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The Journal of Immunology, 2006, 177: 527-537.
Copyright © 2006 by The American Association of Immunologists

Stat3 Is Required for Cytoprotection of the Respiratory Epithelium during Adenoviral Infection1

Yohei Matsuzaki, Yan Xu, Machiko Ikegami, Valérie Besnard, Kwon-Sik Park, William M. Hull, Susan E. Wert and Jeffrey A. Whitsett2

Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3{Delta}{Delta} mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3{Delta}{Delta} mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3{Delta}{Delta} mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3{Delta}{Delta} mice were similar, TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3{Delta}{Delta} mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3{Delta}{Delta} mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-xL, an E1- and E3-deleted, nonproliferative adenovirus expressing the antiapoptotic protein Bcl-xL, protected Stat3{Delta}{Delta} mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signal transducers and activators of transcription include a family of structurally related proteins that play important roles in the intracellular transduction of signals regulated by various cytokines and growth factors. Stat3 was initially identified as a transcription factor that mediated the effects of IL-6 in acute phase response in the liver (1). Stat3 mediates or participates in the signaling pathways of many cytokines (e.g., IL-6, IL-11, IL-10, IL-2, leukemic inhibitory factor, ciliary neurotrophic factor, oncostatin M, leptin, and others) and growth factors (e.g., epidermal growth factor, TGF-{alpha}, hepatocyte growth factor, and G-CSF) in various cells and organs (1, 2, 3). Cytokines and growth factors activate Stat3 via glycoprotein 130 (gp130)-activating phosphorylation by JAKs. Phosphoryl-Stat3 dimerizes and is transported to the nucleus, where it regulates the transcription of target genes. Because systemic deletion of Stat3 in transgenic mice is lethal at E6.5-7.5 (4), the biological roles of Stat3 have been determined in vitro and after conditional deletion of the gene in various cell types and organs of the mouse. Stat3 plays a critical role in the regulation of various biological processes, including cell survival, apoptosis, inflammation, and proliferation (5). Conditional ablation of Stat3 in the respiratory epithelium of mice (Stat3{Delta}{Delta} mice) demonstrated that Stat3 is not required for normal lung morphogenesis or function but is required for maintenance of surfactant homeostasis, lung function, and repair following hyperoxia-induced injury (6). Although IL-6, IL-11, and activated Stat3 protect the lung during oxidant injury in vivo (7, 8, 9), mechanisms by which activation of Stat3 maintains lung homeostasis following injury are poorly understood.

The recent severe acute respiratory syndrome outbreak (10) and the ongoing morbidity and mortality associated with influenza and other pulmonary viral infections has served to raise awareness of the public health consequences of viral pneumonias and the need for further knowledge regarding the pathogenesis of pulmonary viral infections. The mechanisms protecting the respiratory epithelium from acute cellular injury after exposure to respiratory viruses are complex and include induction of antioxidant, antiapoptotic pathways, specific enhancement of cell proliferation, and surfactant production (8, 11, 12). Activation of Stat3 and pre-exposure to IL-6 protects the lung from injury during hyperoxia (7, 9).

The lung is repeatedly subjected to injury caused by viral infection and other pathogens. Maintenance of pulmonary homeostasis requires the continuation of cellular processes and proliferation of cells during viral infection. Infected cells are cleared by acute and immune-mediated apoptosis and necrosis. In this study, we assessed the role of Stat3 in pulmonary homeostasis during adenoviral-mediated lung injury. A conditional system was used to express Cre-recombinase, selectively deleting the Stat3 gene in bronchiolar and alveolar epithelial cells of the mouse lung (6). Intratracheal administration of AV1-GFP, an E1- and E3-deleted, nonproliferative adenovirus, caused severe lung pathology associated with enhanced apoptosis of the respiratory epithelium in Stat3{Delta}{Delta} mice. These results demonstrate that Stat3 plays a critical role in cytoprotection of the lung during the early phases of viral pneumonia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Gene construction and doxycycline administration

SP-C-rtTA/(tetO)7CMV-Cre/Stat3flx/flx triple-transgenic mice were generated as described previously (6). Stat3flx/flx mice were a gift from Dr. Takeda (Hyogo College of Medicine, Hyogo, Japan) (4, 13). In the presence of doxycycline, exon 21 of Stat3 gene was permanently deleted from respiratory epithelial cells before birth (Stat3{Delta}{Delta} mice) (6). To confirm genotype, DNA was purified from tail or lung of experimental mice, and PCR was performed for SP-C-rtTA, (tetO)7CMV-Cre, and Stat3flx genes using the following primers: 5'-GAC ACA TAT AAG ACC CTG GCTA-3' and 5'-AAA ATC TTG CCA GCT TTC CC-3' for surfactant protein (SP)3-C-rtTA; 5'-TGC CAC GAC CAA GTG ACA GCA ATG-3' and 5'-AGA GAC GGA AAT CCA TCG CTCG-3' for (tetO)7CMV-Cre; and 5'-CCT GAA GAC CAA GTT CAT CTG TGT GAC-3' and 5'-CAC ACA AGC CAT CAA ACT CTG GTC TCC-3' for Stat3flx. Stat3-deleted transgenic (Stat3{Delta}{Delta}) and nondeleted littermates (Stat3flx/flx, control mice) were used for the experiments. Doxycycline was administered to the dams in the food at a concentration of 625 mg/kg (Harlan Teklad) from embryonic day 0 (E0) to postnatal day 25 (P25), resulting in extensive deletion of Stat3 in respiratory epithelial cells. Mice were then provided normal food. Body weights of adult control and Stat3{Delta}{Delta} mice were similar, 24.75 ± 3.0 g (n = 89) for control and 24.69 ± 3.0 g (n = 86) for Stat3{Delta}{Delta} mice.

