Am J Physiol Lung Cell Mol Physiol 309: L348–L359, 2015. First published June 19, 2015; doi:10.1152/ajplung.00099.2015.

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IFN-␥-induced JAK/STAT, but not NF-␬B, signaling pathway is insensitive to glucocorticoid in airway epithelial cells Danielle O’Connell,1 Belaid Bouazza,1 Blerina Kokalari,1 Yassine Amrani,2 Alaa Khatib,1 John David Ganther,1 and Omar Tliba1 1

Department of Pharmaceutical Sciences, Thomas Jefferson University, Jefferson School of Pharmacy, Philadelphia, Pennsylvania; and 2Institute for Lung Health, Department of Infection, Inflammation and Immunity, University of Leicester, Leicester, United Kingdom Submitted 2 April 2015; accepted in final form 12 June 2015

O’Connell D, Bouazza B, Kokalari B, Amrani Y, Khatib A, Ganther JD, Tliba O. IFN-␥-induced JAK/STAT, but not NF-␬B, signaling pathway is insensitive to glucocorticoid in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 309: L348 –L359, 2015. First published June 19, 2015; doi:10.1152/ajplung.00099.2015.— Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-␥ (IFN-␥) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-␥ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-␥-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-␥-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN␥-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-␥inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/ STAT vs. NF-␬B signaling pathways with GC-sensitive IIGs being more NF-␬B dependent and GC-insensitive IIGs being more JAK/ STAT dependent. Importantly, transfection of cells with siRNASTAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-␥induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma. asthma; glucocorticoid insensitivity; epithelial cell; inflammation; airway remodeling

airway disease characterized by inflammation, airway hyperresponsiveness, and tissue remodeling (13). Symptoms include cough, wheezing, difficulty breathing, and chest tightness (26). In the United States,

ASTHMA IS A CHRONIC INFLAMMATORY

Address for reprint requests and other correspondence: O. Tliba, 901 Walnut St., Rm. 910, Philadelphia, PA 19107-5233 (e-mail: omar.tliba@jefferson. edu). L348

⬃39.5 million people had asthma in 2011, and 3,404 asthmarelated deaths were reported in 2010 (7). Importantly, the health care costs related to asthma management reached $50.1 billion in 2007 (7) due to, but not limited to, missed work and school days, emergency department visits, hospitalization, and medical expenses (3). Although the majority of patients with asthma can be treated with glucocorticoids (GCs), an estimated 5–10% of patients (mainly patients with severe asthma) fail to properly respond to GCs and fall into the category of patients with GC-insensitive asthma (2). This subset of patients requires high doses of GCs to maintain adequate lung function. High GC doses can lead to complications/side effects such as glaucoma, osteoporosis, and diabetes (33), which significantly increased asthma-related health care costs. In line with this, Sullivan and colleagues (37) found that the annual number of work and school days lost, physician visits, and total costs for patients with uncontrolled asthma was significantly higher than patients with controlled asthma. Consistently, patients with severe persistent asthma had an average of health care costs of $10,121 per patient, whereas patients with mild/moderate asthma had an average of health care costs of $5,011 per patient (20). Collectively, these studies indicate the significant financial burden that GC insensitivity causes. This has led to an increased need to determine the mechanism(s) by which GC insensitivity occurs and, more importantly, ways to restore GC responsiveness in this subset of patients with asthma. Numerous studies reported the important role of airway epithelial cells in the pathogenesis of asthma (18). Indeed, epithelial cells are able to produce a variety of cytokines involved in airway inflammation and remodeling (39). Importantly, the role of epithelial cells as a target for GC therapy is increasing. The epithelium is the site of deposition for inhaled GCs and as such can be exposed to higher concentrations of GCs than any other cell type in the airway, suggesting epithelial cells as key cells in which GC insensitivity may develop. The classical Th2 paradigm suggests that asthma is predominantly driven by Th2-stimulated cytokines, such as interleukins (IL)-4 and -5 (23). More recently however, the Th1 cytokine, interferon-␥ (IFN-␥), has been suggested to also have a prominent role in the pathogenesis of asthma (30). IFN-␥ is a prominent Th1 cytokine that stimulates immune and structural cells, such as macrophages and epithelial cells, respec-

