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Blocking Bcl-2 resolves IL-13–mediated mucous cell hyperplasia in a Bik-dependent manner

2017; Elsevier BV; Volume: 140; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2017.05.038

1097-6825

Hitendra S. Chand, Yohannes A. Mebratu, Philip J. Kuehl, Yohannes Tesfaigzi,

Cell death mechanisms and regulation

Bcl-2 protects cells against a wide range of apoptotic stimuli by preventing mitochondrial permeabilization caused by proapoptotic Bcl-2 family members.1Delbridge A.R. Grabow S. Strasser A. Vaux D.L. Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies.Nat Rev Cancer. 2016; 16: 99-109Crossref PubMed Scopus (495) Google Scholar Although Bcl-2 levels are regulated by various cytokines,2Chand H.S. Harris J.F. Mebratu Y. Chen Y. Wright P.S. Randell S.H. et al.Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.J Immunol. 2012; 188: 4581-4589Crossref PubMed Scopus (22) Google Scholar inflammatory mediators that affect Bcl-2 expression in nonhematopoietic primary cells have been studied poorly. Bcl-2 expression is upregulated in airway mucous cells of patients with chronic bronchitis, asthmatic bronchitis, and cystic fibrosis and in respective animal models to sustain hyperplastic mucous cells.3Harris J.F. Fischer M.J. Hotchkiss J.R. Monia B.P. Randell S.H. Harkema J.R. et al.Bcl-2 sustains increased mucous and epithelial cell numbers in metaplastic airway epithelium.Am J Respir Crit Care Med. 2005; 171: 764-772Crossref PubMed Scopus (43) Google Scholar, 4Chand H.S. Montano G. Huang X. Randell S.H. Mebratu Y. Petersen H. et al.A variant of p53 restricts the mucus secretory phenotype by regulating SPDEF and Bcl-2 expression.Nat Commun. 2014; 5: 5667Crossref PubMed Scopus (20) Google Scholar The purpose of this study was to determine whether identifying and understanding the mechanisms by which cytokines regulate Bcl-2 expression can be used to reduce mucous cell hyperplasia (MCH). Because we have observed repeatedly that Bcl-2 and MUC5AC are coexpressed in airway epithelial cells (AECs),4Chand H.S. Montano G. Huang X. Randell S.H. Mebratu Y. Petersen H. et al.A variant of p53 restricts the mucus secretory phenotype by regulating SPDEF and Bcl-2 expression.Nat Commun. 2014; 5: 5667Crossref PubMed Scopus (20) Google Scholar, 5Tesfaigzi Y. Roles of apoptosis in airway epithelia.Am J Respir Cell Mol Biol. 2006; 34: 537-547Crossref PubMed Scopus (68) Google Scholar we investigated whether IL-13 also induces Bcl-2 expression. We found that IL-13 induced Bcl2 and MUC5AC but did not affect BclxL mRNA levels in differentiated primary human airway epithelial cells (HAECs) from 10 subjects (Fig 1, A), and induction took place in a dose-dependent manner (Fig 1, B). Inhibitors of both epidermal growth factor receptor (AG1478) and extracellular signal-regulated kinase 1/2 (U0126) suppressed IL-13–induced MUC5AC (Fig 1, C) and Bcl-2 mRNA (Fig 1, D) and protein (Fig 1, E) levels, suggesting that Bcl-2 and MUC5AC can be regulated by identical pathways. Interestingly, blocking Bcl-2 expression with a retroviral short hairpin RNA (shRNA) construct (shBcl-2; Fig 1, F) reduced IL-13–induced MUC5AC mRNA levels (Fig 1, F), suggesting Bcl-2 might be upstream of MUC5AC expression. However, when Bcl-2 was constitutively overexpressed (Bcl-2 O/E), MUC5AC mRNA levels remained unchanged (Fig 1, G). Rather, among IL-13–treated cells, the numbers of untransfected and control shRNA with random DNA sequence (shCTRL)–transfected control cells increased, whereas suppression of the antiapoptotic Bcl-2 with a shBcl-2 construct caused reduction of cell numbers (Fig 1, H) by inducing apoptosis (Fig 1, I). To determine the mechanism by which IL-13 induces cell death when Bcl-2 expression is suppressed, we screened for all proapoptotic Bcl-2 family proteins and determined Bik mRNA