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Am J Physiol Lung Cell Mol Physiol 291: L658-L667, 2006. First published April 28, 2006; doi:10.1152/ajplung.00006.2006
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Increased bleomycin-induced lung injury in mice deficient in the transcription factor T-bet

Jianguo Xu,1,2,* Ana L. Mora,1,2,3,* John LaVoy,1,2 Kenneth L. Brigham,1,2,3 and Mauricio Rojas1,2,3

1Division of Pulmonary, Allergy, and Critical Care Medicine, 2Center for Translational Research in the Lung, and 3McKelvey Center for Lung Transplantation, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia

Submitted 4 January 2006 ; accepted in final form 17 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The reasons for variable sensitivity among and within species to lung injury and fibrosis caused by bleomycin (BLM) are unknown. Because T helper (Th) 1 and 2 (Th1 and Th2) polarization of CD4+ T lymphocytes is one of the factors that affects the BLM response, we hypothesized that preventing expression of the Th1 transcription factor T-bet would render BLM-resistant BALB/c mice sensitive to BLM. Wild-type and T-bet-deficient (T-bet–/–) BALB/c mice were treated with BLM or saline solution intratracheally. After BLM treatment, collagen content in the lung increased twofold by day 14 in lungs from T-bet–/– mice but was unaffected in lungs from wild-type BALB/c mice. These findings were confirmed by collagen staining of histopathological sections. BLM treatment significantly increased respiratory frequency and decreased tidal volume by day 14 in T-bet–/– mice but had no effect in wild-type mice. Lung fibrosis in BLM-treated T-bet–/– mice was associated with increased circulating levels of Th2 cytokines and increased expression of the profibrotic factor transforming growth factor-beta1. Depletion of CD4+, but not CD8+, T cells in T-bet–/– mice diminished BLM-induced lung fibrosis and the expression of transforming growth factor-beta1. These data suggest that the T-bet pathway in CD4+ T lymphocytes can confer resistance to BLM-induced lung fibrosis in BALB/c mice.

pulmonary fibrosis; Th2 response; animal models

IDIOPATHIC PULMONARY FIBROSIS (IPF) in humans is characterized by heterogeneous fibrosis with excess extracellular matrix (9, 28, 43) and clusters of activated fibroblasts and myofibroblasts (22, 55). Increasing evidence suggests that IPF results from abnormal repair of persistent epithelial injury (5, 6, 23, 51). This maladaptive repair is characterized by activated fibroblasts, which produce extracellular matrix proteins (13, 27) and a variety of cytokines, including the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) (2, 49, 52). The profibrotic milieu in the lungs includes a predominance of T helper (Th) type 2 (Th2) over Th1 cytokines (46, 47). Th2 cytokines (IL-4, IL-5, and IL-13) activate fibroblasts and induce their production of extracellular matrix, whereas Th1 cytokines (IFN-{gamma}) suppress fibroblast proliferation and production of collagen and fibronectin (35).

Bleomycin (BLM) is commonly used to induce experimental pulmonary fibrosis in rodents (1, 38). BLM causes injury to lung epithelium, presumably secondary to DNA damage induced by reactive oxygen intermediates produced by a metal ion-catalyzed reaction of BLM with oxygen (10, 24). These events are followed by an intense inflammatory response. After 2 wk of BLM administration, the inflammatory response wanes, and collagen deposition can be detected. Accumulation of collagen increases for ≥4–6 wk and then declines (4, 54).

The role of Th2 cytokines in BLM-induced lung fibrosis is not clear. Studies using transgenic and deficient IL-4 mice conclude that IL-4 is not a key profibrotic cytokine (11, 34), but inhibitors of IL-4 and IL-13 attenuate BLM-induced pulmonary fibrosis (14). Recently, a novel transcription factor, T-Box, which is expressed in T cells (T-bet) and transactivates the IFN-{gamma} gene and initiates Th1 lineage development, has been identified (41). T-bet-deficient mice have profound defects in development of the Th1 subset and production of IFN-{gamma} and overproduce Th2 cytokines (41). T-bet also regulates the generation and function of CD8 cytotoxic effector cells. T-bet-deficient mice develop spontaneous susceptibility to Th2-mediated pathophysiology, such as airway hyperresponsiveness and oxalone-induced colitis (8, 26, 50). Recently, it has been reported that a T-bet-deficient hybrid strain of SV129/C57BL/6 mice developed spontaneous airway remodeling associated with an increase of collagen III deposition below the airway basement membrane (8). Further studies in this model indicate that IL-13 is indispensable for airway remodeling in T bet-deficient mice (7). These results support the theory that Th2 cytokines are an important element in the development of pulmonary fibrosis.