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tively, to release chemokines that are responsible for the recruitment and infiltration of inflammatory cells, and therefore amplifies the immune response (29). More specifically, IFN-␥ stimulates proinflammatory activities and prolongs eosinophil survival, which may explain its contribution to allergic inflammation (6). At the cellular level, IFN-␥ activates Janus kinase (JAK)/signal transducers and activators of transcription (STAT)-associated signaling pathway by binding to its two receptor-subtypes, IFN-␥ receptor (IFNGR)-1 and -2. These receptors dimerize and activate JAK1 and JAK2 tyrosine kinases through phosphorylation. Activated JAK1 and JAK2 recruit STAT1 and trigger phosphorylation at its tyrosine 701 residue. Phosphorylated STAT1 then homodimerizes and interacts with importin-␣-1 (NP-1) to translocate to the nucleus through the nuclear pore (34). Upon entering the nucleus, the homodimerized STAT1 becomes phosphorylated at serine residue 727, binds to the IFN-␥-activation site (GAS), and induces the transcription of IFN-␥-inducible genes (IIGs), such as IFN regulatory factor (IRF)-1, CXCL10, CXCL11, and IFIT2 (19, 34). Interestingly, IFN-␥ has been found in high levels in various chronic inflammatory airway diseases. For example, Hens and colleagues (17) showed increased expression of IFN-␥ in nasal secretions of patients with asthma compared with control subjects. Similarly, an increase in IFN-␥ levels has been observed in patients with chronic obstructive pulmonary disease (COPD) and emphysema, two chronic inflammatory airway diseases associated with a high rate of GC insensitivity (14, 25). Importantly, IFN-␥ levels appear to correlate with the disease severity of chronic inflammatory airway diseases. For example, Magnan and colleagues (24) found that IFN-␥ concentrations were elevated in whole blood cell supernatants of patients with asthma compared with healthy subjects and that the percentage of IFN-␥-producing CD8⫹ T cells positively correlated with disease severity. In agreement with this, Abdulamir and colleagues (1) found that, as asthma severity increases, a switch from predominantly Th2 cytokines to predominantly Th1 cytokines occurs. This Th-type switch is marked by a significant increase in IFN-␥ in peripheral blood lymphocytes and exhibited by a decrease in the ratio of IL-4 to IFN-␥ in patients with severe asthma. Similarly, peripheral blood mononuclear cells (PBMC) from patients with COPD expressed a significantly higher percentage of IFN-␥-producing CD8 cells compared with normal subjects, and the levels of which positively correlated with disease severity (41). Lastly, a retrospective pilot study indicated that serum IFN-␥ levels correlated with longitudinal decline in forced expiratory volume in 1 s in patients with asthma over a 3-yr interval (22). Together, these studies suggest a pathogenic role for IFN-␥ in asthma and COPD, especially in severe cases. Even though it is evident that IFN-␥ plays a role in the pathogenesis of chronic inflammatory airway diseases, its involvement in, and correlation with, GC insensitivity remains highly controversial. On one hand, several studies have shown that IFN-␥ levels correlate with GC insensitivity. For example, Bentley and colleagues (4) showed a persistent expression of IFN-␥ mRNA in the bronchial mucosa of patients with asthma despite prednisolone treatment. Similarly, IFN-␥ levels were found to be elevated in bronchoalveolar lavage of patients with COPD and were not significantly inhibited by dexamethasone treatment compared with healthy control subjects and smoking

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patients (21). Other studies went further to show that IFN-␥ can even promote GC insensitivity. Indeed, IFN-␥ has been shown to significantly reduce the sensitivity of alveolar macrophages to the inhibitory effects of dexamethasone (36). In the same study, the authors found that IFN-␥-induced STAT1 phosphorylation was insensitive to dexamethasone (36). Furthermore, microarray analysis of healthy human bronchial epithelial cells demonstrated that IIGs were not significantly affected by dexamethasone after 8 h of stimulation and only partially affected after 24 h of stimulation (29). On the other hand, other studies showed that IFN-␥ levels correlate with GC sensitivity. In fact, Goleva and colleagues (11) found that, whereas treatment of PBMCs with IL-2/IL-4 combination decreases GC receptor (GR) nuclear translocation and the expression of GC-target gene, mitogen-activated protein kinase phosphatase (MKP)-1, the addition of IFN-␥ antagonized these effects and restored GR nuclear translocation and MKP-1 expression. Interestingly, Oehling and colleagues (28) showed that PBMCs from patients with asthma exhibited a significant decrease in phytohemagglutinin-induced IFN-␥ after treatment with oral prednisone. These studies, however, did not address asthma severity when examining IFN-␥ levels. In summary, in acute asthma, IFN-␥ has been found to be beneficial owing to its protective role in suppressing the classical Th2 inflammatory response (19). However, in severe chronic inflammatory airway diseases, IFN-␥ plays a detrimental role by increasing the secretion of proinflammatory responses and by reducing GC cellular responsiveness (4, 21, 36). Therefore, the aim of this study is to determine, in airway epithelial cells, whether IFN-␥-induced JAK/STAT-associated signaling pathway is steroid sensitive and to examine the sensitivity of several IIGs to the inhibitory effects of GCs. The hypothesis is that the IFN-␥-induced JAK/STAT signaling pathway is steroid insensitive and that the expression of IIGs is differentially affected by GC. MATERIALS AND METHODS

Epithelial cell culture. Human A549 lung epithelial cells were purchased from ATCC (Manassas, VA). The culture of A549 cells was performed in F12K Kaighn’s modified medium (ATCC) supplemented with 25 mM HEPES, 12 mM NaOH, 1.7 mM CaCl2, 2 mM L-glutamine, penicillin-streptomycin, and 10% FBS. The cell cultures were incubated at 37°C in 5% CO2. Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Walkersville, MD). The culture of NHBE cells was performed in bronchial epithelial growth medium (BEGM) with supplement bullet kit (Lonza) according to manufacturer’s instructions. The cell cultures were incubated at 37°C in 5% CO2. Culture of epithelial cells in air-liquid interface. Human bronchial epithelial cells cultured at the air-liquid interface (ALI) in MucilAir medium are commercially available and were purchased from Epithelix Sarl (Geneva, Switzerland). Healthy epithelial cells were obtained from a 55-yr-old Caucasian man with no history of smoking, no reported pathology, and negative for HIV-1 and -2 and hepatitis B and C. Diseased epithelial cells were obtained from a 73-yr-old woman with COPD, a history of smoking, and negative for HIV-1 and -2 and hepatitis B and C. Patients were deidentified by Epithelix and therefore use of ALI inserts does not require IRB approval. The cell cultures were incubated at 37°C in 5% CO2 per manufacturer’s instructions. A549 cell transfection. A549 cells were transfected by using FuGENE 6 transfection reagent according to manufacturer’s instructions (Promega, Madison, WI). For the various transfection experi-