to consistently be induced by IL-13 in primary differentiated HAECs (Fig 1, J) and even further increased in shBcl-2 cells (see Fig E1, A, in this article's Online Repository at www.jacionline.org). Bik protein levels were upregulated on days 3 and 4 of IL-13 treatment, whereas Bcl-2 levels remained induced over 6 days (Fig 1, K). Because these findings suggested that IL-13 can be switched from a proliferative to a cell death–inducing cytokine when Bcl-2 is suppressed, we examined the role of the small-molecule BH3 mimetic ABT-263, which blocks Bcl-2 function.6Tse C. Shoemaker A.R. Adickes J. Anderson M.G. Chen J. Jin S. et al.ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor.Cancer Res. 2008; 68: 3421-3428Crossref PubMed Scopus (1432) Google Scholar As a percentage of nontreated controls, IL-13–treated HAECs were reduced when preincubated with 1 μmol/L ABT-263 for 2 hours (see Fig E1, B). Although Bik+/+ primary murine airway epithelial cells (MAECs) showed significant cell death, Bik−/− MAECs were unaffected by the combination treatment of IL-13 and ABT-263 (Fig 1, L). Similar results were observed in HAECs when Bik was suppressed with shBik (see Fig E1, C), confirming that Bik is required for IL-13–induced cell death when Bcl-2 is blocked. To investigate the potential therapeutic value of these findings, we first investigated the efficacy of ABT-263 in suppressing IL-13–induced MCH in differentiated primary HAEC cultures. In IL-13–treated cultures, treatment with ABT-263 compared with vehicle suppressed MUC5AC mRNA levels (see Fig E2, A, in this article's Online Repository at www.jacionline.org) and the percentage of MUC5AC+ and Bcl-2+ cells (Fig 2, A). ABT-263 treatment had no effect on the ciliated cell population but significantly reduced numbers of MUC5AC+ cells (Fig 2, A) without affecting transepithelial electrical resistance (see Fig E2, B). Because IL-13 is a TH2 cytokine central in causing bronchitic asthma,7Wynn T.A. Type 2 cytokines: mechanisms and therapeutic strategies.Nat Rev Immunol. 2015; 15: 271-282Crossref PubMed Scopus (423) Google Scholar we investigated the effect of ABT-263 in a mouse model of allergic asthma (as shown in Fig 2, B). MCH levels were significantly reduced by intranasally administered ABT-263 (2 mg/kg body weight) compared with those seen in vehicle-treated control animals (Fig 2, C), as were Muc5ac+ cell counts (see Fig E2, C). Numbers of AECs positive for cleaved caspase-3 (Fig 2, D) and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL; see Fig E2, D) were increased among Scgb1A1+ secretory cells in ABT-263–treated compared with vehicle-treated mice, indicating reduction of ovalbumin (OVA)–induced MCH by apoptosis. We postulated that delivery of ABT-263 by means of aerosol inhalation will directly target airway mucous cells and thereby minimize the effective dose. Therefore we generated aerosols with respirable particles (<1 μm) with increasing ABT-263 concentrations in a suspension formulation in 0.5% Tween 80 and water using a Pari LC Plus compressed air jet nebulizer8Kuehl P.J. Anderson T.L. Candelaria G. Gershman B. Harlin K. Hesterman J.Y. et al.Regional particle size dependent deposition of inhaled aerosols in rats and mice.Inhal Toxicol. 2012; 24: 27-35Crossref PubMed Scopus (69) Google Scholar and estimated the pulmonary deposited doses (PDDs) for a 30-minute exposure (see Table E1 in this article's Online Repository at www.jacionline.org). Then 3 groups of OVA-sensitized mice were exposed daily by means of nose-only inhalation (as outlined in Fig E3, A, in this article's Online Repository at www.jacionline.org) to either 0 (vehicle only: 0.5% Tween 80 in water) or the average PDD of 44 μg/kg (see Table E2 in this article's Online Repository at www.jacionline.org) or 262 μg/kg (see Table E3 in this article's Online Repository at