Intratracheal administration of adenovirus

AV1-GFP, AV1-Bcl-xL, and AV1-Bcl-2, all E1- and E3-deleted, nonproliferative type 5 adenoviruses sharing an identical viral background, were generated in 293 cells (14, 15). AV1-Bcl-xL and AV1-Bcl-2 were a gift from Dr. Molkentin (Cincinnati Children’s Hospital, Cincinnati, OH) (16). Adenovirus was diluted with HBSS (Invitrogen Life Technologies) or PBS to 2 x 1010 optical particle unit (opu)/mouse and administered intratracheally in a total volume of 80 µl (12). Adenovirus or saline (experimental control) were administered to 8- to 10-wk-old Stat3{Delta}{Delta} and control mice, by intubation with 24-gauge animal feeding needles (Popper & Sons) and a laryngoscope (Bio Research Center) during anesthesia with isoflurane.

Tissue preparation, histology, and immunohistochemistry (IHC)

Lung tissues were prepared as described previously (17). IHC was performed essentially as described previously (18). Tissue sections were stained with H&E. Primary Abs were used at the following dilutions: SP-B (1/2,000, rabbit polyclonal; Chemicon International), pro-SP-C (1/4,000, rabbit polyclonal; Chemicon International), Stat3 (1/400, rabbit polyclonal; Cell Signaling), phosphoryl-Stat3 (Tyr705) (1/50, rabbit polyclonal; Cell Signaling), and cleaved caspase-3 (the active form of caspase-3, 1/500 for immunofluorescence, rabbit polyclonal; R&D Systems). Immunofluorescent, secondary Ab Alexa Flour 568-tagged goat anti-rabbit IgG was used at a dilution of 1/200 (Molecular Probes). All experiments shown are representative of findings from at least four Stat3{Delta}{Delta} mice compared with control mice.

TUNEL assay

The TUNEL assay was performed on tissue paraffin sections and cryosections using In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s protocol.

Morphometric analysis

Severity of adenoviral-induced lung injury was assessed using a system that scored alveolar epithelial injury, bronchiolar injury, inflammation, and alveolar size (Table I). Lung sections from multiple lobes of Stat3{Delta}{Delta} and control mice (14–15 lobes/n = 4 per group) were assessed following saline or AV1-GFP exposure by a blinded reviewer. Kruskal-Wallis one-way analysis and Dunn’s method were performed with individual scores for each characteristic to determine significant differences. Significance was accepted at the 5% level.


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  		Table I. Comparison of histology scoring in control and Stat3{Delta}/{Delta}a

 
Quantification of apoptosis and cleaved caspase-3-reactive cells

To quantify the TUNEL reaction and cleaved caspase-3 signals, 10 random fields per animal were photographed by fluorescence microscopy. Selection of each field was decided by a randomization function of Microsoft Excel. Apoptosis signals were counted manually, and 4',6'-diamidino-2-phenylindole signals were counted by thresholding, using MetaMorph software (Molecular Devices). The ratio of apoptosis-positive cells:total cell number was calculated, and reported as the apoptosis cell index (mean ± SEM). For apoptosis analysis, comparisons among groups were made by ANOVA with Tukey’s test used for post hoc analysis. Results were expressed as mean and SEM. Significance was accepted at the 5% level.

Western blot analysis for SPs

The content of SP-A, SP-B, and SP-C in bronchoalveolar lavage fluid (BALF) was estimated by Western blot analysis. Samples were obtained by pooling three 1-ml lung lavage washes. Recovery volumes were similar in each group of animals. BALF (30 µl) for SP-B, and SP-A and SP-C (10 µl) were diluted in Laemmli buffer and subjected to SDS-PAGE under nonreducing conditions for SP-B (19) and under reducing conditions for SP-A and SP-C (20). The following dilutions of Ab were used: SP-A (1/5000, guinea pig polyclonal; Chemicon International), SP-B (1/5000 rabbit polyclonal; Chemicon International), and SP-C (1/5000, rabbit polyclonal). Peroxidase-conjugated secondary Abs (Calbiochem, EMD Biosciences) were used at 1/5000 dilutions. Immunoreactive bands were detected with ECL reagents (Amersham Health).