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ments we used 2 ␮g of ␥-activated sequence (GAS) luciferase reporter plasmid (Agilent, Santa Clara, CA) and 1 ␮g of ␤-galactosidase vector (to normalize transfection efficiency) (Promega). Lysates were extracted by use of 1⫻ reporter lysis buffer (Promega). The activities of luciferase and ␤-galactosidase were evaluated by using luciferase and ␤-galactosidase assay kits, respectively, according to the manufacturer’s instructions (Promega). The reporter luciferase activities were normalized to ␤-galactosidase activity and expressed as relative light units (38). Small interfering RNA. Cells were transfected by using Cell Line Nucleofector Kit using an Amaxa Nucleofactor II device according to manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany) with program X-001. A549 cells were transfected with 250 nM of small interfering RNA (siRNA) STAT1 (Sigma-Aldrich, St. Louis, MO) or scrambled siRNA control (Dharmacon, Pittsburgh, PA). Western blotting. Proteins were extracted from cells by using RIPA lysis buffer (Thermo Scientific, Rockford, IL) supplemented with 2 mM sodium vanadate, 10 mM sodium fluoride, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 100 ␮M PMSF (Sigma-Aldrich). Protein concentration was determined by a colorimetric detergent-compatible protein assay (Bio-Rad, Hercules, CA). Thirty micrograms of protein, 1⫻ loading buffer (Life Technologies, Grand Island, NY), and RIPA lysis buffer were blotted on 4 –12% SDS-PAGE gel at 150 V for 1.5 h. After electrophoresis, proteins were transferred from the gel to a 0.45 ␮m Whatman nitrocellulose membrane (GE Healthcare Biosciences, Pittsburgh, PA) in a Mini-Protean Tetra cell (Bio-Rad) at 350 mA for 1.5 h. The membranes were blocked for 1 h with 5% milk or 5% BSA for phosphorylated proteins in Tween-Tris-buffered saline to prevent nonspecific protein binding. Immunoblot analyses were then performed as described previously (38) by using anti-phosphoSTAT1 (Tyr701), anti-phospho-STAT1 (Ser727), anti-total-STAT1, anti-phospho-JAK1, anti-phospho-JAK2, anti-phospho-IKK␣/␤ (Cell Signaling, Beverly, MA), anti-total-JAK1, anti-total-JAK2, and antiI␬B␣ (Santa Cruz Biotechnology, Dallas, TX). For normalization purposes, the membranes were stripped and reprobed with antiGAPDH, anti-␣-actinin, anti-␣-tubulin, or anti-lamin A/C (Santa Cruz Biotechnology). A semiquantitative measurement was then performed. To this end, the densitometry of triplicates was assessed with ImageJ image processing software (National Institutes of Health, Bethesda, MD). Cell viability. Cell viability was determined by using MTT absorbance assay purchased from Sigma-Aldrich, according to manufacturer’s instructions. Real-time PCR. Total RNA was extracted from A549 cells using RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Reverse transcription was performed in a Thermocycler (Eppendorf, Hauppauge, NY) using GoScript reverse transcription system according to manufacturer’s instructions (Promega). Real-time PCR analysis was performed on a Mastercycler realplex 2 (Eppendorf) by using a SYBR Green kit according to manufacturer’s instructions (Applied Biosystems, Foster City, CA). Real-time PCR was performed by using predesigned primers for CXCL10, CXCL11, IFIT2, and ␤-actin (Sigma-Aldrich). For PCR amplification, the program consisted of 60 s at 95°C for the initial denaturation period and 40 cycles of 95°C for 1 s, 55°C for 5 s, and 72°C for 20 s. The results were calculated by the comparative cycle threshold method as previously described (38). Immunocytochemistry. A549 cells were fixed with 4% formaldehyde (Sigma-Aldrich) then permeabilized for 20 min with 0.5% Triton X-100 solution (Life Technologies). The cells were then blocked with 0.5% BSA solution to prevent nonspecific protein binding. Cells were incubated at 37°C for 1 h with the primary antibody anti-phosphoSTAT1, then incubated with a secondary antibody conjugated with Alexa Fluor 488 in the dark (Life Technologies). Cells were counterstained with DAPI (Life Technologies) and mounted on slides with Fluoromount G (Fisher Scientific, Pittsburgh, PA). Images were

obtained on a Leica fluorescent microscope (Leica Microsystems, Buffalo Grove, IL). Immunohistochemistry. Epithelial ALI cells were fixed using 4% formaldehyde solution (Sigma-Aldrich) then dehydrated using increasing concentrations of ethanol (Pharmco-Aaper, Brookfield, CT). Xylem (Sigma-Aldrich) was added to the membranes and finally, the membranes were embedded in paraffin with use of the services of Kimmel Cancer Center Translational Research Lab (Philadelphia, PA) for hematoxylin and eosin staining. Materials and reagents. Tissue culture reagents were obtained from ATCC. Human recombinant (r) IFN-␥ was purchased from Roche Diagnostics (Indianapolis, IN). Human rIFN-␤ was purchased from R&D Systems (Minneapolis, MN). Fluticasone propionate (FP) was obtained from Sigma-Aldrich. IKK inhibitor III (BMS-345541) and pan-JAK inhibitor I (DBI) were purchased from Calbiochem EMD Millipore (Darmstadt, Germany). Statistical analysis. Data points from individual assays represent the mean values of triplicate measurements. Significant differences among groups were assessed with t-test analysis. Each set of experiments was performed with a minimum of three different human A549 cell lines. RESULTS