www.jacionline.org) of body weight over 5 days. Aerosolized ABT-263 at both 44 and 262 μg/kg PDD suppressed allergen-induced MCH compared with PDDs in control mice (Fig 2, E, and see the Methods section in this article's Online Repository at www.jacionline.org) and significantly reduced the number of total AECs per millimeter of basal lamina (BL; Fig 2, F). As expected, ABT-263 levels observed in plasma and lung tissues were increased in a dose-dependent manner at 6 and 24 hours after the last ABT-263 dose and decreased at 24 hours for the 44 and 262 μg/kg doses by 4.2- and 10-fold, respectively (see Fig E3, B and C). The importance of Bik in resolution of MCH was assessed by exposing Bik+/+ and Bik−/− mice to ABT-263 aerosols at a PDD of 46 μg/kg (see Table E4 in this article's Online Repository at www.jacionline.org). Nose-only inhalation of ABT-263 suppressed MCH in Bik+/+ but not Bik−/− mice (Fig 2, G), confirming that Bik is crucial in reducing MCH when Bcl-2 function is blocked. The current studies demonstrate that the proinflammatory IL-13 induces Bcl-2 in AECs. Because IL-13 also induces the proapoptotic Bik, targeted blocking of Bcl-2 function switches IL-13 into a cell death inducer. Therefore the small-molecule Bcl-2 inhibitor ABT-263, when delivered by means of nose-only inhalation, reduced allergen-induced MCH levels in a Bik-dependent manner at a 4000-fold lower dose compared with the 200 mg/kg dose that causes reduction in platelets by 50%, as used in clinical trials for treating lymphoma.9Souers A.J. Leverson J.D. Boghaert E.R. Ackler S.L. Catron N.D. Chen J. et al.ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.Nat Med. 2013; 19: 202-208Crossref PubMed Scopus (2028) Google Scholar Whether AECs will be more prone to injury from infection or exposure to noxious substances will be studied in the future. However, as a pleiotropic cytokine, IL-13 affects AECs to produce not only mucins but also the chemokines TGF-β and eotaxin and thereby might perpetuate inflammation. Therefore reducing the number of hyperplastic AECs with aerosolized ABT-263 that is rapidly cleared from the body might be an effective therapy with reduced side effects for reducing inflammation and mucus hypersecretion. We thank Lois Herrera and Susan Fort for assistance on the selected experiments. Pathogen-free wild-type C57BL/6J mice were either purchased from the Jackson Laboratory or were from in-house breeding. Bik−/− mice on the C57BL/6 background were made available by Dr Andreas Strasser (Walter and Eliza Hall Institute) and bred at the Lovelace Respiratory Research Institute and genotyped, as previously described.E1Coultas L. Bouillet P. Stanley E.G. Brodnicki T.C. Adams J.M. Strasser A. Proapoptotic BH3-only Bcl-2 family member Bik/Blk/Nbk is expressed in hemopoietic and endothelial cells but is redundant for their programmed death.Mol Cell Biol. 2004; 24: 1570-1581Crossref PubMed Scopus (100) Google Scholar Mice were housed in isolated cages under specific pathogen-free conditions. All experiments were approved by the Institutional Animal Care and Use Committee and were conducted at the Lovelace Respiratory Research Institute, a facility approved by the Association for the Assessment and Accreditation for Laboratory Animal Care International. HAECs and AALEB (a primary HAEC immortalized through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT) cellsE2Lundberg A.S. Randell S.H. Stewart S.A. Elenbaas B. Hartwell K.A. Brooks M.W. et al.Immortalization and transformation of primary human airway epithelial cells by gene transfer.Oncogene. 