Am J Physiol Lung Cell Mol Physiol 308: L1212–L1223, 2015. First published April 17, 2015; doi:10.1152/ajplung.00042.2014.

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The relative balance of GM-CSF and TGF-␤1 regulates lung epithelial barrier function Christian E. Overgaard,1,2* Barbara Schlingmann,1* StevenClaude Dorsainvil White,1 Christina Ward,1,2 Xian Fan,1,6 Snehasikta Swarnakar,4 Lou Ann S. Brown,2,5 David M. Guidot,1,2,6# and X Michael Koval1,2,3# 1

Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University, Atlanta, Georgia; 2Emory Alcohol and Lung Biology Center, Emory University, Atlanta, Georgia; 3Department of Cell Biology, Emory University, Atlanta, Georgia; 4Drug Development Diagnostics and Biotechnology, CSIR-Indian Institute of Chemical Biology, Kolkata, India; 5Division of Neonatology, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia; 6Atlanta Veterans Affairs Medical Center, Decatur, Georgia Submitted 14 February 2014; accepted in final form 9 April 2015

Overgaard CE, Schlingmann B, Dorsainvil White S, Ward C, Fan X, Swarnakar S, Brown LA, Guidot DM, Koval M. The relative balance of GM-CSF and TGF-␤1 regulates lung epithelial barrier function. Am J Physiol Lung Cell Mol Physiol 308: L1212–L1223, 2015. First published April 17, 2015; doi:10.1152/ajplung.00042.2014.—Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-␤1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-␤1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-␤1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-␤1 alone caused an ⬃25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions (“spikes”) containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ⬃20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-␤1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-␤1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum ␣2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/ TGF-␤1. Critically, patients who survived had significantly higher GM-CSF/TGF-␤1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-␤1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-␤1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

* C. Overgaard and B. Schlingmann shared first authorship; # D. Guidot and M. Koval shared senior authorship. Address for reprint requests and other correspondence: M. Koval, 615 Michael St., Suite 205, Atlanta, GA 30322 (e-mail: [email protected]). L1212

granulocyte/macrophage colony-stimulating factor; lung epithelium; pulmonary edema; claudin; tight junctions ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is a severe form of acute lung injury (ALI) characterized by flooding of terminal lung airspaces with proteinaceous fluid and consequent respiratory failure. Even with comprehensive treatment in the ICU, including a lung-protective ventilator strategy, mortality rates remain unacceptably high at 40 – 60%. Moreover, the incidence of ARDS is increased two- to fourfold in individuals with alcohol use disorders (38, 39). There is compelling experimental evidence that chronic alcohol ingestion increases expression of transforming growth factor-␤1 (TGF-␤1) in the lung and that its release and activation into the airspace during endotoxemia can disrupt the normally tight alveolar epithelial barrier (2, 3). These findings are consistent with other studies that implicate TGF-␤1 in degrading lung epithelial barrier function and playing an active role in the pathophysiology of ALI (2, 3, 49). In contrast to the pathophysiological effects of TGF-␤1 on lung epithelia, granulocyte/macrophage colony-stimulating factor (GM-CSF) production by the alveolar epithelium promotes epithelial barrier integrity and surfactant production. Although more often considered for its ability to promote bone marrow recovery of granulocyte production following cytotoxic chemotherapy, GM-CSF was first identified from mouse lung extracts (4, 5, 50, 53). In fact, when a GM-CSF knockout mouse was generated, the mouse phenotype was completely unexpected; specifically, bone marrow function was completely normal, but the mice developed a lung pathology essentially identical to pulmonary alveolar proteinosis (PAP) in humans (11). This serendipitous finding led to experimental and clinical studies elucidating PAP as an autoimmune disorder in which neutralizing antibodies to GM-CSF interfere with receptor binding and subsequent signaling through its master transcription factor, PU.1. As a result, lung epithelia and macrophages are impaired, which leads to a progressive accumulation of dysfunctional surfactant and eventual death from respiratory failure. Consistent with GM-CSF signaling within

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GM-CSF/TGF-␤1 RATIO AND LUNG BARRIER FUNCTION

the airspace being critically required for epithelial and macrophage function, ARDS survival correlates with the levels of GM-CSF in lung lavage fluid (34). In the same experimental model of chronic alcohol ingestion in which a pathophysiological role for TGF-␤1 was identified, we also determined that GM-CSF signaling is inhibited through downregulation of GM-CSF receptor expression and subsequent dampening of PU.1 expression and nuclear binding (22). Also, recombinant GM-CSF treatment restores lung barrier function to alcoholfed rats and normalizes lung liquid clearance during endotoxemia (45). Taken together, these studies suggest that TGF-␤1 and GM-CSF have opposing effects in the healthy lung, where the effects of GM-CSF dominate and maintain a tight alveolar epithelial barrier but are dysregulated in ARDS/ALI such that TGF-␤1 signaling asserts a dominant role and contributes to the alveolar epithelial permeability and subsequent edema that characterize the syndrome. To test the hypothesis that the relative balance between GM-CSF and TGF-␤1 signaling regulates lung epithelial barrier function, we assessed their separate and combined effects in vitro using two different lung epithelial model systems. GM-CSF was found to antagonize the ability of TGF-␤1 to impair epithelial barrier function. Therefore, we extended these findings to the clinical setting and determined that the higher ratios of GM-CSF/TGF-␤1 in lung lavage fluids of critically ill patients with ventilatorassociated pneumonia (VAP) correlated with improved lung epithelial barrier function, as assessed by leakage of serum proteins into the airspaces. Critically, higher GM-CSF/TGF-␤1 ratios correlated with increased patient survival. Taken together, this study provides novel evidence that the relative balance of GM-CSF and TGF-␤1 regulates lung barrier function and that determining their relative ratios within the airspace may have utility in the diagnosis and treatment of ARDS/ALI. MATERIALS AND METHODS

Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO). Activin receptor-like kinase 5 (ALK5) inhibitors (SB431542, A8301) and rabbit anti-␤-actin IgG were obtained from Sigma. Human GM-CSF and TGF-␤1 were from Preprotech (Rocky Hill, NJ). Calcein, 10-kDa Texas Red dextran, and rabbit anticlaudin-4 (no. 364800), anti-claudin-18 (no. 700178), and zonula occludens (ZO)-1 IgG (no. 339100) were from Life Technologies (Rockville, MD). Mouse anti-actin was obtained from Sigma (no. A53116). Mouse anti-␤-catenin (no. 610153) was from BD Biosciences (San Jose, CA). Horseradish peroxidase-conjugated goat antirabbit (no. 111-035-144) and goat anti-mouse IgG (no. 115-035-166) as well as Cy2-coupled goat anti-rabbit (no. 111-225-1440) and Cy3-conjugated goat anti-mouse IgG (no. 115-165-1660) were from Jackson ImmunoResearch (West Grove, PA). Human subjects. Before implementation, this project was reviewed and approved by both the Emory University Institutional Review Board and the Atlanta Veterans Affairs Medical Center Research and Development Committee. After informed written consent, a bronchoalveolar lavage (BAL) was performed on patients with VAP within the first 24 h of diagnosis. GM-CSF, TGF-␤1, ␣2-macroglobulin, and IgM content of the BAL were determined by ELISA (R & D Systems, Minneapolis, MN). BAL GM-CSF content was in the range of 0.5–113.3 pg/ml; TGF-␤ was in the range of 0.1– 648.8 pg/ml. Samples where either GM-CSF or TGF-␤ were below the limit of detectability were not included in the analysis. BAL concentrations of glutathione (GSH) and glutathione disulfide (oxidized glutathione

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or GSSG) were determined by HPLC as previously described (18, 19). Using these concentrations for GSH and GSSG, the redox potentials (Eh) of the GSH/GSSG pools in the lavage fluid were calculated with the Nernst equation [Eh ⫽ Eo ⫹ RT/nF ln[disulfide]/([thiol1] [thiol2])]. Eo is the standard potential for the redox couple, R is the gas constant, T is the absolute temperature, n is 2 for the number of electrons transferred, and F is Faraday’s constant. The standard potential Eo for the 2 GSH/GSSG couple was ⫺264 mV but was adjusted ⫹5.9 mV per 0.1 change in pH. Cell culture. Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University, as approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Adult male Sprague-Dawley rats were fed the Lieber-DeCarli isocaloric liquid diet containing 36% of the calories as either ethanol or a maltindextrin substitution as previously described (22). Rat type II alveolar epithelial cells (AECs) were isolated as previously described using the method of Dobbs et al. (10) with modifications (1). AECs were cultured in DMEM (Sigma) containing 10% FBS, 100 U/ml penicillin and 10 mg/ml streptomycin (Sigma), and 0.25 ␮g/ml amphotericin B (Life Technologies). Cells were seeded at 7.5 ⫻ 105 cells/well in 12-well 0.4-␮m Transwell permeable supports (Corning Life Sciences, Tewksbury, MA) coated with rat tail type I collagen (Roche Diagnostics, Branchburg, NJ). Cells were cultured for 5 days in DMEM ⫹ 10% FBS before further experimental manipulation to allow the cells to form a model type I cell monolayer (28). Normal immortalized 16HBE14o- human bronchiolar epithelial cells (HBE cells) were acquired from M. Hershenson Laboratory (University of Michigan) (8, 41). HBE cells were maintained on tissue culture plastic in DMEM ⫹ 10% FBS and used at passages 10 –20. For experimental measurements, HBE cells were seeded (5 ⫻ 105 cells/well) on Transwell permeable supports. HBE cells treated with alcohol were incubated for 2 days in medium supplemented with 60 mM ethanol and maintained in a Billups-Rothenberg (Del Mar, CA) isolation chamber that was vapor equilibrated using ethanol-containing medium (16). Control cells were placed in an identical chamber without alcohol. Barrier function measurements. Transepithelial resistance (TER) was measured using an EVOM Ohmmeter (World Precision Instruments, Sarasota, FL), as previously described (9). For examining the effects of GM-CSF and TGF-␤ on HBE barrier function, cells were cultured for 4 days in DMEM ⫹ 10% FBS, switched to DMEM ⫹ 1% FBS for 2 days, and then incubated in serum-free DMEM for 16 h before further treatment. Changing from 1% FBS to serum-free medium decreased TER of HBE cells from 659.0 ⫾ 25.1 to 493.3 ⫾ 24.2 Ohm ⫻ cm2. Changing from 10% FBS to 1% FBS media decreased TER of AEC from 541.7 ⫾ 25.8 to 335.7 ⫾ 19.1 Ohm ⫻ cm2. For AECs, cells were cultured for 4 days in DMEM ⫹ 10% FBS, switched to DMEM ⫹ 1% FBS for 1 day, and then incubated with cytokines in DMEM ⫹ 1% FBS for 16 h. In experiments using pharmacological agents or cytokines, agents were added to both the apical and basolateral media. Paracellular dye permeability was assessed by simultaneous measurement of the diffusion of two differentsized fluorescent dyes across the cell monolayer. Briefly, the medium in the bottom chamber was replaced with DPBS ⫹ 0.675 mM CaCl2 ⫹ 0.2 mM MgCl2, and the medium in the top chamber was replaced with DPBS ⫹ Ca2⫹ ⫹ Mg2⫹ containing 0.1 mg/ml calcein (0.62 kDa) and 1.0 mg/ml Texas Red dextran (10 kDa). The cells were further incubated at 37°C for 2 h. At varying intervals, DPBS from the lower chamber was removed, and the amount of fluorophore that diffused into the lower chamber was measured using a multiwell plate fluorimeter (PE Biosystems, Foster City, CA). Claudin-18 siRNA treatment. AECs were seeded at 2 ⫻ 105 cells/well in 12-well 0.4-␮m Transwell permeable supports (Corning Life Sciences). On day 1, the cells were transfected as described (14) using HiPerFect (Qiagen, Gaithersburg, MD) with 30 nM siRNA duplexes from Sigma-Aldrich that were either specific for rat clau-