IFN-␥- but not IFN-␤-induced JAK/STAT signaling pathway is insensitive to GC treatment in primary bronchial epithelial cells. First we sought to determine the sensitivity of JAK and STAT1 phosphorylation to GC treatment in NHBE cells. To this end, cells were treated with IFN-␥ for 8 h in the presence or absence of FP added 2 h after, and protein expression was examined by Western blot analysis. Interestingly, we found that the ability of IFN-␥ to induce the phosphorylation of JAK1 and JAK2 (Fig. 1, A and B) and of STAT1 at both Tyr701 and S727 residues was not inhibited by FP (Fig. 1, C and D). We next sought to determine whether the insensitivity of STAT1 phosphorylation to GC was specific to IFN-␥ or could be extended to other JAK/STAT inducers, such as IFN-␤ (19). To this end, cells were treated with IFN-␤ for 8 h, with FP added 2 h after. As shown in Fig. 1, C and D, the ability of IFN-␤ to induce STAT1 phosphorylation at both Y701 and S727 residues was significantly lower than that of IFN-␥. Surprisingly, although STAT1 phosphorylation at Y701 and S727 was insensitive to FP treatment in IFN-␥-treated cells, such phosphorylation was partially sensitive to FP treatment in IFN-␤treated cells (Fig. 1, C and D). In agreement with this, JAK1 phosphorylation was insensitive to FP treatment in IFN-␥treated, but not IFN-␤-treated, cells (data not shown). Taken together, these data suggested that the insensitivity of JAK/ STAT signaling pathway to GC effects was specific to IFN-␥. IFN-␥-induced STAT1 nuclear translocation is steroid insensitive in airway epithelial cells. We next examined whether IFN-␥-induced STAT1 nuclear translocation was affected by GC treatment. As shown in Fig. 2A, STAT1 Y701 phosphorylation was increased in the nuclear fraction of IFN-␥-treated NHBE cells. Interestingly, the addition of FP was not able to reduce STAT1 nuclear accumulation. However, STAT1 Y701 phosphorylation was not detected in the cytosolic fractions in either the presence or absence of IFN-␥ (data not shown). Similarly, immunocytochemistry studies showed that although no Y701 phosphorylation of STAT1 was observed in untreated A549 cells, such phosphorylation was significantly increased only in the nucleus of IFN-␥-treated A549 cells (Fig. 2B). Furthermore, the addition of FP was unable to inhibit the

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Fig. 1. IFN-␥- but not IFN-␤-induced JAK/STAT signaling pathway is insensitive to glucocorticoid (GC) effects. A and B: normal human bronchial epithelial (NHBE) cells were treated for 8 h with IFN-␥ (1,000 IU/ml) in the presence or absence of fluticasone propionate (FP) (100 nM) added 2 h after. Protein expression of phosphorylated JAK1 (A) and phosphorylated JAK2 (B) was measured by Western blot analysis. C and D: NHBE cells were treated with IFN-␥ (1,000 IU/ml) or IFN-␤ (1,000 IU/ml) for 8 h in the presence or absence of FP added 2 h after. Protein expression of phosphorylated STAT1 at Y701 residue (C) and S727 residue (D) was measure by Western blot analysis. **P ⬍ 0.01 compared with untreated cells, ***P ⬍ 0.001 compared with untreated cells. NS, nonsignificant compared with cells treated with IFN-␥ alone. #P ⬍ 0.05 compared with cells treated with IFN-␤.

increase of Y701 phosphorylation of STAT1 in the nuclear fractions. These findings indicated that the ability of IFN-␥ to promote the nuclear accumulation of active STAT1 was insensitive to steroid treatment. The expression of IIGs is differentially sensitive to GC in airway epithelial cells. We next investigated whether the insensitivity of STAT1 activation to GC effects, as shown above, was also associated with the insensitivity of STAT1dependent gene expression to GC. To this end, STAT1-dependent gene transcription and the expression of well-defined STAT1-inducible genes, IFIT2, CXCL11, and CXCL10, was assessed. Interestingly, whereas IFN-␥ treatment increased GAS-reporter activity, FP failed to significantly inhibit such induction in both A549 and NHBE cells (Fig. 3, A and B). Moreover, real-time PCR analyses showed that although IFN-␥ treatment increased the expression of all STAT1-dependent genes examined, FP differentially affected their expression (Fig. 4). Indeed, although the expression of IFIT2 and CXCL11 was completely insensitive to GC treatment, the expression of CXCL10 was partially inhibited by GC in both A549 and NHBE cells (Fig. 4, A and B). Together, these results

suggested that whereas IFN-␥-induced GAS-reporter activity was insensitive to GC effects, IIGs were differentially sensitive. Steroid-insensitive IIGs are more STAT1 dependent. Since IFN-␥-induced STAT1 activation was insensitive to GC effects, we next sought to determine whether STAT1 dependency of IIGs determines their sensitivity to GC. To this end, the effect of various concentrations of a pan-JAK inhibitor, DBI, on the expression of IIGs was tested. Real-time PCR analysis showed that DBI was able to completely inhibit the expression of CXCL11 and IFIT2 in IFN-␥-treated cells at 250 nM (⬎97% inhibition) (Fig. 5, A and B). Interestingly, at a similar concentration (250 nM), DBI was unable to completely inhibit IFN-␥-induced CXCL10 expression (⬍70% inhibition) (Fig. 5C). Figure 5D shows that cell viability was not significantly affected by the use of DBI. These data suggest that the steroid-insensitive IIGs studied here are more STAT1 dependent than steroid-sensitive IIGs. NF-␬B dependency of IIGs determines their differential sensitivity to GC. Previous studies showed that NF-␬B and STAT1 cooperatively regulate the expression of some IIGs (9,