2002; 21: 4577-4586Crossref PubMed Scopus (199) Google Scholar were maintained in bronchial epithelial growth medium (Lonza, Walkersville, Md), and NCI-H292 cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with FBS. Primary HAECs were either purchased from Clontech (Walkersville, Md) or provided kindly by Dr Scott Randell and were obtained from lungs under the protocol and consent form approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. For air-liquid interface culture, primary HAECs were seeded on Transwell membranes (Corning, Corning, NY) and differentiated for 14 days. After treatment, membrane quarters were used for quantitative RT-PCR, and membrane halves were embedded in paraffin for immunostaining. AALEB cells and HAECs were treated with human IL-13 (10 ng/mL; R&D Systems, Minneapolis, Minn). Cells were also pretreated with U0126 (Santa Cruz Biotechnology, Santa Cruz, Calif), an inhibitor of extracellular signal-regulated kinase 1/2, or the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (Santa Cruz Biotechnology) 2 hours before IL-13 treatment. pcDNA3.0–Bcl-2 plasmid was provided by Dr Stanley Korsmeyer (Harvard Medical School, Boston, Mass). Transfections were carried out with the FuGENE-6 reagent (Roche, Mannheim, Germany), according to the manufacturer's protocol. Cell viability was determined by using trypan blue exclusion. MAECs were isolated essentially as described previously.E3Mebratu Y.A. Schwalm K. Smith K.R. Schuyler M. Tesfaigzi Y. Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.Am J Respir Crit Care Med. 2011; 183: 1531-1538Crossref PubMed Scopus (38) Google Scholar Briefly, tracheas from mice were excised, connective tissue was cleared, and tracheas were cut open lengthwise. Cleaned tracheas were incubated in Pronase solution (DMEM, 1.4 mg/mL Pronase, and 0.1 mg/mL DNase) overnight at 4°C to dissociate AECs from BL. Enzymatic activity was stopped with 10% FBS (Invitrogen, Carlsbad, Calif), and cells were collected by gently rocking trachea in DMEM/Ham H12 media (Invitrogen), followed by centrifugation at 400g for 10 minutes at 4°C. Cell clumps were dissociated by using 5 mL of declumping solution (DMEM and 2 mmol/L EDTA) and plated at 100,000 cells/well on collagen-coated plates. HAECs or AALEB cells were transfected with Bcl-2 shRNA containing retroviral or control vectors (Origene Technologies, Rockville, Md), according to the manufacturer's instructions. After infection with shBcl-2 or control shRNAs, cells were treated with 10 ng/mL human IL-13, and 48 hours later, they were assessed for Bcl-2 expression by means of immunofluorescence or Western blotting. RNA was isolated from snap-frozen right lungs of animals by using TRIzol, as described previously,E4Chand H.S. Woldegiorgis Z. Schwalm K. McDonald J. Tesfaigzi Y. Acute inflammation induces IGF-1 to mediate Bcl-2 and Muc5ac expression in airway epithelial cells.Am J Respir Cell Mol Biol. 2012; 47: 784-791Crossref PubMed Scopus (24) Google Scholar whereas RNA from cultured cells was extracted with the RNeasy kit (Qiagen, Valencia, Calif), and the concentration was determined with the Thermo Scientific Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, Mass). The primer/probe sets for Bcl2, BclXL, MUC5AC, and CDKN1B were obtained from Life Technologies (Carlsbad, Calif), and RNA was amplified by using quantitative PCR with the RT-PCR Master Mix (Life Technologies) in the ABI PRISM 7900HT Real-Time PCR System. Relative quantities were calculated by normalizing averaged cycle threshold values to CDKN1B to obtain the Δ cycle threshold value, and the relative standard curve method was used for determining the fold change, as described previously.E5Schwalm K. Stevens J.F. Jiang Z. Schuyler M.R. Schrader R. Randell S.H. et al.Expression of the pro-apoptotic protein bax is reduced in bronchial mucous cells of asthmatics.