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din-18 (cldn-18 siRNA 1: 5=- GUGAUGAUGUGCAUUGCUU - 3=; siRNA 2: 5=- CCAUUUACAGGUUAAGAGA - 3=) or control siRNA (Sigma MISSION Universal siRNA negative control; no. SIC001). The cells were cultured for 4 days in DMEM ⫹ 10% FBS, switched to DMEM ⫹ 1% FBS for 1 day, and then incubated with 25 ng/ml GM-CSF in DMEM ⫹ 1% FBS for 16 h. TER was measured before and after cytokine treatment. To measure claudin-18 mRNA, total RNA was extracted using the Qiagen RNeasy Mini kit. Reverse transcription and real-time PCR (Bio-Rad, Hercules, CA) were performed as previously described using 5=-TGTGGAGCACTCAAGACCTG-3= and 5=-AGATGGACACGAGGATACCG-3= as forward and reverse primers, respectively (14). Immunoblot. Cells on permeable supports were harvested and lysed in 2⫻ sample buffer containing 50 mM DTT, resolved by SDSPAGE, transferred to Immobilon membranes (Millipore, Billerica, MA), and blotted using primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG and chemiluminescence reagent (GE Healthcare, Pittsburgh, PA). All blots were loaded with equal protein determined by DC Protein Assay (Bio-Rad). Additional normalization for protein content was done using parallel samples analyzed for actin. Blots were imaged and quantified using a ChemiDoc XRS⫹ Molecular Imaging System (Bio-Rad) and analyzed using Image Lab 2.0.1 software (Bio-Rad). Immunofluorescence. Immunofluorescence staining was performed as previously described (9, 54). Cells on Transwells were washed with DPBS with Ca2⫹/Mg2⫹ three times, fixed in MeOH/acetone 1:2 for 2 min at room temperature, washed three times with DPBS with Ca2⫹/Mg2⫹, once with DPBS with Ca2⫹/Mg2⫹ ⫹ 0.5% Triton X-100, and then once with DPBS with Ca2⫹/Mg2⫹ ⫹ 0.5% Triton X-100 ⫹ 5% normal goat serum. Cells were incubated with primary anti-rabbit and/or anti-mouse antibodies in DPBS with Ca2⫹/Mg2⫹ ⫹ 5% normal goat serum for 1 h, washed, incubated with Cy2-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) in DPBS with Ca2⫹/Mg2⫹ ⫹ 5% normal goat serum, washed, and then mounted in MOWIOL (Kuraray, Houston, TX) under a glass coverslip. Cells processed for labeling of F-actin with rhodamine phalloidin were processed in a comparable manner but were also fixed in 4% paraformaldehyde for 15 min at room temperature before MeOH/acetone treatment. Cells were imaged by phase-contrast and fluorescence microscopy using an Olympus IX70 with a U-MWIBA filter pack (BP460-490, DM505, BA515550) or U-MNG filter pack (BP530-550, DM570, BA590-800⫹) or by confocal immunofluorescence microscopy using an Olympus Fluoview FV1000 system. Minimum and maximum intensities were adjusted for images in parallel so that the intensity scale remained linear to maximize dynamic range. To quantify tight junction spikes, AECs immunostained for claudin-18 were scored as the percentage of cells containing five or more claudin-18-positive spikes perpendicular to the tight junction that were oriented toward the cell nucleus. Data were from eight fields containing 25–51 cells/field. Statistical analyses. Survival outcome for human subject data was determined, and statistical significance was assessed using the MannWhitney U-test (n ⫽ 16 for subjects who did not survive and n ⫽ 13 for subjects who survived). Simple comparisons were made using unpaired two-tailed Student’s t-test for parametric data or MannWhitney test for nonparametric data. Multiple comparisons were made using one-way ANOVA with Student-Newman-Keuls posttest. Linear correlation was determined using Pearson’s correlation coefficient. P ⱕ 0.05 was considered statistically significant. All data are presented as means ⫾ SD. RESULTS

Inhibitors of TGF-␤ signaling antagonize the deleterious effects of alcohol on barrier function. Previously, we found that rats on a chronic alcohol diet have impaired lung barrier function, and this persists in isolated primary AECs (18, 19).

Here, we confirmed that primary AECs from alcohol-fed rats had lower TER in vitro than monolayers derived from rats on a control diet (Fig. 1, A and B). Given the central role of TGF-␤1 in alcoholic lung disease (2, 3, 37), we examined the effect of two different TGF-␤1 signaling inhibitors that target ALK5 kinase. As shown in Fig. 1, alveolar epithelial monolayers from alcohol-fed rats treated in vitro with either A8301 or SB431542 showed a dose-dependent increase in TER, implicating TGF-␤1 in alcoholinduced barrier dysfunction. To extend these observations to a human lung epithelial cell line, we tested whether cultured HBE cells were sensitive to alcohol treatment in vitro. HBE cells exposed to 60 mM (0.24%) alcohol in medium for 3 days showed an 18.0 ⫾ 1.8% to 23.6 ⫾ 1.9% decrease in TER (Fig. 1, C and D, respectively). Consistent with primary AECs, treatment with either ALK5 inhibitor restored the TER of alcohol-exposed HBE cells to control levels, indicating that bronchiolar epithelial tight junctions were also sensitive to TGF-␤1 (Fig. 1, C and D). Taken together, these findings implicate TGF-␤1 signaling in the pathophysiological effects of alcohol on lung epithelial barrier function. Differential effects of TGF-␤1 and GM-CSF on lung epithelial cell barrier function. To assess the effects of TGF-␤1 on lung epithelial barrier function, HBE cells and AECs were treated with varying amounts of recombinant TGF-␤1 or GM-

Fig. 1. Transforming growth factor (TGF)-␤1 signaling mediates alcoholinduced decreases in lung epithelial barrier function. A and B: primary alveolar epithelial cells (AECs) isolated from control-fed (light gray bars) or alcoholfed rats (dark gray bars) were cultured as monolayers on Transwell supports and treated for 24 h with vehicle control or an activin receptor-like kinase 5 (ALK5) inhibitor [either A8301 (A) or SB43152 (B)], after which transepithelial resistance (TER) was measured. Cells from alcohol-fed rats had significantly lower TER than monolayers derived cells from control-fed rats (#P ⬍ 0.05). In contrast, treatment with either of the ALK5 inhibitors increased barrier function of monolayers derived from alcohol-fed rats in a dosedependent manner (*P ⬍ 0.05 vs. untreated alcohol cells; n ⫽ 4 – 8). C and D: human bronchiolar epithelial (HBE) cells cultured on Transwells were incubated in control medium or medium containing 60 mM alcohol for 2 days and then treated with either a vehicle control or an ALK5 inhibitor A8301 (C) or SB43152 (D) for an additional 24 h. Consistent with the findings in primary AEC monolayers (A and B), alcohol-treated HBE cell monolayers had significantly lower TER than untreated HBE cells or alcohol-treated HBE cells that were further treated with ALK5 inhibitors (*P ⬍ 0.05).