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Fig. 2. IFN-␥-induced phosphorylated STAT1 nuclear translocation is insensitive to GC effects. A: NHBE cells were treated with IFN-␥ (1,000 IU/ml) for 8 h in the presence or absence of FP (100 nM) added 2 h after, and Western blot analysis was used to examine expression of phosphorylated STAT1 at Y701 in the nuclear fraction, in which lamin A/C is used as a positive control. B: A549 cells stimulated with IFN-␥ in the presence or absence of FP were fixed, permeabilized, and incubated with DAPI to stain the nucleus and antiphosphorylated STAT1 at Y701 residue, then incubated with Alexa Fluor 488, a secondary fluorescein antibody. ***P ⬍ 0.001 compared with untreated cells.

27). Therefore, we next sought to determine whether NF-␬B dependency of IIGs determines their differential sensitivity to GC. To this end, various concentrations of the NF-␬B inhibitor BMS345541 were tested. Interestingly, we found that the

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steroid-sensitive IIG (CXCL10) was most sensitive to NF-␬B inhibition (Fig. 6C). Indeed, in IFN-␥-treated cells, although BMS345541 was able to reduce the expression of CXCL10 at a concentration as low as 30 nM, it was only able to signifi-

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Fig. 3. IFN-␥-induced ␥-activated sequence (GAS)-luciferase reporter activity is insensitive to GC effects. A549 (A) and NHBE cells (B) were cotransfected with 2 ␮g of GAS-luciferase reporter plasmid and 1 ␮g of ␤-galactosidase vector, then treated with IFN-␥ (1,000 IU/ml) for 8 h in the presence or absence of FP (100 nM) added 2 h after. Luciferase activity was measured and normalized to ␤-galactosidase levels. **P ⬍ 0.01 compared with untreated cells, ***P ⬍ 0.001 compared with untreated cells. NS, nonsignificant compared with cells treated with IFN-␥ alone. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00099.2015 • www.ajplung.org

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Fig. 4. Effects of FP on IFN-␥-inducible genes’ expression. A549 (A) and NHBE cells (B) were treated with IFN-␥ (1,000 IU/ml) for 8 h in the presence or absence of FP (100 nM) added 2 h after. mRNA expression of CXCL11, IFIT2, and CXCL10 was assessed by real-time PCR analysis. Real-time PCR results were normalized to ␤-actin and expressed as percent of IFN-␥ induction. NS, nonsignificant compared with cells treated with IFN-␥ alone. ##P ⬍ 0.01 compared with cells treated with IFN-␥ alone, ###P ⬍ 0.001 compared with cells treated with IFN-␥ alone.

cantly reduce the expression of steroid-insensitive IIGs (CXCL11 and IFIT2) at much higher concentration of 3 ␮M (Fig. 6, A and B). Figure 6D shows that cell viability was not significantly affected by the use of BMS345541. These results suggest that NF-␬B dependency of IIGs determines their differential sensitivity to GC, with steroid-sensitive IIGs being more NF-␬B dependent. NF-␬B inhibition interferes with IFN-␥-induced STAT1-dependent gene transcription, but not with STAT1 phosphorylation. Since NF-␬B inhibition reduced the expression of steroid-sensitive IIGs, we next sought to determine whether NF-␬B inhibition interferes with IFN-␥-induced STAT1 phosphorylation and

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Fig. 5. Effects of pan-JAK inhibitor on IFN␥-inducible genes expression. A549 cells were treated with various concentrations of DBI for 1 h before treatment with IFN-␥ (1,000 IU/ml) for 8 h. mRNA expression was examined by real-time PCR analysis for steroid-insensitive IFN-␥-inducible genes (IIGs) CXCL11 (A) and IFIT2 (B) and steroid-sensitive IIG CXCL10 (C). Real-time PCR results were normalized to ␤-actin and expressed as percent of IFN-␥ induction. D: cell viability was determined by MTT absorbance assay. #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 compared with cells treated with IFN-␥ alone.

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IFN-␥-dependent gene transcription, which were shown above to be steroid insensitive (Fig. 1, D and E, and Fig. 3). As expected, the ability of IFN-␥ to induce GAS-reporter activities was completely abrogated when cells were treated with BMS345541 (Fig. 7A). Surprisingly, STAT1 phosphorylation at Y701 and S727 residues in IFN-␥-treated cells was completely insensitive to BMS345541 effects (Fig. 7, B and C). These data suggest that NF-␬B modulates STAT1 activation at the nuclear/promoter level. IFN-␥ induces NF-␬B activation in A549 lung epithelial cells in a steroid-sensitive manner. Studies previously demonstrated the ability of IFN-␥ to activate NF-␬B (12). However,

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Fig. 6. Effects of IKK/NF-␬B inhibitor on IFN-␥-inducible genes expression. A549 cells were treated with various concentrations of BMS345541 for 1 h before treatment with IFN-␥ (1,000 IU/ml) for 8 h. mRNA expression was examined by real-time PCR analysis for steroid-insensitive IIGs CXCL11 (A) and IFIT2 (B), and steroid-sensitive IIG CXCL10 (C). Real-time PCR results were normalized to ␤-actin and expressed as percent of IFN-␥ induction. D: cell viability was determined by MTT absorbance assay. #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 compared with cells treated with IFN-␥ alone.

