of FF showed significant increases whereas at 50 nM and 100 nM the SOCS-3 expression decreased when compared with control (Fig. 1C). MF induced SOCS-3 expression in BAEpCs was weaker when compared to FF induced response (Fig. 1D). These results demonstrate the superior efficacy of FF over MF as it relates to the expression of the master anti-inflammatory protein, the SOCS-3 in BAEpCs. These data indicate that both FF and MF are capable of inducing SOCS-3

2.8. Lung histology The lungs were perfused via pulmonary artery with heparinized normal saline. The lungs were inflated with 0.5% soft agar at 30 cm gravity, harvested and treated in 4% paraformaldehyde for 24 h and transferred into 70% ethyl alcohol, processed for histology. Left lung lobe was embedded in paraffin and 5-μm transverse sections were cut. Lung tissue samples were stained with H&E and examined by light microscopy. The photomicrographs were obtained using SPOTcamera on a Nikon inverted microscope. 2.9. Statistical analysis The statistical analyses were performed by using SigmaStat 3.5 (SYSTAT Software, Inc., San Jose, CA). Comparisons between the groups were made using Kruskal–Wallis one-way analysis of variance by ranks. The significance of difference between the 2 groups was tested by using the all-pairwise multiple comparisons (Student–Newman– Keuls method). Differences were considered significant when P values were b 0.05. Results were expressed as mean ± SEM. 3. Results 3.1. FF and MF induce SOCS-3 expression in BAEpCs in a concentration dependent manner BAEpCs were treated with varying concentrations of FF (1, 5, 10, 25, 50 and 100 nM) and MF (10, 25, 50, 100, 250 and 500 nM) for 4 h and 24 h. At these concentrations neither FF nor MF caused any cytotoxicity in BAEpCs. The expression of SOCS-3 was determined by quantitative real time PCR analysis and Western blot analysis. FF induced SOCS-3 expression in BAEpCs in a concentration and time dependent manner. At lower concentrations (1 nM and 5 nM) FF induced marginal expression of SOCS-3 in BAEpCs. However, at 10 nM concentration FF induced a seven fold higher SOCS-3 expression when compared to control after 4 h. On the other hand, following 24 h the FF mediated expression of SOCS-3 decreased when compared to the response at 4 h

Fig. 1. FF- and MF induced SOCS-3 expression in BAEpCs. SOCS-3 mRNA expression in BAEpCs treated with varying concentrations of FF (Panel A) and MF (Panel B). SOCS-3 mRNA expression was determined by quantitative real time PCR analysis compared with 18 s RNA). Data presented is the mean ± SEM of three separate experiments. Statistical significance *p b 0.001 compared to respective control. Panels C and D: The SOCS-3 expression was determined by Western blot analysis and β-actin was detected to demonstrate equal sample loading. These are representative blots of three similar observations each performed at different times. The densitometry data presented is the mean ± SEM of three separate experiments. Statistical significance *p b 0.001 compared to respective control.

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expression in BAEpCs in vitro. Besides, among these two corticosteroids FF is more potent in SOCS-3 induction in BAEpCs compared to MF. 3.2. FF and MF induced Jak1 and Stat-3 phosphorylation in BAEpCs BAEpCs were treated with either FF or MF over time (10, 20, 30, 60 and 120 min) and Jak1, Stat-3 phosphorylation was determined by Western blot analysis (Fig. 2). FF induced relatively higher phosphorylation of Jak1 and Stat-3 which remained high up to 120 min when compared to untreated BAEpCs (Fig. 2A and C), whereas MF induced Stat-3 phosphorylation was noted up to 60 min and declined at 120 min in BAEpCs when compared to control. The MF induced phosphorylation of Jak1 was lower compared to FF (Fig. 2A and B). These data indicate that FF and MF induced SOCS-3 expression is mediated through Jak1/Stat-3 signaling pathways in BAEpCs. 3.3. FF mediated up-regulation of SOCS-3 expression in BAEpCs is dependent on Jak1 signaling pathway In order to determine the mechanisms whereby the FF and MF induce the expression of SOCS-3 in BAEpCs, BAEpCs were treated with Jak1 inhibitors before FF and MF treatment and the SOCS-3 expression was measured by q-PCR after 4 h. Some cell cultures were exposed to either room air or 2 EU of TS and subsequently treated with either FF or MF or left untreated. Our data indicate that room air exposed BAEpCs showed SOCS-3 expression whereas in TS exposed cells SOCS-3