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GM-CSF is a critical cytokine previously shown to promote lung epithelial barrier function (22, 45). Consistent with these previous findings, GM-CSF treatment increased the TER of HBE cell monolayers by ⬃20% over control (Fig. 2A) and increased the TER of AECs by ⬃30 – 40% (Fig. 2B). This effect was saturable because HBE cells treated with 100 ng/ml GM-CSF alone showed a 22.3 ⫾ 16.1% (n ⫽ 7) increase in TER, which was comparable to the increase for HBE cells treated with GM-CSF/TGF-␤1 at ratios of 10:1 (20.7 ⫾ 15.9%, n ⫽ 6) and 50:1 (22.2 ⫾ 11.4%, n ⫽ 6). AECs showed increased TER at lower dosages (25 ng/ml) than that required to see an effect on HBE cells (100 ng/ml), indicating that GM-CSF is more effective at promoting AEC barrier function than for HBE cells. TER measures the electrophysiological barrier including both paracellular and transcellular components. By contrast, paracellular flux to small molecules is another measure of epithelial barrier function (25). Consistent with the effects of TGF-␤1 and GM-CSF on TER, TGF-␤1 increased the paracellular flux of two fluorescent molecules, calcein (⬃0.6 kDa) and Texas Red dextran (10 kDa), compared with control, whereas GM-CSF treatment decreased paracellular flux (Fig. 3). Taken together, these results further demonstrate that TGF-␤1 and GM-CSF have opposite effects on lung epithelial barrier function.

Fig. 2. Granulocyte/macrophage colony-stimulating factor (GM-CSF) antagonizes the effects of TGF-␤1 to promote barrier function. HBE cell monolayers (A) or AEC monolayers (B) were treated overnight with varying levels of TGF-␤1 and GM-CSF as described in MATERIALS AND METHODS and then assessed for changes in TER. TGF-␤1 had a significant effect on TER, with HBE cell monolayers being more sensitive to lower doses of TGF-␤1 than AEC monolayers (#P ⬍ 0.05; n ⫽ 3– 4). Addition of GM-CSF significantly increased TER regardless of whether or not TGF-␤1 was present (*P ⬍ 0.05; n ⫽ 3– 4). AEC monolayers were more sensitive to the protective effects of GM-CSF than were HBE cell monolayers. C: combined HBE () and AEC () monolayer data plotting % control TER vs. GM-CSF/TGF-␤1 ratio. By regression analysis, there was a linear relationship between % TER and GM-CSF/TGF-␤ ratio in the range of 0.0 –12.5, which plateaued at higher GM-CSF/TGF-␤ ratios (e.g., 50:1). Pearson’s correlation coefficient (R) showed a significant positive correlation (P).

CSF, as described in MATERIALS AND METHODS (Fig. 2). TGF-␤1 alone decreased the TER of HBE cells by 20 –25% in a dose dependent manner (Fig. 2A) comparable to the effect of alcohol. TGF-␤1 also decreased the TER of AECs by 10 –15%, suggesting that AECs were less sensitive to TGF-␤1 than HBE cells (Fig. 2B). In contrast to the deleterious effects of TGF-␤1,

Fig. 3. Reciprocal effects of TGF-␤1 and GM-CSF on paracellular flux across HBE cell monolayers. Paracellular dye permeability was assessed by simultaneous measurement of the diffusion of 2 different-sized fluorescent dyes across the cell monolayers, calcein (0.6 kDa; A) and Texas Red dextran (10 kDa; B). Overnight treatment with TGF-␤1 (20 ng/ml) significantly increased paracellular flux of both calcein and Texas Red dextran vs. untreated controls (*P ⬍ 0.05). In contrast, overnight treatment with GM-CSF (100 ng/ml) decreased paracellular flux of calcein and Texas Red dextran (*P ⬍ 0.05).

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Although GM-CSF improved lung epithelial barrier function, it was not known whether GM-CSF could directly antagonize the effects of TGF-␤1 on TER. Therefore, we examined changes in TER to determine barrier function of HBE cells and AECs following treatment with different combinations of GMCSF and TGF-␤1. As shown in Fig. 2, GM-CSF was effective in increasing TER in the presence of TGF-␤1 for both HBE cells and AECs, even at concentrations of TGF-␤1 that when used alone significantly decreased barrier function. Note that, in the range where the GM-CSF/TGF-␤ ratio was ⬍12.5, we found that there was a linear relationship with increased TER (Fig. 2C; R ⫽ 0.914), suggesting that the GM-CSF/TGF-␤ ratio is a critical parameter in regulating lung epithelial barrier function regardless of overall cytokine concentration. In addition, at GM-CSF/TGF-␤ ratios ⬎15, the beneficial effect of GM-CSF plateaued to a level that was comparable to cells treated with GM-CSF alone (Fig. 2, A and B). Taken together these data suggest the hypothesis that the relative signaling influences of GM-CSF to TGF-␤1 have the capacity to regulate lung epithelial barrier function. Claudin family tight junction proteins form the structural basis for paracellular permeability barrier (25). Because AECs are the major lung epithelial cells required for the permeability barrier needed to prevent airway edema and flooding, we examined the effects of TGF-␤1 and GM-CSF on two prominent AEC claudins, claudin-4 and claudin-18, by immunoblot (Fig. 4). TGF-␤1 caused a decrease in total claudin-18, which correlated with decreased TER (Fig. 2B). In contrast, claudin-18 increased in response to GM-CSF; this was particularly significant in AECs treated with 5 ng/ml TGF-␤1 and 25 ng/ml GM-CSF. On the other hand, total claudin-4 in AECs was unchanged in response to either TGF-␤1, GM-CSF, or both (Fig. 4C). As there was increased claudin-18 protein associated with GM-CSF, we determined whether depleting claudin-18 using siRNA would antagonize the effects of GM-CSF on AEC barrier function. Consistent with previous reports (31), specific siRNAs decreased claudin-18 mRNA and concomitantly decreased the barrier function of AECs at baseline (Fig. 5). Nonetheless, when AECs treated with claudin-18 siRNA were subsequently treated with 25 ng/ml GM-CSF, there was still a significant increase in TER that was comparable to control cells. Surprisingly, GM-CSF treatment was associated with an unexpected decrease in claudin-18 mRNA, even in control siRNA-treated cells (Fig. 5B), despite the observation that GM-CSF increased claudin-18 protein (Fig. 4). Given that the effects of GM-CSF on claudin mRNA and protein differed, these data support a posttranslational mechanism for the effects of GM-CSF (and TGF-␤1) on lung epithelial barrier function. To address this possibility, we examined the morphological changes to tight junctions induced by TGF-␤1 using AECs immunostained for claudin-18 and for the tight junction scaffold protein ZO-1. As shown in Fig. 6, claudin-18 and ZO-1 showed a high level of colocalization at areas where cells were in direct contact. Moreover, there were areas where claudin-18 was organized into structures we refer to as spikes (55); cells containing spikes were particularly prevalent when cells were treated with TGF-␤1 (Fig. 6, D–F and J–L). This was confirmed by quantitative analysis, where overnight treatment with 5 ng/ml TGF-␤1 resulted in a significant increase in the fraction of cells with spikes containing claudin-18 [16.9 ⫾ 1.5% spike-containing cells (n ⫽ 8) vs. untreated controls,