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L1102-L1109Crossref PubMed Scopus (16) Google Scholar For cytometry, cells were grown on Lab-Tek-II 8-chamber slides (Nalgene Nunc International, Rochester, NY), treated with 10 ng/mL of IL-13 or vehicle, fixed with 3% paraformaldehyde and 3% sucrose in PBS, and processed for immunostaining, as described previously.E6Tesfaigzi Y. Fischer M.J. Daheshia M. Green F.H. De Sanctis G.T. Wilder J.A. Bax is crucial for IFN-gamma-induced resolution of allergen-induced mucus cell metaplasia.J Immunol. 2002; 169: 5919-5925Crossref PubMed Scopus (30) Google Scholar Quantification of Bcl-2+ and MUC5AC+ cells per millimeter of BL was performed with the Visiomorph system (Visiopharm A/S, Hørsholm, Denmark). Cells cultured at the air-liquid interface were immunostained similarly for Bcl-2 and MUC5AC. Tissue sections were deparaffinized, hydrated in graded ethanol and deionized water, and then washed in 0.05% v Brij-35 in PBS (pH 7.4). Antigens were unmasked by treating with the Digest-All kit (Zymed Laboratories, San Francisco, Calif) at a 1:3 dilution of trypsin to diluent at 37°C for 10 minutes. Sections were incubated in 0.2% Triton X-100 with 0.2% Saponin in a blocking solution containing 3% IgG-free BSA, 1% gelatin, and 2% normal donkey serum, followed by anti–Bcl-2 (#sc-492, Santa Cruz Biotechnology), anti-MUC5AC (#MAB2011; Millipore, Temecula, Calif), or isotype controls. The immunolabeled cells were detected by using F(ab)2 fragments of respective secondary antibodies conjugated to either Dylight-549 or Dylight-649 (Jackson ImmunoResearch, West Grove, Pa) and mounted with 4′,6-diamidino-2-phenylindole containing Fluormount-G (SouthernBiotech, Birmingham, Ala) for nuclear staining. Immunofluorescence was imaged with the ×63 objective of a Zeiss Axioplan 2 Imaging microscope as 0.3-μm increment z-stacks with multiple images and deconvoluted with a nearest neighbors algorithm with the Everest Imaging System with SlideBook software (Intelligent Imaging Innovation). Epithelial integrity was determined by measuring the transepithelial electrical resistance of differentiated cultures grown on Transwell membranes. HBSS (200 μL) was added to the apical surfaces of cell cultures, and transepithelial electrical resistance was measured by using the Millicell ERS-2 Volt-ohm meter (EMD Millipore, Billerica, Mass). C57BL/6 mice at 6 to 8 weeks of age were immunized by means of intraperitoneal injection with a mixture of 10 μg of OVA and 2 mg of aluminum hydroxide gel adjuvant in a total volume of 0.5 mL of sterile water on days 1 and 7.E7Shi Z.O. Fischer M.J. De Sanctis G.T. Schuyler M.R. Tesfaigzi Y. IFN-gamma, but not Fas, mediates reduction of allergen-induced mucous cell metaplasia by inducing apoptosis.J Immunol. 2002; 168: 4764-4771Crossref PubMed Scopus (53) Google Scholar On day 14, mice were exposed to OVA aerosols at a concentration of 2 mg/m3 for 5 consecutive days. Control animals were housed in similar exposure chambers and exposed to filtered air. Treatment groups received ABT-263 (2 mg/kg in 0.05 mL of 0.9% pyrogen-free saline) intranasally 2 hours before OVA exposures starting on day 13. Control mice were instilled with 0.05 mL of 0.9% pyrogen-free saline. Mice were killed 24 hours after the last exposure. ABT-263 was aerosolized with a commercial compressed air jet nebulizer (Pari LC Plus) into a 24-port nose-only inhalation chamber (In-Tox Products, Albuquerque, NM). The inhalation exposure system was developed to support a range of PDDs.E8Alexander D.J. Collins C.J. Coombs D.W. Gilkison I.S. Hardy C.J. Healey G. et al.Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals.Inhal Toxicol. 