expression was significantly inhibited. FF induced a seven fold increase whereas MF induced a three fold increase in SOCS-3 expression in BAEpCs (Fig. 3). Furthermore, pretreatment with Jak1 inhibitors significantly blocked FF and MF induced SOCS-3 expression in BAEpCs (Fig. 3AB). These data suggest that FF and MF mediated induction of SOCS-3 expression in BAEpCs is dependent on Jak1/Stat3 signaling pathway. 3.4. FF and MF mediated up-regulation of SOCS-3 expression in BAEpCs is dependent on Stat-3 signaling pathway In order to determine the mechanisms whereby FF and MF induce the expression of SOCS-3 in BAEpCs, inhibitor for Stat-3 was also used before treatment of BAEpCs with FF and MF. Our data indicate that FF and MF treatments significantly enhanced the SOCS-3 expression in BAEpCs and pretreatment with Stat-3 inhibitor significantly blocked FF- and MF induced SOCS-3 expressions in BAEpCs (Fig. 4A and B). These data indicate that FF- and MF mediated induction of SOCS-3 expression in BAEpCs is Stat-3 activation dependent. 3.5. FF and MF restore TS mediated inhibition of SOCS-3 expression in BAEpCs in vitro In order to determine if FF and MF restore TS inhibited SOCS-3 expression in BAEpCs, some cell cultures were exposed to either room air or 2EU of TS and subsequently treated with either FF or MF for 4 h or left untreated and SOCS-3 expression was determined by q-PCR

Fig. 2. FF- and MF activated Jak1 and Stat-3 signaling in BAEpCs. BAEpCs treated with FF and MF for 10 to 120 min and total, phosphorylated Jak-1 and Stat-3 expression were detected by Western blot analysis. Panels A and C: FF induced Jak-1 and Stat-3 activation in BAEpCs over time. Panels B and D: MF induced Jak-1 and Stat-3 activation in BAEpCs over time. β-actin was probed to demonstrate equal loading of the test samples. Data presented is a single representative of three similar separate experiments. Statistical significance *p b 0.001 compared to respective control.

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Fig. 3. FF- and MF induced SOCS-3 mRNA expressions in BAEpCs is Jak1 dependent. BAEpCs were exposed to various conditions for 4 h and SOCS-3 mRNA expression was measured by quantitative PCR analysis. Panel A: FF mediated SOCS-3 expression in BAEpCs; Panel B: MF induced SOCS-3 expression in BAEpCs. Data presented is mean ± SEM of three separate experiments. Statistical significance *p b 0.001 vs Control; $p b 0.001 vs FF; and ¥p b 0.05 vs MF.

analysis and Western Blot analysis. Our data indicate that resting BAEpC expressed low levels of SOCS-3 and TS exposure significantly down regulated SOCS-3 expression in BAEpCs (Fig. 5). However, FF and MF treatments restored TS inhibited SOCS-3 mRNA expression (Fig. 5A) as well as SOCS-3 protein expression (Fig. 5B) in BAEpCs. FF has relatively higher levels of SOCS-3 expression in BAEpCs when compared to MF. In addition FF restored the TS mediated suppression of SOCS-3 expression more effectively compared to MF. These data indicate that FF and MF treatments restore TS mediated inhibition of SOCS-3 expression in BAEpCs.