Fig. 4. Reciprocal effects of TGF-␤1 and GM-CSF on alveolar epithelial claudins. A: AEC monolayers were treated overnight with varying concentrations of TGF-␤1 or GM-CSF and then harvested. Claudin-18 and claudin-4 proteins were determined by immunoblot, quantified by densitometry, and normalized to actin. TGF-␤1 treatment significantly decreased AEC claudin-18 (B) but had no effect on claudin-4 (C) (#P ⬍ 0.05; n ⫽ 4). Conversely, GM-CSF treatment increased claudin-18 protein in AEC monolayers (*P ⬍ 0.05; n ⫽ 4).

11.7 ⫾ 1.8% spike containing cells (n ⫽ 8), P ⬍ 0.05]. A comparable effect on tight junction morphology associated with paracellular leak has previously been observed in AECs isolated from alcohol-fed rats (14) and AEC cells treated with BMS-345541 (55). Note that GM-CSF did not significantly decrease spike formation by AECs either in the absence or presence of TGF-␤1 (10.6 ⫾ 0.9%, n ⫽ 8, vs. 14.8 ⫾ 1.3%, n ⫽ 8). Thus, under these experimental conditions, GM-CSF was unable to fully antagonize the effects of TGF-␤1 on AEC tight junction morphology despite the ability to improve barrier function. As alterations in F-actin have been shown to affect alveolar barrier function and morphology (30, 32) and F-actin previously was associated with tight junction spikes (55), we examined whether TGF-␤1 and GM-CSF affected the organization of F-actin in AECs. As shown in Fig. 7, AECs treated overnight with 5 ng/ml TGF-␤1 alone showed prominent actin

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00042.2014 • www.ajplung.org

GM-CSF/TGF-␤1 RATIO AND LUNG BARRIER FUNCTION

Fig. 5. Increased alveolar barrier function by GM-CSF is not linked to an increase in claudin-18 mRNA. AEC monolayers were transfected with either control siRNA or siRNA directed against rat claudin-18. The cells were then either treated with vehicle control (untreated) or 25 ng/ml GM-CSF for 16 h, and TER was measured (A). In the absence of GM-CSF, claudin-18 siRNA significantly decreased TER (*P ⬍ 0.05 vs. cells transfected with control siRNA). GM-CSF caused an increase in TER regardless of whether the cells were treated with control or claudin-18 siRNA (#P ⬍ 0.05 vs. untreated, matched cells). The cells were then harvested, and claudin-18 mRNA was measured (B). In monolayers that were not treated with GM-CSF, siRNA knockdown significantly decreased claudin-18 mRNA (*P ⬍ 0.05 vs. cells transfected with control siRNA). Surprisingly, GM-CSF also caused a significant decrease in claudin-18 mRNA (#P ⬍ 0.05 vs. untreated, matched cells), suggesting posttranslational control of claudin-18 protein by AECs.

stress fibers (Fig. 7B) organized into aggregates reminiscent of those seen in cells isolated from claudin-18-deficient mice (32). This contrasts with untreated AECs that typically favor cortical actin over filamentous actin (30, 55). Moreover, the tight junction spikes in cells treated with TGF-␤1 alone showed F-actin colocalizing with claudin-18 (Fig. 7, G–I). By contrast, simultaneous treatment with 5 ng/ml TGF-␤1 and 25 ng/ml GM-CSF attenuated the appearance of F-actin stress fibers (Fig. 7E). Moreover, spikes containing claudin-18 showed much weaker colocalization with F-actin in cells treated with GM-CSF (Fig. 7, J–L). As actin plays a critical role in enhancing tight junction protein turnover (21, 57), our data suggest a mechanism whereby GM-CSF increased barrier function by stabilizing tight junctions as opposed to changing the relative amount of protein, analogous to the effects of keratinocyte growth factor on AECs (30, 32). We also examined cells immunostained for claudin-4 and ␤-catenin (Fig. 8). In contrast to claudin-18, AECs showed