2008; 20: 1179-1189Crossref PubMed Scopus (141) Google Scholar Key parameters that were modulated to achieve different pulmonary doses were the suspension concentration in the nebulizer and system flow rates. Aerosols were generated with suspension formulations at ABT-263 concentrations ranging from 2 to 60 mg/mL and prepared in 0.5% Tween 80 and water. ABT-263 aerosol concentrations were measured at the breathing zone of the exposure chamber by collecting onto Pallflex filters (Pall Life Sciences, Port Washington, NY). The filters were analyzed by using gravimetric and chemical methods. Chemical analysis used liquid extraction, 0.1% TFA in water/ACN (50:50 vol/vol), and HPLC-UV analysis. Inhalation exposures with ABT-263 and vehicle (0.5% Tween 80 in water) were conducted in separate flow-past, nose-only, mouse inhalation exposure systems. The suspension concentrations used were 6, 20, and 60 mg/mL in 0.5% Tween 80 and water. Under these conditions, the actual average PDDs for the groups were 44 and 262 μg/kg, respectively. The particle size (mass median aerodynamic diameters [MMADs]) ranged between 0.78 and 3.92 μm, with geometric SDs (GSDs) of less than or equal to 2.71. Groups of OVA-sensitized mice were exposed daily to ABT-263 aerosols or vehicle before exposure to OVA for 6 hours by using nose-only exposure tubes (as outlined in Fig E3, A). The aerosolized ABT-263 had a particle size ranging from 0.68 to 1.12 μm MMADs, with an average of 0.80 μm, and the GSDs ranged from 1.84 to 2.65, with an average of 2.08. Particle size distribution was determined with a 7-stage Mercer-style Impactor (In-Tox Products, Albuquerque, NM), as described previously.E8Alexander D.J. Collins C.J. Coombs D.W. Gilkison I.S. Hardy C.J. Healey G. et al.Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals.Inhal Toxicol. 2008; 20: 1179-1189Crossref PubMed Scopus (141) Google Scholar The aerosol was withdrawn directly from the breathing zone of the exposure chamber 4 times during each exposure period. Impactor stages were assayed based on differential weight and processed to determine the MMAD. MMADs ranged between 0.78 and 3.92 μm, with GSDs of less than or equal to 2.71. Histochemical staining for Alcian blue and periodic acid–Schiff were carried out, as previously described.E9Tesfaigzi Y. Harris J.F. Hotchkiss J.A. Harkema J.R. DNA synthesis and Bcl-2 expression during the development of mucous cell metaplasia in airway epithelium of rats exposed to LPS.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L268-L274Crossref PubMed Scopus (30) Google Scholar AEC and mucous cell numbers per millimeter of BL were measured with the Visiomorph system (Visiopharm A/S, Hørsholm, Denmark) by counting the number of nuclei and mucous cells, respectively, and dividing by the length of the BL. In all cases quantification and morphometry were carried out by a person unaware of slide identity. After airway hyperreactivity (AHR) measurements and at scheduled necropsies, each animal was killed by means of intraperitoneal injection of an overdose of a pentobarbital-based anesthetic (1:9 diluted Euthasol; [Virbac Ah Inc, Fort Worth, Tex] 0.2 mL/20 g body weight). Immediately after death, blood was collected by means of cardiac stick in an EDTA tube and centrifuged to isolate plasma, and samples were stored at −80°C. For detection of apoptotic cells, deparaffinized lung sections were stained with the TACS 2 TdT Fluorescein Kit (Trevigen, Gaithersburg, Md), and fluorescent TUNEL+ cells were detected for fluorescent staining, as described earlier. In all cases quantification of TUNEL positivity was carried out by a person unaware of slide identity. Grouped results were expressed as means ± SEMs. Data were analyzed by using GraphPad Prism software (GraphPad Software, San Diego, Calif). Grouped results were analyzed by using 2-way ANOVA. When significant main effects were detected (P < .05), the Fisher least significant difference test was used to determine differences between groups.Fig E2ABT-263 reduces MUC5AC expression without affecting the epithelial integrity of differentiated air-liquid interface cultures. A, Relative MUC5AC mRNA levels in differentiated HAECs treated with IL-13 and 1 μmol/L ABT-263 for 2 hours and quantified by using quantitative PCR. B, After differentiation of primary HAECs for 28 days, transepithelial electrical resistance (TEER) values were measured in air-liquid interface cultures treated with or without 10 ng/mL IL-13, 1 μmol/L ABT-263, or both. Data are shown as means ± SEMs (n = 3-5 per group). C, Micrographs showing Muc5ac+ (green) and Scgb1a1+ (red) airway cells in allergic mice treated with vehicle or ABT-263. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). D, Micrographs showing TUNEL positivity (green) in Scgb1a1+ (red) secretory cells in axial airways and relative fold change in TUNEL+ secretory cells. Nuclei were stained with DAPI (blue). *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Change in weight and clearance of ABT-263 from the lungs and blood of OVA-sensitized mice. A, Experimental design for ABT-263 aerosol exposure of allergic mice. B, ABT-263 concentrations in the lungs of mice at 6 and 24 hours after ABT-263 aerosol exposure, as determined by using HPLC. C, Blood plasma levels of ABT-263 at 6 and 24 hours after ABT-263 aerosol exposure, as determined by using HPLC. Data are shown as means ± SEMs (n = 20 per group).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Characterization of ABT-263 aerosolized particles generated from various formulationsFormulation (mg/mL)Total aerosol concentration (mg/L)ABT-263 aerosol concentration (μg/L)Total MMAD (GSD)Pulmonary dose (μg/kg)20.0799.40.75 μm (1.74)2040.08313.270.95 μm (2.39)2860.09820.090.95 μm (2.62)42100.15945.510.76 μm (1.67)96200.10749.821.18 μm (2.92)106400.13084.681.73 μm (3.11)180600.247183.362.06 μm (2.81)389 Open table in a new tab Table E2Characterization of ABT-263 aerosols for the targeted PDD of 50 μg/kgDayTotal aerosol concentration (mg/L)ABT-263 aerosol concentration (μg/L)Pulmonary dose (μg/kg)10.10230.13220.06725.35430.07725.65540.06922.84950.05014.030ABT-263 aerosols were generated with suspension concentrations of 6 and 20 mg/mL, and deposited doses were achieved through modulation of the exposure duration with 30 and 15 minutes of exposure, respectively. The particle size was determined 4 times during the study, with MMADs of 0.78 to 3.92 μm and GSDs of 2.71 μm or less. Open table in a new tab Table E3Characterization of ABT-263 aerosols for the targeted PDD of 250 μg/kgDayTotal aerosol concentration (mg/L)ABT-263 aerosol concentration (μg/L)Pulmonary dose (μg/kg)10.10256.624420.242228.124630.220209.522640.396375.540550.234176.0190ABT-263 aerosols were generated with suspension concentrations of 20 and 60 mg/mL with 60 and 15 minutes of exposure time, respectively. Open table in a new tab Table E4Characterization of ABT-263 aerosols in OVA-challenged Bik-deficient (Bik−/−) and Bik-sufficient (Bik+/+) miceDayTotal aerosol concentration (mg/L)ABT-263 aerosol concentration (μg/L)Total MMAD (GSD)PDD (μg/kg)10.07219.540.68 μm (1.87)4220.07520.570.69 μm (1.94)4430.08521.731.12 μm (2.65)4740.08723.240.69 μm (1.84)5050.08321.81NA47Particle size ranged from 0.68 to 1.12 μm MMAD, with an average of 0.80 μm. GSD ranged from 1.84 to 2.65 μm, with an average of 2.08 μm. Overall average PDD was 46 μg/kg (7% deposition fraction).NA, Not assessed. Open table in a new tab ABT-263 aerosols were generated with suspension concentrations of 6 and 20 mg/mL, and deposited doses were achieved through modulation of the exposure duration with 30 and 15 minutes of exposure, respectively. The particle size was determined 4 times during the study, with MMADs of 0.78 to 3.92 μm and GSDs of 2.71 μm or less. ABT-263 aerosols were generated with suspension concentrations of 20 and 60 mg/mL with 60 and 15 minutes of exposure time, respectively. Particle size ranged from 0.68 to 1.12 μm MMAD, with an average of 0.80 μm. GSD ranged from 1.84 to 2.65 μm, with an average of 2.08 μm. Overall average PDD was 46 μg/kg (7% deposition fraction). NA, Not assessed.