In the present study, we show that BALB/c mice lacking the T-bet transcription factor are more susceptible to BLM-induced lung fibrosis than a BLM-resistant (BALB/c wild-type) strain of mice. In BALB/c T-bet-deficient (T-bet–/–) mice, interstitial fibrosis does not develop spontaneously but, rather, develops only after BLM treatment and in association with increased expression of TGF-beta1 and Th2 cytokines. Similar to wild-type animals, depletion of CD4+ T lymphocytes rendered T-bet–/– mice resistant to BLM, but depletion of CD8+ T lymphocytes did not. Our data suggest that, in BALB/c mice, polarization of CD4+ T cells to a Th1 phenotype as a result of activation of the transcription factor T-bet reduces susceptibility of the lungs to BLM-induced fibrosis.


   METHODS
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 METHODS
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Experimental animals. Six- to eight-week-old female wild-type C57BL/6, BALB/c mice, and BALB/c mice genetically modified to lack expression of T-bet (T-bet–/– mice) were obtained from Jackson Laboratories (Bar Harbor, ME). Wild-type BALB/c mice are well characterized as resistant to BLM-induced lung fibrosis. Before inoculation, the mice were weighed, and a blood sample was collected. All the animals were maintained in the Division of Animal Resources at Emory University, an American Association for Accreditation of Laboratory Animal Care-approved facility, under pathogen-free conditions with water and food provided ad libitum. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee.

Induction of pulmonary fibrosis by BLM instillation. Mice were anesthetized by isoflurane inhalation. The trachea was exposed using sterile techniques, and BLM (6 U/kg; Sigma, St. Louis, MO) was dissolved in 75 µl of PBS and instilled into the tracheal lumen. After inoculation, the incision was closed and the animals were allowed to recover.

Collagen assay. Samples of lung tissue were collected at the time of death, immediately frozen in liquid nitrogen, and stored at –80°C. Collagen content was determined by measurement of the hydroxyproline content of whole lung homogenates. Hydroxyproline was assessed by hydrolysis of the lung tissue with 6 N HCl for 16 h at 120°C followed by colorimetric analysis using Ehrlich's solution (Sigma) read at 550 nm.

Histopathology. The animals were euthanized, the trachea was cannulated, and the lungs were fixed at constant pressure with 4% paraformaldehyde. After overnight fixation, the tissue was embedded in paraffin. Sections were cut on a microtome, mounted on slides, and stained with hematoxylin-eosin or Masson's trichrome (Sigma). A fluorescence microscope (model EX41, Olympus America, Melville, NY) with x20 and x4 lenses and an Olympus MagnaFire camera were used to obtain photomicrographs.

Morphometric analysis. The photomicrographs were quantitatively assessed for lung tissue damage. Scion Image software was used for quantification of tissue density as an indicator of interstitial thickness. Ten images at x2 magnification were analyzed to include the total lung section.

Immunohistochemistry. Tissue sections (5 µm) from formalin-fixed paraffin-embedded lung tissue were deparaffinized and rehydrated by consecutive changes of xylene-alcohol. Endogenous peroxidase activity was quenched by incubation of the sections with 3% hydrogen peroxide for 30 min. Vectastain procedures were performed according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Briefly, the sections were incubated for 30 min with 2% normal serum from the species in which the secondary antibody was made to reduce nonspecific binding. After excess serum was blotted, the sections were incubated with rat anti-CD4 (catalog no. sc-13573), rat anti-CD8 (catalog no. sc-20041), goat anti-phosphorylated Smad 2/3 (catalog no. sc-11769), or rabbit anti-TGF-beta1 (catalog no. sc-146) antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at room temperature. Then the sections were washed and probed with diluted biotinylated secondary antibody for 30 min. The sections were incubated with Vectastain ABC reagent for 30 min and then with peroxidase substrate solution until dark brown stain became obvious. Finally, the sections were counterstained with hematoxylin, cleared with alcohol-xylene, and mounted.