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little, if any, change in claudin-4 localization, regardless of whether they were cytokine treated or not. Consistent with this, HBE cells did not exhibit changes in claudin-4 localization in response to either TGF-␤1 or GM-CSF (Fig. 9). ␤-Catenin, an adherens junction marker, also showed no changes in response to cytokine treatment (Fig. 8). Note also that claudin-4 and ␤-catenin did not extensively colocalize, indicating that these two proteins represented distinct AEC junctions. Also, this is consistent with our previous study where we found that junctional adhesion molecule A depletion had no effect on alveolar claudin-4 in situ, whereas assembly of claudin-18 into alveolar junctions was specifically impaired (36). The GM-CSF/TGF-␤1 ratio in BAL fluid of patients with VAP correlates with lung barrier function and patient survival. Overall, our results in vitro suggest that there are reciprocal effects on lung epithelial barrier function by TGF-␤1 and GM-CSF. To extend these experimental findings to a relevant clinical setting, we tested whether the ratio of GM-CSF to TGF-␤1 correlated with patient outcome in critically ill patients with documented VAP. BALs were collected and analyzed by ELISA for GM-CSF and TGF-␤1 and for two different serum proteins as markers of compromised lung barrier: ␣2-macroglobulin (a tetramer with total molecular weight of 812 kDa) and IgM (molecular weight ⫽ 900 kDa). As shown in Fig. 10, A and B, there was an inverse correlation between the GM-CSF/TGF-␤1 ratio and the BAL levels of ␣2-macroglobulin and IgM, consistent with the hypothesis that relatively increased GM-CSF has a protective effect and preserves lung barrier function. We also examined whether there was a relationship between the GM-CSF/TGF-␤1 ratio and the glutathione redox potential in the BAL by HPLC (Fig. 10C). There was a significant correlation between the redox potential and the GM-CSF/TGF-␤1 ratio. Specifically, a relatively more oxidized airspace correlated with relatively lower GM-CSF/ TGF-␤1. The GM-CSF/TGF-␤1 ratios were then correlated with patient survival. As shown in Fig. 10, the median GM-CSF/ TGF-␤1 ratio for those patients with VAP that survived was 0.175 with a 25–75% range of 0.0610 to 0.3410. In contrast, the median GM-CSF/TGF-␤1 ratio for those patients with VAP that did not survive was 0.0310 with a 25–75% range of 0.0073 to 0.0640. This difference in survival was statistically significant, as determined using the Mann-Whitney U-test, where P ⱕ 0.001 between the two groups (n ⫽ 16 for subjects who did not survive and n ⫽ 13 for subjects who survived). Although patient survival is admittedly a surrogate marker of the severity of ALI and is obviously affected by other comorbid factors, these results are nevertheless provocative and taken together are consistent with the hypothesis that the relative signaling influences of GM-CSF to TGF-␤1 within the lung airspace regulate the lung epithelial barrier. DISCUSSION

In this translational study, we determined that GM-CSF antagonizes the barrier-disrupting effects of TGF-␤1 on the lung epithelium and that the relative balance of these cytokines regulates lung barrier integrity. Although previous studies had shown that TGF-␤1 and GM-CSF individually have opposite effects on lung epithelial barrier function (2, 3, 22, 40, 44 – 46), no studies had evaluated whether or not these cytokines had

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Fig. 6. TGF-␤1 induced changes to alveolar epithelial tight junctions. AEC monolayers were treated overnight with either 5 ng/ml TGF-␤ (D–F), 25 ng/ml GM-CSF (G–I), or both (J–L). Untreated AEC monolayers are in A–C. The cells were then fixed and immunostained for claudin-18 (A, D, G, J) and zonnula occludens (ZO)-1 (B, E, H, K). Merged images are shown in C, F, I, and L, where claudin-18 is green and ZO-1 is red. AEC monolayers showed junction-localized claudin-18 and ZO-1 as well as spikes enriched for claudin-18 (arrowheads), which were more prominent in monolayers treated with TGF-␤1 (D–F and J–L). Bar ⫽ 10 ␮m.

opposing effects within the airspace and, in parallel, how the relative balance between them regulated epithelial barrier function. Importantly, GM-CSF antagonized the ability of TGF-␤1 to impair barrier function in model human epithelial monolayers (Fig. 2). In parallel, in a relevant clinical population of critically ill patients, the relative ratio of GM-CSF to TGF-␤1 within the alveolar space was significantly higher for patients who survived compared with nonsurviving patients (Fig. 10). Taken together, these experimental and clinical results suggest that the relative balance of these opposing cytokines has profound implications for lung epithelial barrier function. Their relative ratio in the airspace, which is a dilution-independent measurement that can be performed in critically ill patients, may have diagnostic and/or prognostic utility. Alcohol abuse has been identified in multiple studies to independently increase the risk of ARDS/ALI (33, 38, 39, 52). Experimentally, chronic alcohol ingestion causes lung epithelial barrier dysfunction both in vitro and in vivo (18), and

increased permeability is associated with significant perturbations in tight junction proteins (15). Clinically, otherwise healthy alcoholics have evidence of increased lung airway edema (7, 39). Although there are multiple mechanisms by which alcohol abuse disrupts lung epithelial barrier function, there is compelling evidence that aberrant expression and activation of TGF-␤1 play a major role (7, 39). Furthermore, TGF-␤1 causes epithelial barrier disruption and lung injury even in the absence of prior alcohol ingestion (46, 49). Therefore, we first determined that blocking TGF-␤1 signaling with inhibitors of the ALK5 kinase subunit of the TGF-␤ receptor complex improved epithelial barrier function in cell monolayers derived from alcohol-fed rats and in human bronchial epithelial cell monolayers exposed to alcohol in vitro (Fig. 1). We next tested the hypothesis that TGF-␤1 and GM-CSF signaling interact in a dynamic and inverse manner in regulating lung barrier function. Specifically, we had previously

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Fig. 7. TGF-␤1 increased localization of F-actin to spikes containing claudin-18, and this effect was antagonized by GM-CSF. AEC monolayers were cultured and treated overnight with either 5 ng/ml TGF-␤ (A–C, G–I), or both 5 ng/ml TGF-␤1 and 25 ng/ml GM-CSF (D–F, J–L). The cell monolayers were then fixed and immunostained for claudin-18 (A, D, G, J), and F-actin was imaged using rhodamine-phalloidin (B, E, H, K). Merged images are shown in C, F, I, and L, where claudin-18 is green and actin is red. Images in G–I and J–L represent magnifications of the images in A–C and D–F, respectively as indicated by the inset box. AEC monolayers treated with TGF-␤1 showed an enhancement of actin stress fibers (B, arrowheads) as well as F-actin associated with claudin-18 in spikes (G–I, arrowhead). By contrast, claudin-18 in AEC monolayers cotreated with TGF-␤1 and GM-CSF had less prominent actin stress fibers, and tight junction spikes showed little colocalization with F-actin (arrow). Bar ⫽ 10 ␮m (A–F); bar ⫽ 5 ␮m (G–L).