RNA extraction and RNA analysis for SPs

Lung tissues were excised and homogenized with TRIzol reagent (Invitrogen Life Technologies). RNA concentration was measured by spectrophotometer. S1 nuclease assays for SP-B, SP-C, and L32 mRNA were performed as described previously (21).

Isolation alveolar type II epithelial cells

Alveolar type II cells were isolated from 6-wk-old control and Stat3{Delta}{Delta} mice 2 h after isolation using collagenase and differential plating as described by Rice et al. (22). Type II cells were used for FACS and type II cell culture.

Apoptosis analysis following AV1-GFP exposure in vitro

Isolated type II cells were counted with a hemacytometer. Equivalent numbers of cells were cultured on 100% Matrigel. After 4 days of culture on the gel, cells were exposed to saline or AV1-GFP (2 x 107 PFU/well, 2 x 108 PFU/well, 2 x 109 PFU/well). Twenty-four hours after exposure, cells were removed from the Matrigel with Cell Recovery Solution (BD Biosciences). The recovered type II cells were rinsed with cold PBS and centrifuged. The cell pellets were resuspended in 50 µl of Laemmli buffer and sonicated. Samples (20 µl) were analyzed by Western blot of cleaved caspase-3 (1/1,000, rabbit polyclonal; Cell Signaling). beta-Actin (1/10,000, mouse polyclonal; Seven Hills Bioreagents) was used as a control. Peroxidase-conjugated secondary Abs (Calbiochem, EMD Biosciences) were used at 1/2,000 for cleaved caspase-3 and 1/10,000 for actin.

RNA microarray analysis

mRNA was extracted from pools of alveolar type II epithelial cells isolated from Stat3{Delta}{Delta} and control mice. Each group had three males and three females, 6 wk of age. The cRNA was hybridized to the murine genome MOE430 chip (consists of {approx}45,000 gene entries) (Affymetrix) according to the manufacturer’s protocol. Affymetrix Microarray Suite 5.0 was used to scan and quantitate the gene chips under default scan setting. Normalization was performed using the Robust Multichip Average model (23, 24). Data were further analyzed using Linear Models for Microarray Data (25). Differentially expressed genes were selected with the threshold of Student’s t test (p < 0.05; false discovery rate <10% and fold change >1.5). Unknown genes/expressed sequence tags and duplicated gene entries were temperately removed from further functional analysis. Genes associated with apoptosis were identified by PubMed Browser. Gene Ontology (GO) analysis was performed using public available web-based tool DAVID (database for annotation, visualization and integrated discovery) (26).

Validation of mRNAs

Real-time RT-PCR was used to cross-validate changes in several mRNAs detected by IHC and microarray analysis. Malt-1-1, rtn4, reg3g, and Bcl-xL were detected using primers listed (see Table IV). Changes in mRNA were determined in type II cells isolated from Stat3{Delta}{Delta} and controls (n = 3–4/group). Student t test was performed. Significance was accepted at the 5% level. The following primers were used: Malt1: forward, TAT CCA GGA GGA CCC CAT GT and reverse, TCT GAT CAA AGC CAG TTA GCA TCAT; Rtn4: forward, AAG TGG AAG GAG TTT GAG AGA GCA and reverse, CTG TCT CAA AGC AGA TGT GAA AGC; Reg3g: forward, TGC CAA AAG AGC CCT CAG GA and reverse, TGC CTG AGG AAG AGG AAG GAT TCG; Bcl-xL: forward, TCT CTC TCC TCT GTC CAC CCT TG and reverse, TGC CCC TCA GAA GCC AGA AC.


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  		Table IV. Comparison of mRNAs by RT-PCR and RNA microarraya

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Stat3{Delta}{Delta} mice were susceptible to adenoviral-mediated lung injury

Lung histology was assessed 1–7 days following intratracheal administration of AV1-GFP. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in lungs of Stat3{Delta}{Delta} mice as early as 1 day after exposure to the virus (Figs. 1 and 2 and Table I). In contrast, airspace abnormalities were not observed in control mice, consistent with previous studies with this virus (18, 27). Severe airspace enlargement related to loss of alveoli, bronchiolar sloughing, and focal hemorrhage were observed 1, 2, 5, and 7 days after infection in Stat3{Delta}{Delta} mice. In contrast, lung inflammation was substantially resolved in control mice during the same time period. Thus, Stat3{Delta}{Delta} mice were more susceptible to lung injury and alveolar simplification following adenoviral infection. Consistent with the importance of Stat3 during the early phase of infection, activation of Stat3 was indicated by nuclear staining of phospho-Stat3 observed 30 min after adenoviral infection in control mice (Fig. 3).