3.6. TS attenuates SOCS-3 expression and FF- and MF inhalation restores TS mediated inhibition of SOCS-3 expression in the lungs of mice with COPD In room air exposed mice (control mice) SOCS-3 expression significantly increased in the lungs and TS exposure significantly suppressed SOCS-3 expression. However, subsequent FF or MF inhalation for 2 and 4 weeks restored TS inhibited SOCS-3 expression in the lungs (Fig. 6A). FF- and MF induced SOCS-3 expression was time dependent. Maximum induction of SOCS-3 was noticed with FF following 4 weeks of inhalation. MF treatment induced relatively less SOCS-3 expression compared to FF induced response. The room air exposed mice bronchial airway epithelium stained densely positive for SOCS-3 expression whereas the TS exposed mice bronchial airway epithelium was negative for SOCS-3 expression (Fig. 6B). FF and MF treatments restored TS

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Fig. 4. FF- and MF induced SOCS-3 expression in BAEpCs is Stat-3 dependent. BAEpCs were exposed to various conditions for 4 h and SOCS-3 mRNA expression was measured by quantitative PCR analysis. Panel A: FF mediated SOCS-3 expression in BAEpCs; Panel B: MF induced SOCS-3 expression in BAEpCs. Data presented is mean ± SEM of three separate experiments. Statistical significance *p b 0.001 vs Control; $p b 0.001 vs FF and ¥p b 0.001 vs MF.

mediated inhibition of SOCS-3 expression in bronchial airway epithelium. Besides, in FF- and MF treated TS exposed group of mice the leukocyte infiltration and interstitial edema were significantly ameliorated compared to TS exposed mice. Among the FF- and MF treated mice, the bronchial airway epithelium was highly positive for SOCS-3 expression in FF treated mice compared to MF treated group. These data indicate that TS inhibits the SOCS-3 expression in bronchial airway epithelium and FF and MF treatments restore TS inhibited SOCS-3 expression in COPD. Besides, among these two corticosteroids FF is more effective in restoring SOCS-3 expression in mice with COPD compared to MF. In addition, the lungs in room air exposed mice were characterized by normal interstitial structure whereas in TS exposed mice, substantial inflammation, interstitial edema and leukocyte infiltration were noticed in the airways (Fig. 7). However, the airway inflammation was significantly lower in mice that inhaled MF and FF. In order to determine the anti-inflammatory effect of FF and MF in the lungs of COPD mice, bronchoalveolar lavage (BAL) fluids were collected and differential leukocyte counts were performed on cytospin slides. The BAL differential leukocyte counts were determined after Geimsa staining. The differential leukocyte counts data indicate that the COPD mice showed 7.2 × 105 inflammatory leukocytes in the BAL fluids similar to COPD patients. On the other hand, FF and MF treatments resulted in significant inhibition of inflammatory cell influx into lungs in mice with COPD (Table 1). In FF treated mice BAL fluid 3.65 leukocytes were noticed whereas in MF treated mice 5.4 × 105 leukocytes were present. Taken

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Fig. 5. FF and MF treatments restored TS inhibited SOCS-3 expression in BAEpCs. BAEpCs were exposed to 2EU of TS, subsequently treated with either FF or MF for 4 h and the SOCS-3 expression was measured. Panel A: SOCS-3 mRNA expression in BAEpCs as measured by quantitative PCR analysis. The PCR data presented is the mean ± SEM of three separate experiments; Panel B: SOCS-3 protein expression in BAEpCs as measured by Western blot analysis. This is a representative blot of three similar observations performed at different times. The densitometry data presented is the mean ± SEM of three blots. Statistical significance *p b 0.001 compared to respective Control and $p b 0.001 vs TS.