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such induction was highly cell-specific (12). Since NF-␬B seems to be involved in IFN-␥-induced STAT1-dependent gene transcription, we next sought to determine whether IFN-␥ is able to induce NF-␬B activation. As shown in Fig. 8A, IFN-␥ induced the phosphorylation of IKK␣/␤ as early as 5 min of treatment. Similarly, I␬B degradation was observed as early as 10 min of IFN-␥ treatment, with complete degradation at 60 min of IFN-␥ treatment (Fig. 8B). Interestingly, treatment with FP decreased IKK␣/␤ phosphorylation and I␬B degradation in IFN-␥-treated cells (Fig. 9, A and B). These results clearly indicate the steroid-sensitive induction of NF-␬B by IFN-␥ in A549 cells. Steroid sensitivity of IIGs can be restored by silencing STAT1. Since we here found that IFN-␥-induced JAK/STATassociated signaling pathway, but not NF-␬B signaling pathway, is insensitive to GC effects, we hypothesized that inhibiting JAK/STAT signaling pathway would restore GC responsiveness. Therefore, using siRNA-STAT1 we here found that mRNA expression of steroid-insensitive IIGs (CXCL11 and IFIT2) was reduced with inhibition of STAT1 (Fig. 10, A and B). Importantly, when STAT1 was inhibited, these steroidinsensitive IIGs became responsive to GC effects (Fig. 10, A and B). When CXCL11 protein secretion was assessed, similar results were obtained (Fig. 10C). Taken together, these results indicate that steroid sensitivity of IFN-␥-induced JAK/STAT signaling pathway can be restored by the inhibition of STAT1. Epithelial cells derived from patients with COPD display higher levels of STAT1 phosphorylation. Lastly, we sought to validate these results using primary ALI human bronchial epithelial cells obtained from healthy and COPD patients. The epithelial cells cultured on ALI were treated with IFN-␥ for 8 h in the presence or absence of FP added 2 h after. As shown

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in Fig. 11A, IFN-␥ induction of STAT1 phosphorylation at the Y701 residue was significantly higher in cells obtained from patients with COPD compared with cells obtained from healthy patients (Fig. 11A, lane 6 vs. 5 and lane 8 vs. 7). Interestingly, IFN-␥-induced STAT1 phosphorylation at Y701 was insensitive to FP treatment (Fig. 11A, lane 7 vs. 5 and lane 8 vs. 6). ALI human bronchial epithelial cells used were cultured in a condition that recapitulates the pseudostratified mucociliary phenotype observed in vivo. ALI cells are physiologically relevant because they display all features of lung epithelial cells found in the human airway, including the presence of highly motile cilia, production and secretion of mucus, and the development of stable transepithelial electrical resistance (Fig. 11B). DISCUSSION

Although the role of IFN-␥ in chronic inflammatory airway diseases is controversial, numerous studies have shown increased levels of IFN-␥ in severe asthmatic patients and COPD patients (14, 17, 24, 25). Interestingly, constitutive activation of STAT1 was found in lung epithelial cells obtained from asthmatic patients, resulting in higher expression levels of IIGs, e.g., IRF-1 (31). Although the level of IFN-␥ was not increased in those patients, the degree of disease severity from which the cells were obtained was not addressed (31). In the present study, we found that the levels of activated STAT1 in lung epithelial cells differentiated in ALI after IFN-␥ treatment were higher in patients with COPD compared with control patients (Fig. 11A). We also found that IFN-␥-induced JAK/ STAT-associated signaling pathway was insensitive to GC

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Fig. 7. DBI inhibits IFN-␥-induced JAK/STAT pathway, whereas BMS345541 only inhibits STAT1-dependent gene transcription. A549 cells were treated with DBI (25 ␮M) or BMS345541 (30 ␮M) for 1 h before treatment with IFN-␥ (1,000 IU/ml) 8 h. A: cells were cotransfected with 2 ␮g of GAS-luciferase reporter plasmid and 1 ␮g of ␤-galactosidase vector before treatment. Luciferase activity was measured and normalized to ␤-galactosidase levels. Protein expression of phosphorylated STAT1 at Y701 residue (B) and S727 residue (C) was measured by Western blot analysis. ***P ⬍ 0.001 compared with untreated cells, ###P ⬍ 0.001 compared with cells treated with IFN-␥ alone. NS, nonsignificant compared with cells treated with IFN-␥ alone.