Figure 1
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  		FIGURE 1. Lung histology following pulmonary adenoviral infection (day 2). Histology was assessed with H&E staining 2 days after exposure of 8-wk-old control (A, C, E, and G) and Stat3{Delta}{Delta} (B, D, F, and H) mice to AV1-GFP. Control and Stat3{Delta}{Delta} mice were administered 2 x 1010 opu of AV1-GFP or saline. Severe airspace enlargement, loss of alveolar septae, and sloughing of airway epithelium were observed in Stat3{Delta}{Delta} mice following AV1-GFP exposure (D and H). Figures are representative of n ≥ 5 per genotype. Scale bar, 100 µm.

 

Figure 2
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  		FIGURE 2. Lung histology following pulmonary adenoviral infection (day 7). Lung sections were prepared 7 days after exposure of control (A, C, E, and G) and Stat3{Delta}{Delta} (B, D, F, and H) mice to 2 x 1010 opu of AV1-GFP or saline and stained with H&E. More airspace loss was observed in Stat3{Delta}{Delta} mice following AV1-GFP. Figures are representative of n ≥ 5 per genotype. Scale bar, 100 µm.

 

Figure 3
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  		FIGURE 3. Immunohistochemical staining of phosphoryltyrosine Stat3 following adenoviral infection of control mice. Lung tissues were inflation fixed 30 min (A), 60 min (B), 120 min (C), and 1 day (D) following intratracheal administration of 2 x 1010 opu of AV1-GFP, and 1 day (E) following saline. Tissue was immunostained with phosphoryl-Stat3 (Tyr705). Stat3 activation was observed in the conducting airways 30 min after adenovirus administration and in alveolar epithelial cells within 60 min. Scale bar, 100 µm.

 
SP expression was maintained in Stat3{Delta}{Delta} mice

The concentration of SPs (SP-A, SP-B, and SP-C) was analyzed in BALF 48 h after administration of adenovirus (Fig. 4A). In both Stat3{Delta}{Delta} and control mice, the levels of SPs in BALF increased following adenovirus administration. SP content in BALF was not different in Stat3{Delta}{Delta} and control mice after exposure to the virus, at times during which severe lung injury was observed in the Stat3{Delta}{Delta} mice. Thus, the early susceptibility of the Stat3{Delta}{Delta} mice to lung injury was not likely mediated by changes in SP expression. SP mRNAs in whole lung were analyzed by S1 nuclease assay (Fig. 4B). Levels of SP mRNAs were not altered by infection or deletion of Stat3, consistent with the lack of change in SP levels in BALF.


Figure 4
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  		FIGURE 4. SP expression and SP-B mRNA were not altered by deletion of Stat3. A, SP-A, SP-B, and SP-C were quantitated in BALF by Western blot analysis 48 h after intratracheal instillation of 2 x 1010 opu of AV1-GFP. AV1-GFP increased SPs in BALF of both Stat3{Delta}{Delta} and control mice. Deletion of Stat3 did not alter SP content following treatment with AV1-GFP. B, SP-B and SP-C mRNAs were quantitated by S1 nuclease assay. SP-B and SP-C mRNAs were similar in control and Stat3{Delta}{Delta} mice following adenoviral exposure.

 
IHC for SP-B and pro-SP-C was performed 48 h after intratracheal administration of adenovirus. Although SP staining in the alveoli was not different in Stat3{Delta}{Delta} mice, SP-B and pro-SP-C-stained cells were fewer, likely representing the generalized loss of alveolar tissue following adenovirus (Fig. 5). Because there was no difference in the levels of SP in BALF after adenoviral exposure, we hypothesized that lung injury and alveolar loss observed in Stat3{Delta}{Delta} mice was not mediated by changes in extracellular SP homeostasis. Airspace enlargement and alveolar loss was observed in the Stat3{Delta}{Delta} mice rather than atelectasis, the latter generally associated with surfactant deficiency.


Figure 5
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  		FIGURE 5. IHC for SP-B and pro-SP-C. SP-B (A–D) and pro-SP-C (E–H) immunohistochemical staining was performed in control and Stat3{Delta}{Delta} mice 48 h after exposure to 2 x 1010 opu of AV1-GFP (D). Intracellular staining for SP-B was slightly decreased in Stat3{Delta}{Delta} mice following AV1-GFP. SP-B was detected in the alveolar spaces and in type II epithelial cells in the Stat3{Delta}{Delta} mice following AV1-GFP. Cellular staining for pro-SP-C (E–H) in Stat3{Delta}{Delta} and control mice was used to identify type II epithelial cells. Photomicrographs are representative of n ≥ 4. Scale bar, 100 µm.

 
Apoptosis increased in Stat3{Delta}{Delta} mice following adenoviral infection

Cleaved caspase-3 IHC and TUNEL assay was used to determine whether epithelial cell apoptosis might mediate the extensive loss of the alveolar epithelium seen in the Stat3{Delta}{Delta} mice following adenoviral infection. The TUNEL assay was performed on lung tissue from Stat3{Delta}{Delta} and control mice 24 h after exposure to AV1-GFP or saline (Fig. 6). Consistent with this observation, cleaved caspase-3-positive cells were increased in lung epithelial cells of Stat3{Delta}{Delta} mice following adenovirus exposure (Fig. 6). The number of caspase-3 and TUNEL-positive cells was significantly increased in Stat3{Delta}{Delta} mice following AV1-GFP compared with controls (Fig. 7).