Detection of cytokines and chemokines. Serum concentrations of cytokines, growth factors, and chemokines were determined using a Luminex system (Luminex, Austin, TX) with a single anti-mouse kit (Linco, St. Charles, MO). Well filters were prewashed, and samples (1:1 dilution, 50 µl final volume) were applied to each well. Specific antibody-coated beads were added to the wells, and the samples were incubated for 18 h at 4°C. After incubation, the plate was washed twice. Biotinylated antibodies against the different cytokines and chemokines were added, and the mixture was incubated for 1 h. Then the cytokine-antibody complexes were detected by addition of streptavidin coupled to phycoerythrin. For determination of the number of positive complexes, each sample was read in a Luminex XYP platform. Data were analyzed using MasterPlex 1.2 (Mai-Rabio) and expressed in picograms per milliliter.

Western blot. Whole cell extracts from lung tissue samples were prepared using ELB lysis buffer [50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, and 10% NP-40] (33). Aliquots of lung lysates (10 µg) and bronchoalveolar lavage fluid (12 µl) were resuspended in Laemmli sample buffer, resolved using 4–20% SDS-polyacrylamide gels (Bio-Rad), and transferred overnight using Tris-glycine buffer [20 mM Tris, 150 mM glycine (pH 8.0), and methanol to a final concentration of 20%] onto polyvinylidene difluoride membranes (GE, Piscataway, NJ). The blots were blocked in 5% skim milk in PBS for 1 h at room temperature. Western blot analysis for TGF-beta1, Smad 3, and Smad 7 was performed according to the manufacturer's recommendations (Santa Cruz Biotechnology). Proteins were visualized after incubation of the blots in Super Signal chemiluminescent substrate solution and exposure to X-Mat film (Kodak, Rochester, NY). Filters were stripped and reprobed with an antiserum against beta-actin (Santa Cruz Biotechnology) as a loading control.

In vivo depletion of CD4+ and CD8+ cells. CD4+ and CD8+ cells were depleted by intraperitoneal administration of monoclonal antibodies against CD4 and CD8. GK 1.5, a rat IgG2b monoclonal antibody against mouse CD4 (American Type Culture Collection, Manassas, VA), and the hybridoma 2.43, a rat IgG2b antibody against mouse CD8 (American Type Culture Collection), were purified using a protein G column (Amersham Biosciences). Purified antibodies (0.1 mg) were administered by intraperitoneal injection every 3 days during the week before BLM and once a week thereafter. Depletion of CD4+ and CD8+ cells was corroborated by fluorescence-activated cell sorting (FACS) analysis before BLM instillation. Rat IgG was used as a control.

FACS analysis. White cell suspensions were prepared from venous blood. Red blood cells were lysed by hypotonic shock; cells were stained with specific antibodies using phycoerythrin-conjugated anti-CD11b, peridinin chlorophyll protein-anti-CD4, and FITC-conjugated anti-CD8 (PharMingen). Analysis was performed on a FACScan cytometer using Cellquest software (Becton Dickinson, San Jose, CA), and the data were further analyzed using FlowJo software (Tree Star, San Carlos, CA). Forward angle and side light scatter were used to exclude dead cells.

Statistical methods. For comparisons between groups, paired Mann-Whitney tests or unpaired t-tests with or without Welch's correction and one-way ANOVA were used. P < 0.05 was considered significant. GraphPad Prism and GraphPad InStat statistical packages were used to make these calculations.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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T-bet–/– mice are more susceptible to BLM-induced pulmonary fibrosis. At 14 days after BLM administration, hematoxylin and eosin-stained lung sections from T-bet–/– mice showed loss of the normal pulmonary architecture, with inflammatory cell infiltration, interstitial fibrosis, and edema (Fig. 1, A and B). In contrast, lungs from wild-type BALB/c mice collected at the same time after BLM administration showed minimal histological changes (Fig. 1, C and D). To demonstrate that the effect of BLM was specific, lungs from T-bet–/– BALB/c mice treated with saline solution (SS) did not present alveolar remodeling or interstitial fibrosis (Fig. 1, E and F). In contrast, histological sections from BLM-susceptible C57BL/6 mice showed extensive areas of inflammation and interstitial matrix deposition (Fig. 1, G and H). Morphometric analysis to determine the increase of interstitial thickness by inflammation and fibrosis confirmed significantly worse pathology in lungs from T-bet–/– mice than in lungs from wild-type mice 14 days after BLM administration (Fig. 1I).