actions, an insensitivity that was reversed by STAT1 inhibition. The effect of GC on JAK/STAT1 activation varies depending on the interferon used for cell treatment. Indeed, we found here that whereas IFN-␥-induced STAT1 phosphorylation was GC insensitive, IFN-␤-induced STAT1 phosphorylation was GC sensitive (Fig. 1, C and D). Similarly, GCs were found to inhibit IFN-␤-induced STAT1-STAT2 activation in macrophages (10). These data indicate that GC insensitivity of the JAK/STAT-associated signaling pathway is specific to IFN-␥. Interestingly, IFN-␥, through the phosphorylation and the heterodimerization of JAK1 and JAK2, activates STAT1 and promotes its homodimerization, whereas IFN-␤ through the phosphorylation and the heterodimerization of JAK1 and TYK2, activates STAT1 and STAT2 and their subsequent heterodimerization. Therefore, the GC insensitivity of STAT1 activated by IFN-␥, but not IFN-␤, may lie in the mechanisms upon which JAKs are regulated. Interestingly, several studies showed that suppressors of cytokine signaling (SOCS) proteins inhibit IFN signal transduction by binding to phosphorylated tyrosine residues on IFN receptors and JAKs via the SH2 domain and subsequently inhibit JAKs by promoting their

ubiquitination (32). Interestingly, while the induction of SOCS1 by GC has been shown to mediate GC inhibitory effects on IFN-␤ actions (5), the involvement of SOCS1 in mediating GC effects on IFN-␥ actions is only transient (15). However, further studies are still needed to delineate the role of SOCS in mediating the lack of steroid responsiveness of IFN-␥- vs. IFN-␤-associated signaling pathways. IFN-␥ is a pleiotropic cytokine and able to induce several signaling pathways besides JAK/STAT pathways, such as the NF-␬B signaling pathway (12). Indeed, numerous studies show that JAK/STAT and NF-␬B pathways cooperatively induce gene expression and promote inflammatory responses (9, 27). For example, in estrogen-treated murine macrophages, the activation of both JAK/STAT and NF-␬B pathways was required for inducible NO synthase and NO production, and the inhibition of either pathway significantly decreased IFN-␥ actions (9). Furthermore, in macrophages/microglia, Nguyen and colleagues (27) showed that NF-␬B activation mediates the induction of CD40 by IFN-␥. Consistently, we found here that IFN-␥ induces IKK␣/␤ phosphorylation and I␬B degradation (Fig. 8, A and B). In addition, we also found that NF-␬B pharmacological inhibition using BMS345541 significantly in-

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Fig. 8. IFN-␥ induces NF-␬B-associated signaling pathway through IKK␣/␤ phosphorylation and I␬B degradation. A549 cells were treated with IFN-␥ (1,000 IU/ml) at various times points. Protein expression was measured by Western blot analysis for phosphorylated IKK␣/␤ (A) and I␬B (B). *P ⬍ 0.05 compared with untreated cells, **P ⬍ 0.01 compared with untreated cells, ***P ⬍ 0.001 compared with untreated cells; #P ⬍ 0.05 compared with untreated cells, ##P ⬍ 0.01 compared with untreated cells, ###P ⬍ 0.001 compared with untreated cells.

hibited IFN-␥-induced GAS-reporter activity (Fig. 7A) and the expression of IIGs (Fig. 6, A–C). Interestingly, microarray analysis of human bronchial epithelial cells showed that IFN-␥ treatment induced TNF-␣ expression (29). Because TNF-␣ is known to activate the NF-␬B-associated signaling pathway in nearly all cell types (16), it is therefore legitimate to speculate that IFN-␥ effects on NF-␬B shown here may be due to an autocrine action of TNF-␣; however, further studies are needed to validate such hypothesis. Interestingly, we found that IIGs are differentially sensitive to GC effects. Indeed, whereas CXCL10 was sensitive to GC

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actions, CXCL11 and IFIT2 were insensitive (Fig. 4, A and B). In agreement with this and in murine macrophages, three IIGs, i.e., Fc␥RI, Fc␥RIII␣, and Ia antigen, were found to be differentially affected by dexamethasone treatment (35). It is tempting to speculate that the variable sensitivity of IIGs to GC is due to their dependency to signaling pathways that also show variable sensitivity to GC. Since we here show that IFN-␥ not only activated JAK/STAT signaling pathway, but also NF-␬B signaling pathway, we hypothesized that the differential sensitivity of IIGs to GCs is due to their variable dependencies to different signaling pathways induced by IFN-␥. Indeed, we

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Fig. 9. IFN-␥-induced NF-␬B-associated signaling pathway is sensitive to GC effects. A549 cells were treated for 15 or 30 min with IFN-␥ (1,000 IU/ml) in the presence or absence of FP (100 nM) added 1 h before. Protein expression of phosphorylated IKK␣/␤ (A) and I␬B (B) was measured by Western blot analysis. A: **P ⬍ 0.05 compared with untreated cells, ##P ⬍ 0.01 compared with cells treated with IFN-␥ alone. B: ␦P ⬍ 0.05 compared with untreated cells, **P ⬍ 0.01 compared with cells treated with IFN-␥ alone. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00099.2015 • www.ajplung.org

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Fig. 10. Silencing STAT1 reduces the expression of steroid-insensitive IIGs and restores GC responsiveness. A549 cells were transfected with siRNA-STAT1 (250 nM) then treated with IFN-␥ (1,000 IU/ml) 8 h in the presence or absence of FP (100 nM) added 2 h after. mRNA expression was examined by real-time PCR analysis for steroid-insensitive IIGs CXCL11 (A) and IFIT2 (B). Real-time PCR results were normalized to ␤-actin and expressed as percent of IFN-␥ induction. C: A549 cells were transfected with silencing RNA-STAT1 (siSTAT1) or silencing Control (siControl) (250 nM) for 48 h then treated with IFN-␥ for 24 h in the presence or absence of FP (100 nM) added 2 h after. The secretion of CXCL11 in cell supernatants was examined by ELISA. #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 compared with siControl cells treated with IFN-␥ alone.