Figure 6
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  		FIGURE 6. Increased cleaved caspase-3 and TUNEL staining in Stat3{Delta}{Delta} mice. Immunostaining for cleaved caspase-3 and the TUNEL assay were performed 24 h after intratracheal administration of 2 x 1010 opu of AV1-GFP in vivo. Caspase-3 (red, arrow) and TUNEL (green) were increased in Stat3{Delta}{Delta} mice following adenoviral infection. Photomicrographs are representative of n ≥ 5. Scale bar, 100 µm.

 

Figure 7
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  		FIGURE 7. Quantification of TUNEL and cleaved caspase-3. TUNEL assay was assessed in lung sections from mice receiving 2 x 1010 opu of AV1-GFP. The proportion of TUNEL-positive cells in Stat3{Delta}{Delta} mice following AV1-GFP was increased significantly. Statistical differences were assessed by ANOVA test (*, p < 0.05).

 
Increased caspase-3 activity following AV1-GFP exposure in type II cells from Stat3{Delta}{Delta} mice in vitro

To further assess whether Stat3 plays a role in protection of the epithelial cells from apoptosis, primary alveolar type II cells from Stat3{Delta}{Delta} and control mice were isolated and treated with AV1-GFP (2 x 107 – 2 x 109 PFU/well) or saline. Changes in caspase-3 were analyzed by Western blot analysis (Fig. 8). Cleaved caspase-3 was significantly increased in type II cells isolated from Stat3{Delta}{Delta} mice 24 h after exposure to the adenovirus in vitro.


Figure 8
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  		FIGURE 8. Increased cleaved caspase-3 in alveolar type II cells from Stat3{Delta}{Delta}. Western blotting of cleaved caspase-3 was performed in primary type II cells 24 h after saline (a), AV1-GFP 2 x 107 PFU/well (b), 2 x 108 PFU/well (c), and 2 x 109 PFU/well (d) in vitro. Cleaved caspase-3 was increased in type II cells from Stat3{Delta}{Delta} after AV1-GFP exposure was increased relative to dose of AV1-GFP.

 
Stat3 regulates expression of genes modulating apoptosis and other cellular processes

The mRNA expression profiles were compared in type II epithelial cells isolated from Stat3{Delta}{Delta} and control mice using Affymetrix murine genome MOE430 gene chips. mRNAs derived from n = 1425 genes were identified as significantly altered using the criteria described in Materials and Methods. Expression of 887 mRNAs was increased while that of 538 mRNAs was decreased in response to the deletion of Stat3. Differentially expressed genes were further classified according to GO classification on Biological Process using public available web-based tool DAVID (26). The Fisher exact test was used to calculate the probability that category was overrepresented using the entire MOE430 mouse genome as reference dataset (Table II). Genes regulating protein transport (p = 8.30E-12), transcription (p = 4.41E-07), and programmed cell death/apoptosis (p = 8.59E-07) were significantly increased after deletion of Stat3, while lipid, carboxylic acid, and organic acid metabolism (with p values of 8.34E-07, 3.5E-07, and 3.54E-07, respectively) were selectively decreased by deletion of Stat3. Thus, despite the lack of observable abnormalities in the lungs of Stat3{Delta}{Delta} mice before challenge with virus, expression of numerous genes was altered by the deletion of Stat3. Of particular interest, a number of genes regulating cell survival and apoptosis were influenced by deletion of Stat3 in type II epithelial cells (Table III). Selected mRNAs identified by microarray analysis including Malt-1-1, Rtn4, Reg3a, and Bcl-xL were assessed by real-time RT-PCR, demonstrating similar changes in expression of a number of genes influencing cell survival (Table IV).


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  		Table II. Functional classification of categories of genes influenced by deletion of Stat3 in type II epithelial cellsa

 

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  		Table III. mRNA expression profiles were compared in type II epithelial cells 2 h after isolation from Stat3{Delta}/{Delta} and control micea