together these data indicate the greater anti-inflammatory efficacy of FF over MF. We also noticed similar inflammatory responses in the lung histology sections obtained from TS exposed and TS + FF, and TS + MF treated groups of mice (Fig. 7). Control mice had no inflammatory cells in airways whereas TS exposed mice showed high number of leukocytes in the airways. This inflammatory leukocyte infiltration significantly decreased in FF- and MF treated mice (Fig. 7). 4. Discussion Glucocorticoids are commonly used for the treatment of respiratory inflammatory disorders including COPD. FF is a novel glucocorticoid which mediates its effect through a glucocorticoid receptor. FF showed greater effect on several key pathways, such as NF-κB and inhibited the expression of pro-inflammatory cytokine TNF-α [31,39]. However, whether FF induced anti-inflammatory effects mediated through the SOCS-3 is unknown. In this study we report that FF and MF induces SOCS-3 expression in BAEpCs and restores TS inhibited SOCS-3 expression in BAEpCs both in vitro and in vivo. In addition, FF induced phosphorylation of Jak1, and Stat-3 in BAEpCs. FF induced SOCS-3 expression in BAEpCs is dependent on Jak1 and Stat-3 whereas MF induced SOCS-3 expression was Stat-3 dependent. TS inhibited SOCS-3 in bronchial airway epithelial cells both in vitro and in vivo. Besides, FF and MF were less effective on SOCS-3 induction in BAEpC in very low concentrations. Interestingly, the higher concentrations turned inhibitory on SOCS-3 induction in BAEpCs. The inhibitory effect noticed at

Fig. 6. SOCS-3 expression in the lungs of mice with COPD after FF and MF treatments. Panel A: SOCS-3 mRNA expression in mice lungs as measured by quantitative PCR analysis. Mice were exposed to TS for 6 months and subsequently treated with either FF or MF for 2 and 4 weeks. Data presented is the mean ± SEM of six animals in each group. Statistical significance *p b 0.001 compared with Control; $p b 0.001 compared with TS; and # p b 0.001 compared with FF; Panel B: SOCS-3 expression in bronchial airway epithelium in situ. Room air = Control mice; TS = mice exposed to TS for 6 months; TS + FF = mice exposed to TS for 6 months plus 4 weeks of FF inhalation; TS + MF = mice exposed to TS for 6 months plus 4 weeks of MF inhalation as described in methods. Brown color (arrows) in the tissues indicates SOCS-3 positive staining in the airway epithelium. Magnification 400×.

high concentration of drugs may be due to their cytotoxic effect. In TS exposed mice FF- and MF induced anti-inflammatory effects may be due to induction of SOCS-3 expression in the airway epithelium. FF induced several folds higher expression of SOCS-3 compared to MF and it was concentration dependent. In addition FF showed greater efficacy compared to MF in mice with COPD. Earlier studies demonstrated that FF acts faster when compared to other glucocorticoids by an enhanced affinity of the receptor interactions as determined by X-ray crystal structure analysis [39]. In the present study FF showed seven fold increase in SOCS-3 expression in BAEpCs compared to three fold increase observed with MF. In other studies FF also showed more potent effect in BAEpCs compared to fluticasone propionate [22,30]. Besides, in studies involving mechanical and elastase induced cell damage FF demonstrated more effective cellular protection than other clinically used glucocorticoids, including MF and FP [40]. This indicates that via induction of anti-inflammatory protein SOCS-3 FF may likely contribute to the anti-inflammatory response in the airway epithelium. In addition, TS exposed mice showed high number of leukocytes in the airways. FF and MF treatments resulted in significant inhibition of inflammatory cell influx into lungs in mice with COPD. In FF treated mice BAL fluid the anti-inflammatory response was more prominent compared to that of MF treated mice. We also noticed similar inflammatory responses in the lung histology sections obtained from TS exposed and TS + FF and TS + MF treated

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Fig. 7. Lung histology showing airway inflammation in mice with COPD after 4 weeks of FF, MF treatments. Mice were exposed to TS for 6 months and subsequently treated with either FF or MF 4 weeks as discussed in the methods. Room air = Control mice; TS = Mice exposed to TS for 6 months; TS + MF = mice exposed to TS for 6 months plus 4 weeks of MF inhalation; TS + FF = Mice exposed to TS for 6 months plus 4 weeks of FF inhalation. Data presented is representative of six mice from each group and arrows in the airways points to inflammatory cells. Magnification 400×.