found that GC-sensitive IIGs, i.e., CXCL10, were more dependent on NF-␬B (Fig. 6C) than the GC-insensitive IIGs, i.e., CXCL11 and IFIT2 (Fig. 6, A and B). We also found that GC-insensitive IIGs were more STAT1-dependent (Fig. 5, A–C), suggesting that STAT1 dependency of IIGs determines their lack of GC responsiveness. However, additional IIGs need to be explored in epithelial and other airway cells to

A

generalize such observation, since our study focused only on three IIGs. Interestingly, using STAT1 Tyr701 mutant, Walter and colleagues (40) were able to reduce STAT1 activation and subsequently the expression of IIG, i.e., ICAM1. Furthermore, the pharmacological inhibition of JAK/STAT1 signaling pathway in human airway smooth muscle cells has been shown to kDa -97

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Fig. 11. IFN-␥-induced STAT1 phosphorylation at Y701 residue is significantly higher in epithelial cells from patients with chronic obstructive pulmonary disease (COPD) than in epithelial cells from control patients. A: epithelial cells from healthy and COPD patients cultured in air-liquid interface (ALI) were treated with IFN-␥ (1,000 IU/ml) for 8 h in the presence or absence of FP (100 nM) added 2 h after. IFN-␥-induced STAT1 phosphorylation at Y701 residue was also insensitive to FP treatment. ***P ⬍ 0.001 compared with healthy patients. B: hematoxylin and eosin staining of epithelial cells from a healthy patient cultured at ALI. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00099.2015 • www.ajplung.org

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restore steroid responsiveness of GC-insensitive genes (8). In agreement with this, we here show that siRNA STAT1 not only reduced the expression of all IIGs but also increased the responsiveness of GC-insensitive IIGs to GC actions (Fig. 10, A and B). Indeed, although GC was unable to inhibit the expression of steroid-insensitive IIGs in siRNA control-transfected cells, it significantly reduced the expression of such genes in siRNA STAT1-transfected cells (Fig. 10, A and B). These findings suggest that targeting STAT1 could restore steroid responsiveness in patients with severe asthma or COPD where the levels of IFN-␥ and its target genes (IIGs) are high (4, 21, 36). In conclusion, our study demonstrated that IFN-␥-induced JAK/STAT-associated signaling pathway was insensitive to GC actions in airway epithelial cells, and that targeting STAT1 restores GC responsiveness of different IIGs. This could help in the design of novel drugs that will act as steroid-sparing agents, especially in patients requiring high doses of GC therapy. However, further studies are still needed to determine whether IFN-␥induced STAT1 activation, in turn, affects GR functions. GRANTS This work was funded by National Heart, Lung, and Blood Institute Grant R01HL111541. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS D.O., B.B., B.K., A.K., and J.D.G. performed experiments; D.O. and O.T. analyzed data; D.O. prepared figures; D.O. drafted manuscript; D.O., Y.A., and O.T. edited and revised manuscript; Y.A. and O.T. interpreted results of experiments; O.T. conception and design of research; O.T. approved final version of manuscript. REFERENCES 1. Abdulamir AS, Hafidh RR, Abubakar F, Abbas KA. Changing survival, memory cell compartment, and T-helper balance of lymphocytes between severe and mild asthma. BMC Immunol 9: 73, 2008. 2. Adcock IM, Ito K. Steroid resistance in asthma: a major problem requiring novel solutions or a non-issue? Curr Opin Pharmacol 4: 257– 262, 2004. 3. Barnett SB, Nurmagambetov TA. Costs of asthma in the United States: 2002–2007. J Allergy Clin Immunol 127: 145–152, 2011. 4. Bentley AM, Hamid Q, Robinson DS, Schotman E, Meng Q, Assoufi B, Kay AB, Durham SR. Prednisolone treatment in asthma. Reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa. Am J Respir Crit Care Med 153: 551–556, 1996. 5. Bhattacharyya S, Zhao Y, Kay TW, Muglia LJ. Glucocorticoids target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate Toll-like receptor-induced STAT1 activation. Proc Natl Acad Sci USA 108: 9554 –9559, 2011. 6. Borish L, Rosenwasser L. TH1/TH2 lymphocytes: doubt some more. J Allergy Clin Immunol 99: 161–164, 1997. 7. CDC. Asthma Facts. In: CDC’s National Asthma Control Program Grantees, edited by Department of Health and Human Services. Atlanta, GA: Centers for Disease Control and Prevention, 2013. 8. Clarke DL, Clifford RL, Jindarat S, Proud D, Pang L, Belvisi M, Knox AJ. TNFalpha and IFNgamma synergistically enhance transcriptional activation of CXCL10 in human airway smooth muscle cells via STAT-1, NF-kappaB, and the transcriptional coactivator CREB-binding protein. J Biol Chem 285: 29101–29110, 2010. 9. Dai R, Phillips RA, Karpuzoglu E, Khan D, Ahmed SA. Estrogen regulates transcription factors STAT-1 and NF-kappaB to promote inducible nitric oxide synthase and inflammatory responses. J Immunol 183: 6998 –7005, 2009.

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AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00099.2015 • www.ajplung.org

STAT, but not NF-κB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells.

Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive ...
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