 
Bcl-xL prevents adenoviral-mediated lung injury in vivo

TUNEL-positive cells and cleaved caspase-3 were increased in Stat3{Delta}{Delta} mice following exposure to AV1-GFP, indicating that Stat3-deficient cells may be susceptible to apoptosis. Bcl-xL and Bcl-2, Bcl-2 family members, are known to inhibit apoptosis (28, 29, 30, 31). To assess whether Bcl-xL or Bcl-2 prevents adenovirus-induced apoptosis, AV1-Bcl-xL and AV1-Bcl-2, E1- and E3-deleted, nonproliferative adenoviruses, of identical genetic background to AV1-GFP, was administered intratracheally to Stat3{Delta}{Delta} and control mice. Lung histology was assessed 2 days after infection with AV1-Bcl-xL and AV1-Bcl-2. Lung histology was substantially improved in mice receiving AV1-Bcl-xL compared with AV1-GFP (Figs. 9 and 10). Airspace enlargement, loss of alveolar septae, and sloughing of airway epithelial cells seen after AV1-GFP exposure were not observed following AV1-Bcl-xL administration. Expression of Bcl-xL in 293 cells was increased 24 h after exposure to AV1-Bcl-xL, demonstrating the activity of the virus in vitro. The TUNEL assay was performed on lung tissue from Stat3{Delta}{Delta} and control mice 24 h after exposure to AV1-GFP, AV1-Bcl-xL, or saline. Although TUNEL signals increased following AV1-GFP exposure, TUNEL signals after AV1-Bcl-xL were similar to that seen after saline exposure (Fig. 10). In contrast, treatment of the Stat3{Delta}{Delta} mice with the same titer of AV1-Bcl-2 did not ameliorate viral-induced injury in vivo (data not shown).


Figure 9
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  		FIGURE 9. Bcl-xL protects Stat3{Delta}{Delta} mice from pulmonary adenoviral infection. Lung histology was assessed after intratracheal administration of saline (A and B), 2 x 1010 opu of AV1-GFP (C and D), or 2 x 1010 opu of AV1-Bcl-xL (E and F) in control (A, C, and E) and Stat3{Delta}{Delta} (B, D, and F) mice. Severe enlargement of airspace, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3{Delta}{Delta} mice following AV1-GFP (C and D). Lung injury was ameliorated by expression of Bcl-xL in the same adenoviral vector (E and F). Photomicrographs are representative of n ≥ 5. Scale bar, 100 µm.

 

Figure 10
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  		FIGURE 10. Bcl-xL reduces TUNEL staining. TUNEL assay was performed 24 h after intratracheal instillation of saline (A), AV1-GFP (B), or AV1-Bcl-xL (C). TUNEL-positive cells were increased following AV1-GFP in Stat3{Delta}{Delta} mice (B). TUNEL signal was similar to that of saline-treated mice (A) following AV1-Bcl-xL to Stat3{Delta}{Delta} mice (C). Photomicrographs are representative of n ≥ 5. Scale bar, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Stat3{Delta}{Delta} mice were highly susceptible to AV1-GFP

Although respiratory epithelial cell-specific deletion of Stat3 did not alter lung morphogenesis or function (6), Stat3{Delta}{Delta} mice were highly susceptible to lung injury and apoptosis and alveolar loss following intratracheal administration of a nonproliferative E1- and E3-deleted adenovirus, AV1-GFP. Histological studies demonstrated remarkable airspace enlargement, rapid loss of alveolar septae, sloughing of the respiratory epithelium, and increased inflammation in the Stat3{Delta}{Delta} mice. Increased TUNEL staining and enhancement of cleaved caspase-3 expression were observed after exposure of Stat3{Delta}{Delta} mice to the replication-defective adenovirus. Thus, Stat3 is critical for cytoprotection of the respiratory epithelium following adenoviral infection. Although Stat3 plays an essential role in maintenance of pulmonary surfactant homeostasis during oxygen-induced injury (6, 7, 8, 9), the early and severe epithelial cell injury seen following adenoviral infection in Stat3{Delta}{Delta} mice was not associated with decreased SPs in the airspace. The present findings support that Stat3 is required for survival of the respiratory epithelium during early phases of pulmonary viral infection.

Severe lung injury seen in Stat3{Delta}{Delta} mice was not due to surfactant deficiency or dysfunction

Because Stat3 is known to regulate expression of SP-B (Sftpb) and to be critical for the maintenance of lung function during hyperoxia (6, 9, 32), we assessed SPs content in BALF after adenoviral exposure in the Stat3{Delta}{Delta} mice. SP content in lung lavage fluid was similar in Stat3{Delta}{Delta} and control mice following adenovirus administration. Likewise, SP-B and SP-C mRNAs were not influenced by Stat3 deletion or AV1-GFP exposure, 24 and 48 h after exposure to the virus. However, the number of cells and the intracellular staining for SP-B and pro-SP-C were slightly decreased in Stat3{Delta}{Delta} mice 24 and 48 h after adenoviral exposure, at times when lung injury and airspace enlargement were extensive. The decrease in intracellular immunohistochemical staining for SP-B and pro-SP-C may represent changes in intracellular content, including synthesis, processing, routing, or secretion that may be directly or indirectly influenced by Stat3, or represent that numbers of stained cells per residual alveoli appeared decreased, because the virus caused extensive loss of alveolar cells per se. The finding that SP-B and SP-C mRNAs were unchanged suggest that the change in staining is not mediated by direct effects of Stat3 on their transcription. Because the half-life of SP-B protein is ~6–8 h in the adult mouse lung (33), the observed differences in intracellular SP-B staining were not reflected by changes in alveolar SP-B concentrations. Pathologic findings in the lung of AV1-GFP-infected mice were not consistent with findings associated with surfactant deficiency or dysfunction that are generally indicated by atelectasis and edema hemorrhage. In contrast to the present findings, increased alveolar hemorrhage and atelectasis were observed during 4–5 days of hyperoxia that were mediated primarily by surfactant deficiency and decreased SP-B (6). The severe alveolar loss presently observed after virus exposure was not observed in the Stat3{Delta}{Delta} mice during oxygen exposure, supporting the requirement for Stat3 for prevention of alveolar injury after adenoviral exposure was distinct from its role in the regulation of surfactant homeostasis during hyperoxia.