determined that chronic alcohol ingestion dampens GM-CSF receptor expression and signaling (22) and that treatment with recombinant GM-CSF restores barrier function in the alveolar epithelium of alcohol-fed animals both in vitro and in vivo (45). These previous studies suggested a dynamic balance between TGF-␤1 and GM-CSF in mediating lung barrier function that is perturbed by chronic alcohol ingestion. Therefore, we examined the opposing effects of TGF-␤1 and GMCSF on lung epithelial cells in the absence of alcohol priming. We determined that TGF-␤1 treatment predictably decreased barrier function and that GM-CSF treatment tightened the barrier more than that of control monolayers (Fig. 2). In parallel, cotreatment with TGF-␤1 and GM-CSF showed that it is the relative balance of these cytokines more than their individual concentrations that modulate epithelial barrier function (Fig. 2C). Finally, we extended these experimental findings and determined that the relative GM-CSF/TGF-␤1 ratio in the alveolar space of patients with VAP correlated positively

with improved lung epithelial barrier function to serum proteins and patient survival (Fig. 10). These findings provide novel evidence that the relative balance of GM-CSF and TGF-␤ signaling regulates the epithelial barrier in the healthy lung and that a shift from GM-CSF to TGF-␤1 signaling during severe stresses may play a critical role in determining the severity of ARDS/ALI. Lung barrier function involves both a tight paracellular barrier to solute flow and active transcellular efflux of salt and water to maintain an air-liquid interface well suited for gas exchange. Note that chronic alcohol ingestion actually increases the active transport of salt and water across the alveolar epithelium (17, 42), which likely counterbalances the increased paracellular permeability in the alcoholic lung. Thus alcohol abuse alone does not cause pulmonary edema but rather renders the lung susceptible to acute edematous injury. Although paracellular permeability is regulated by multiple mechanisms, it is clear that claudins are required to regulate the tight

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Fig. 8. TGF-␤1 and GM-CSF had little effect on claudin-4 and ␤-catenin localization. AEC monolayers were cultured and treated overnight with either 5 ng/ml TGF-␤1 (D–F), 25 ng/ml GM-CSF (G–I), or both (J–L). Untreated monolayers are in A–C. The cell monolayers were then fixed and immunostained for claudin-4 (A, D, G, J) and ␤-catenin (B, E, H, K). Merged images are shown in C, F, I, and L, where claudin-4 is green and ␤-catenin is red. AEC monolayers showed differential localizations of claudin-4 and ␤-catenin that were unchanged by treatment with either GM-CSF or TGF-␤ (D–F and J–L). Bar ⫽ 10 ␮m.

junction barrier, particular to regulation of paracellular flux of macromolecules. Claudin-18 is central to alveolar barrier function. We found that the relative amount of claudin-18 protein in AECs increased as the relative ratio of GM-CSF to TGF-␤1 increased (Fig. 4). However, this was not regulated at the level of transcription, as claudin-18 mRNA actually decreased in cells treated with GM-CSF (Fig. 5). This pointed to a posttranslational mechanism in which GM-CSF regulated claudin-18 protein content, most likely attributable to a change in claudin-18 turnover. Consistent with this model, Ramierez et al. (47) have shown that glycogen synthase kinase-3␤ inhibitors increased claudin-5 protein in endothelial cells by increasing protein half-life while having no effect on claudin-5 mRNA. The factors that regulate claudin-18 half-life in AECs remain to be determined. Claudin turnover is primarily controlled by changes in tight junction assembly (26). Alveolar tight junctions that are dis-

ordered by chronic alcohol exposure exhibit strand breaks and other morphological abnormalities, suggesting a defect in tight junction assembly (13, 15). In addition, treatment of uninflamed AECs with an NF-␬B inhibitor, BMS-345541, induces formation of tight junction spikes associated with barrier dysfunction (55). In BMS-345541-treated AECs, claudin-18-enriched tight junction spikes were aligned with F-actin, indicating that they were sites of active tight junction remodeling, a process that is known to increase paracellular leak (21). Here we found that TGF-␤1 treatment also induced the formation of tight junction spikes where claudin-18 colocalized with actin, suggesting a comparable mechanism (Figs. 6 and 7). This observation is consistent with the work of Lafemina et al. (30), who found that keratinocyte growth factor increases alveolar barrier function by stimulating the cytoskeleton to form cortical actin filaments parallel to tight junctions, which stabilizes them (27), as opposed to altering alveolar claudin expression.

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Fig. 9. TGF-␤1 and GM-CSF had little effect on claudin-1 and claudin-4 localization by HBE cells. HBE cell monolayers were cultured and treated overnight with either 20 ng/ml TGF-␤ (B and E) or 100 ng/ml GM-CSF (C and F). Untreated monolayers are in A and D. The cells were then fixed and immunostained for claudin-1 (A–C) or claudin-4 (D–F). For HBE cells, neither claudin-1 nor claudin-4 showed changes in localization in response to treatment with either GM-CSF or TGF-␤. Bar ⫽ 10 ␮m.

Interestingly, the effects of TGF-␤1 on the cytoskeleton were similar to the effects of claudin-18 deficiency, which was associated with an increase in formation of actin stress fibers in AECs (32), raising the possibility that decreased claudin-18 amplifies the effects of TGF-␤1 on AECs. GM-CSF reversed the effects of TGF-␤1 on actin remodeling by AECs, further underscoring a role for GM-CSF/TGF-␤1 balance in regulating AEC function. Moreover, if the main effects of GM-CSF and TGF-␤1 are to alter tight junction turnover, this provides a potential link between the effects of these cytokines on the alveolar epithelial barrier, which contains claudin-18, and the airway epithelial barrier, which does not. Claudin-4 protein and localization were unchanged by GMCSF and TGF-␤1 (Fig. 8). Previous studies have demonstrated a protective role for claudin-4, where it was clinically associated with enhanced lung fluid clearance and improved patient outcomes in ventilator-induced lung injury (32, 35, 48, 56). Our data suggest that retention of claudin-4 alone is not sufficient to support normal lung epithelial barrier function. Consistent with this observation, claudin-4-deficient mice have a relatively mild lung phenotype (23). Moreover, claudin-4 upregulation does not rescue the decrease in AEC barrier function found in claudin-18 knockout mice (31, 32). These data further underscore the need to understand how multiple components of the tight junction proteome interact to form a fully functional lung epithelial barrier. Our findings have likely implications for patient outcome in ARDS/ALI, especially when GM-CSF and TGF-␤1 are imbalanced. TGF-␤1 and GM-CSF had direct and opposing effects on lung epithelial permeability by differentially regulating tight junction protein expression. The opposing effects of GM-CSF and TGF-␤1 depended on the relative ratio of these two cytokines, and the determination of their ratio in the airways of critically ill individuals may have clinical relevance. We confirmed that the relative ratio of GM-CSF and TGF-␤1 correlated with markers of lung barrier dysfunction in the lung lavage fluid of critically ill individuals with pneumonia. Provocatively, surviving patients had a significantly higher GMCSF/TGF-␤1 ratio than nonsurvivors. Although we favor a model where GM-CSF is protective by directly regulating