groups of mice. Taken together these data indicate the greater antiinflammatory efficacy of FF over MF. In a recent clinical study on patients with moderate to severe COPD, FF in combination with Vilanterol (VI) once daily dose showed improved lung function along with safety and tolerability [17]. Glucocorticoids mediate their effects by switching off activation of multiple inflammatory genes [1,2,8]. In animal models of lung injury glucocorticoids reduced leakage of surfactant proteins from the lungs to systemic compartment indicating attenuation of inflammation associated with injury [11]. In our mouse model of COPD, FF and MF treatment restored TS mediated inhibition of SOCS-3 expression and significantly reduced airway inflammation when compared to TS exposed untreated mice. It is evident that corticosteroids regulate gene expression either at transcriptional or translational level. Inhibition of inflammatory gene expression by corticosteroids is due to the interaction with transcription factors such as NF-κB and AP-1. NF-κB is intimately involved in the synthesis of inflammatory cytokines, and glucocorticoids modulate NF-κB expression during therapeutic intervention in inflammatory diseases [10,38]. Moreover, corticosteroids bind to the receptors of inflammatory cytokines in the cytoplasm and translocate to the nucleus directly inhibiting the transcription of cytokines thereby repressing the expression of proinflammatory genes. In the present study FF showed significant effect on BAEpCs expression of SOCS-3 compared to MF indicating that FF is more effective than MF. This suggests that FF alone may have a beneficial effect on the airway epithelium in COPD. However

Table 1 FF and MF treatment down regulated the TS induced airway inflammation in COPD mice. Mice treatment

BAL total leukocyte

Room air TS only TS + MF TS + FF

2.6 7.2 5.4 3.6

× × × ×

105 105 105 105

BAL differential counts 98% AM, 2% L 70% AM, 18% L, 12% PMN 84% AM, 9% L,7.0% PMN 92% AM, 4.6% L,3.6% PMN

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the FF with combination of LABA such as salmeterol or vilanterol (VI) may show a synergistic effect on the expression of SOCS-3 which needs to be further investigated. It is now evident that SOCS proteins play a potential role in inflammatory diseases. Recent reports have detailed the involvement of SOCS proteins in inflammatory diseases such as rheumatoid arthritis, heart failure, and cerebral and renal injury [12,27,32]. The inhibition of SOCS-3 proteins resulted in tissue damage, whereas its over expression effectively reduced disease progression. Besides, inflammatory cytokines activate Stat signaling during the course of inflammation SOCS-3 suppresses cytokine receptor signaling [36]. Moreover, over expression of SOCS-3 resulted in suppression of erythropoiesis in fetal liver and deletion of SOCS-3 resulted in embryonic lethality [18]. In monocytes, SOCS-3 protein has inhibited 3 independent signaling pathways, Stat-3, ERK1/2 and AKT thus constituting a negative feedback circuit for monocyte hypertrophy and survival [41]. Furthermore, SOCS proteins were shown to mediate negative crosstalk between different signaling pathways. FF induced phosphorylation of Jak1, and Stat-3 in BAEpCs. Besides, FF mediated up-regulation of SOCS-3 expression in BAEpCs was dependent on Jak1 and Stat-3 signaling. SOCS-3 is known to attenuate cytokine induced inflammation via inhibition of Jak/Stat signaling [36]. Therefore, it is possible that thus induced SOCS-3 may also negatively regulate Jak/Stat signaling pathway in BAEpCs. In conclusion, the present investigation demonstrates that FF has a greater effect on SOCS-3 induction compared to MF in BAEpCs. FF induced SOCS-3 expression was dependent on Jak1/Stat-3 signaling pathway. The higher induction of SOCS-3 may represent a greater efficacy of FF therapy over MF and a novel mechanism for the antiinflammatory action of FF in the treatment of obstructive lung diseases including COPD.

Acknowledgments This work was supported by grant funding from GlaxoSmithKline. Zita Burkhalter's technical assistance is acknowledged. This research work was done using resources and facilities at the NF/SG VHS, Malcom Randall VA Medical Center, Gainesville, FL.

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