TUNEL staining was increased in epithelial cells in Stat3{Delta}{Delta} mice following exposure to adenovirus. Variability in TUNEL staining was observed and that may represent variability of the Stat3 gene targeting, in the distribution of the virus after intratracheal administration or efficacy of clearance of apoptotic cells from the lung. Immunohistochemical staining for cleaved caspase-3 and increased caspase activity were observed in Stat3-deficient mice and in type II cells isolated from Stat3{Delta}{Delta} mice. Type II cells, identified by pro-SP-C staining, represented 85% of the cells isolated from adult mouse lungs by flow cytometry. Because, in this model, deletion of Stat3 occurs only in epithelial cells, increased caspase-3 activation likely indicates the increased susceptibility of the epithelial cells rather than macrophages or fibroblasts. This study demonstrates that viral-induced apoptosis was controlled by Stat3 in the respiratory epithelium in a cell-autonomous manner.

Mechanism of adenoviral-induced apoptosis

When infected with adenovirus, cells express viral proteins on the cell surface mediated by MHC class 1 TCR. CTLs recognize the exposed viral proteins causing apoptosis via granzyme or FAS ligand-mediated pathways. Activation of CTL generally occurs 4–7 days after viral infection (34). In this study, however, apoptosis was observed 1–2 days after adenoviral exposure, before the activation of CTL-mediated apoptosis. These findings support the likelihood that adenoviral proteins per se induce apoptosis in the absence of Stat3. Although E1a protein of the adenovirus is an initiator of apoptosis (35, 36, 37), the E1 region is deleted in the AV1-GFP construct (14). Adenovirus also induces apoptosis via the pro-death gene product, E4ORF4 (38, 39), although the mechanisms by which this protein is unknown at present. Although E4ORF4 is intact in the AV1-GFP construct and is likely expressed following infection with this adenoviral vector, the mechanism by which AV1-GFP induces epithelial cell loss in the Stat3{Delta}{Delta} mice remains unclear.

Role of Stat3 during adenoviral infection

The present finding demonstrates that Stat3 plays a critical role in protection of the lung from an early apoptotic effect of the virus, thus avoiding catastrophic loss of lung structure. In this study, phospho-Stat3 staining increased in epithelial cells within 30 min after exposure to adenovirus (40, 41, 42), indicating that Stat3 is likely to play an important role in cytoprotection early in the process of infection, perhaps limiting injury by sustaining cell survival until repair processes are initiated. The observed cytoprotective effect of Stat3 is consistent with previous findings demonstrating that loss of Stat3 in macrophages and neutrophils renders cells or mice susceptible to endotoxin administration (43), impairs proliferative response in T cell lymphocytes (13, 44), delays wound healing in keratinocytes (45), and enables susceptibility to injury, failure of survival in hepatocytes (46), cell death in motoneurons (47), and hypersensitivity of thymic epithelial cell to apoptosis (48).

Stat3 regulates expression of genes regulating cell survival

RNA microarray analysis demonstrated that there were many genes regulating apoptosis/cell survival that were altered after deletion of Stat3 in respiratory epithelial cells, in both positive and negative directions, and at multiple levels (Table II). Interestingly, RNAs that play an important role in proapoptotic pathways, including caspase-3, amyloid beta 4A precursor protein (APP), and Cox7c, were increased, perhaps consistent with the observed increased sensitivity of the Stat3{Delta}{Delta} mice to viral-induced cell loss.