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assembly of claudin-18 in alveolar tight junctions, changes in barrier function induced by GM-CSF could also be downstream of a more general effect on cytoskeletal dynamics (21, 29, 55). It is also possible that part of the protective effect of GM-CSF could be attributable to its role as an alveolar epithelial growth factor (20, 44) or by inducing an antiapoptotic effect (51). Note that the GM-CSF/TGF-␤1 ratio measured in human BAL is based on an assay where latent TGF-␤1 is activated before analysis. Thus it is important to consider that, even in lungs predisposed to increased TGF-␤1 expression and activity, it is likely that only a fraction of BAL TGF-␤1 is activated via proteolytic cleavage. Consistent with this, the active pool of TGF-␤1 in patients with idiopathic pulmonary fibrosis, ranges from 17.6% in the upper lobe to 78.4% in the lower lobe (24). Active TGF-␤1 has been detected in BALs from patients with ARDS using bioactivity assays (6, 12). However, the extent of activation was not determined in these studies. Thus the ratios of total GM-CSF/TGF-␤1 we measured in human BALs in the range 0.0 – 0.5 more likely reflect ratios of active GM-CSF and TGF-␤1 in a higher range (e.g., 0 –3), which is in line with our experiments in vitro on the effects of active GM-CSF and TGF-␤1 on lung epithelial barrier function (Fig. 2). Given these experimental and clinical translational results, it is tempting to hypothesize that manipulating the ratio of GM-CSF to TGF-␤1 could be of clinical value. Specifically,

Fig. 10. The GM-CSF/TGF-␤1 ratio in the bronchoalveolar lavage (BAL) fluid of critically ill patients correlates with decreased leak of large serum proteins and patient survival. BAL was collected from patients with ventilator-associated pneumonia (VAP), and the ratio of GM-CSF to TGF-␤1 was determined by ELISA. In parallel, BAL samples were assessed for 2 serum proteins, ␣2-macroglobulin (A) and IgM (B). BAL ␣2-macroglobulin and IgM concentrations were inversely proportional to the GM-CSF/TGF-␤1 ratio. Specifically, at high levels of TGF-␤1, BAL concentration of these serum proteins was high, whereas, at high levels of GM-CSF, the BAL concentration of these serum proteins was low. BAL glutathione (GSH) redox potential was determined by HPLC and calculated using the Nernst equation (C). Increased GM-CSF/TGF-␤1 ratio correlated with a relatively more reduced GSH redox potential. In A–C, for each parameter, there was a significant negative correlation (P) with GM-CSF/TGF-␤1 ratio as determined by Pearson’s correlation coefficient (R). Patients who survived VAP (n ⫽ 13) had, on average, significantly higher GM-CSF/TGF-␤1 ratio in their BAL than patients who did not survive (D) (n ⫽ 16) (*P ⬍ 0.05).

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treating individuals suffering from ALI with recombinant GMCSF, either alone or in combination with inhibitors of TGF-␤1 signaling, may improve lung epithelial barrier integrity and decrease the severity of acute edematous injury. Recombinant GM-CSF treatment has been studied in the context of established ARDS with no efficacy in at least one relatively small trial (43). It is certainly possible that the treatment was given too late (i.e., after ARDS was established) and/or that the steady-state levels of GM-CSF were inadequate to counterbalance TGF-␤1 to have a meaningful protective effect. Our data suggest that a more efficacious approach would be to combine GM-CSF with TGF-␤1 inhibitors. Treating ARDS/ALI has proven to be a daunting task, as the injury can evolve over a longer period of time than can be modeled experimentally and may be exacerbated or propagated by mechanical ventilation and other complications. We believe that the experimental and translational findings in this study provide the novel observation that GM-CSF and TGF-␤1 have opposing effects on lung epithelial barrier function and that it is the relative ratio of the two as opposed to total cytokine concentration that is the critical parameter to control the relative signaling dominance of either cytokine. This dynamic balance has important implications for our understanding of how epithelial barrier function is regulated in the healthy and the injured lung and may eventually lead to therapeutic strategies that could limit the severity of acute edematous injury. Whether or not manipulating the GM-CSF/TGF-␤1 signaling ratio within the airway, combined with lung-protective ventilation and other interventions, would be an effective therapeutic strategy to treat ARDS/ALI remains to be determined in clinical trials. Because we have no effective pharmacological therapy for a syndrome that kills tens of thousands of individuals each year in the U.S. alone, such a strategy merits consideration. Moreover, the relative ratio of GM-CSF to TGF-␤1 in the BAL fluid could prove to be a dilutionindependent biomarker with diagnostic and/or prognostic applications in identifying critically ill individuals at relatively higher risk for developing severe lung injury. ACKNOWLEDGMENTS We thank Dr. M. Hershensen (University of Michigan) for the kind gift of 16HBE14o- cells and Samuel Molina for critical reading of the manuscript. GRANTS This work was supported by the Emory Alcohol and Lung Biology Center through P50-AA013757 (D. Guidot, L. Brown, and M. Koval), R01AA017627 (D. Guidot), R01-HL083120 (M. Koval), R01-HL116958 (M. Koval), T32-AA-013528 (C. Overgaard), and R25-GM099644 (S. Dorsainvil White), the German Academic Exchange Service (DAAD) (B. Schlingmann), the Fulbright Scholar Program (S. Swarnakar), the Veterans Administration through a Merit Review (D. Guidot), and the Emory University Research Committee (M. Koval). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: C.E.O., B.S., C.W., S.S., L.A.S.B., D.M.G., and M.K. conception and design of research; C.E.O., B.S., S.D.W., C.W., X.F., S.S., and M.K. performed experiments; C.E.O., B.S., S.D.W., C.W., X.F., S.S., L.A.S.B., D.M.G., and M.K. analyzed data; C.E.O., B.S., and M.K. prepared figures; C.E.O., L.A.S.B., D.M.G., and M.K. drafted manuscript; C.E.O., B.S., C.W., S.S., L.A.S.B., D.M.G., and M.K. approved final version of manuscript;

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