The relationship between Stat3 and genes regulating apoptosis/cell survival were complicated (Table III). Deletion of Stat3 altered type II cells resulted in changes in mRNAs influencing multiple components mediating cell death processes including Bcl-2 family members (Bcl-2, Bcl211, Mc11, Bcl2111, Bak1, and BAD), caspase-3, caspase-8 and Fas-associated death domain protein-like apoptosis regulator (Cflar, also known as Flip), voltage-dependent anion channel 1 (Vdac1), and cytochrome C (Cox7c). Taken together, these findings support the concept that Stat3 influences cell survival at multiple levels (i.e., direct transcriptional regulation or indirectly through the regulation of other transcription factors or posttranslational modifications of other proteins). Because our experimental data demonstrated susceptibility to apoptosis, we focused on the analysis of cell survival pathways (Table II). Stat3 is known to serve in an antiapoptotic role by enhancing transcription of Bcl-2 and Bcl-xL (49, 50, 51, 52, 53, 54), Bcl-xL being a direct transcription target of Stat3 (52). The decreased expression of Bcl-xL in Stat3-deficient cells may represent a potential direct transcriptional effect of the deletion of Stat3. Increased Bcl-2 and Bcl-2a1a mRNA may indicate compensatory responses serving to protect cells from apoptosis. Stat3 induces apoptosis by regulating PI3K-Akt-mediated survival signaling (52, 55). The PI3K regulatory subunits, p55{alpha} and p50{alpha}, are known as targets of Stat3 (55, 56). PI3K controls the activity of Akt by regulating its location and activation (57), Akt, in contrast, inhibits STAT3 transcriptional activity and phosphorylation (58). Several PI3K subunits were increased, whereas Akt2 was reduced, following deletion of Stat3 as indicated by the microarray data that is likely to represent a compensatory response to the lack of Stat3. The PI3K-Akt pathway influences a wide spectrum of downstream targets that influence apoptosis (58). For example, Akt phosphorylates multiple Bcl-2 family members including BAD and Bcl-xL (59), blocks cytochrome C release from mitochondria (60), and inhibits caspase-3 activation (61). Components of the apoptotic machinery interact at multiple levels. Bcl-xL interacts with Vdac1 to regulate this outer mitochondrial membrane channel opening and membrane potential that controls production of reactive oxygen species and release of cytochrome C, both of which are the potent inducers of cell apoptosis (62, 63). In the present analysis, cleaved caspase-3 staining and caspase-3 mRNA were increased. Caspase-3 mediates cleavage of APP (64). APP mRNA was also increased after deletion of Stat3.

Taken together, the present findings suggest that lack of Stat3 causes complex changes in expression of both pro- and antiapoptotic proteins that regulate balance of pro- and antiapoptotic activities that determine cell survival. Because changes in gene expression represent the net outcome of complicated balances, it is unlikely that changes in a single or even a few genes determines the susceptibility of type cells to viral-induced injury. Rather, Stat3 plays a remarkable and critical role in the regulation of multiple components of the cell survival pathways that maintains cellular homeostasis following viral infection (Fig. 11).


Figure 11
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  		FIGURE 11. Model of apoptotic pathways influenced by Stat3. A simplified model is proposed by which the lack of Stat-3 influences the expression of genes that regulate cell survival.

 
Bcl-xL protects the Stat3{Delta}{Delta} mice from adenoviral-induced injury

Rapid loss of alveolar-bronchiolar epithelial cells, increased TUNEL, and cleaved caspase-3 following treatment with adenovirus strongly supported the concept that susceptibility to apoptosis played a role in severe injury observed in the Stat3{Delta}{Delta} mice. Likewise, mRNA array studies support the likelihood that diverse changes in expression of genes influencing cell survival were perturbed by deletion of Stat3. Expression of Bcl-xL in the same viral vector conferred cytoprotection following adenoviral infection, supporting the concept that deletion of Stat3 increased susceptibility to cell death. Expression of Bcl-xL in Av1-Bcl-xL, but not AV1-Bcl-2, protected the Stat3{Delta}{Delta} mice from viral-induced lung injury. Although Bcl-xL mRNA was also decreased in type II epithelial cells as assessed by the mRNA array data, many mRNAs influencing cell survival were altered in Stat3-deficient cells before viral infection. Thus, the observed cytoprotection by AV1-Bcl-xL strongly supports the important role of Stat3 in cytoprotection, but does not indicate that changes in Bcl-xL alone account for the observations. AV1-Bcl-2 did not protect the lung as did Bcl-xL. Bcl-2 has both antiapoptotic activity and proapoptotic activities at different concentration (65). It is unclear whether Bcl-2 is less active than Bcl-xL or that Bcl-2 was not expressed at appropriate levels to influence cytoprotection in our experiments.

In summary, the present study demonstrates that Stat3 plays a critical role in the regulation of a number of genes in the respiratory epithelium that support cell survival during the early phases of adenoviral infection of the lung.


   Acknowledgments
 
We thank Ann Maher for secretarial assistance. We also thank K. Takeda for the gift of Stat3flx/flx mice, B. C. Trapnell for the gift of AV1-GFP, and J. D. Molkentin for the gift of AV1-Bcl-xL and AV1-Bcl-2.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL61646 (to M. Ikegami, S.E. Wert, and J.A. Whitsett) and HL38859 (to J.A. Whitsett). Back

2 Address correspondence and reprint requests to Dr. Jeffrey A. Whitsett, Cincinnati Children’s Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: jeff.whitsett{at}cchmc.org Back

3 Abbreviations used in this paper: SP, surfactant protein; opu, optical particle unit; IHC, immunohistochemistry; BALF, bronchoalveolar lavage fluid; APP, amyloid beta 4A precursor protein. Back

Received for publication December 13, 2005. Accepted for publication April 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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