Figure 1
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  		Fig. 1. Deficiency of T-bet transcription factor renders bleomycin (BLM)-resistant BALB/c mice susceptible to BLM-induced lung injury and fibrosis. Wild-type (WT) BALB/c and T-bet deficient (T-bet–/–) BALB/c mice were infused intratracheally with BLM (6 U/kg) and killed on day 14. A and B: extensive tissue remodeling with matrix deposition and increased cellularity in hematoxylin and eosin-stained sections from T-bet–/– BALB/c mice; arrows, areas of inflammation and remodeling. C and D: small areas of inflammation without significant tissue remodeling in lungs from wild-type BALB/c mice. E and F: no tissue remodeling in T-bet–/– BALB/c mice treated with saline solution (SS, control). G and H: extensive areas of inflammation and extracellular matrix deposition in BLM-treated C57BL/6 mice (positive control). I: quantitative measurement of lung tissue abnormalities by morphometric analysis of lung sections. Low-magnification (x2) photomicrographs (10 for each lung section) were analyzed to quantify tissue density as a measurement of lung interstitial thickness. Values are means ± SE of 4 lung histological sections per group. Increase of interstitial thickness in T-bet–/– mice reflects early inflammatory response on day 7 and later fibrotic phase on day 14.

 
The presence of collagen is highlighted in photomicrographs of Masson's trichrome-stained lung sections 1 and 2 wk after BLM instillation in wild-type and T-bet–/– mice in Fig. 2. At 1 wk after BLM instillation, modest amounts of collagen, along with small inflammatory infiltrates around airways, were present in T-bet–/– mice (Fig. 2, A and B). At 2 wk after BLM instillation, collagen staining, including multiple foci of interstitial dense collagen, was clearly increased in lungs from T-bet–/– mice (Fig. 2, C and D). In sharp contrast, lungs from BALB/c wild-type mice harvested at the same time points showed nearly normal lung architecture (Fig. 2, E–H).


Figure 2
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  		Fig. 2. A–H: Masson's trichrome-stained (blue) histological sections showing that collagen deposition was responsible for the increase in tissue density. Low-magnification (x4) and higher-magnification (x20) images show increased cellularity and substantial amounts of collagen in lungs from T-bet–/– mice 7 days (A and B) and 14 days (C and D) after BLM and modest inflammation 7 days (E and F) and almost normal histology 14 days (G and H) after BLM in lungs from wild-type BALB/c mice. I: amount of collagen in lungs determined by measurement of hydroxyproline (OH-proline) 14 days after BLM. Collagen content was greater in lungs of T-bet–/– than control animals.

 
Measurements of collagen content in lungs from both groups of mice are summarized in Fig. 2I. Hydroxyproline content was significantly greater in lungs from T-bet–/– mice 14 days after BLM instillation than in lungs from wild-type control animals at the same time.

Serum concentrations of cytokines and growth factors. The time course of serum concentrations of Th1 and Th2 cytokines and proinflammatory factors implicated in the pathogenesis of fibrosis after BLM administration is summarized in Fig. 3. The greater increase in concentrations of the Th2 cytokines IL-5, IL-9, and IL-13 in T-bet–/– than in wild-type mice (Fig. 3A) was expected, because CD4+ T cells in T-bet–/– mice are biased to differentiate into a Th2 phenotype. The increase in circulating IL-10, a cytokine that cooperates in generation of polarized Th2 cell responses (18, 25), was also greater in T-bet–/– mice (Fig. 3A). Plasma concentrations of granulocyte-macrophage colony-stimulating factor also increased more in T-bet–/– mice than in controls; granulocyte-macrophage colony-stimulating factor has been implicated in the generation of Th2 responses by its effects on antigen-presenting cells and by its enhancement of Th2 cell function and survival (30). These results confirm a profound bias to Th2 responses in T-bet–/– mice after BLM administration.


Figure 3
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  		Fig. 3. Time course of serum concentrations of cytokines after BLM-induced lung injury in wild-type and T-bet–/– mice at 3, 7, and 14 days. Values are means ± SE. Serum was analyzed in a multiplex system to determine levels of different cytokines (n = 3 for each time point in each group). A: type 2 cytokines; B: type 1 cytokines and IP-10 chemokine; C: other proinflammatory cytokines and chemokines. MIP-1{alpha} and MCP-1, monocyte and macrophage attractant chemokines; GM-CSF, granulocyte-macrophage colony-stimulating factor; KC, keratinocyte-derived chemokine. *Significantly different from BALB/c WT (P < 0.05).

 
Plasma concentrations of IFN-{gamma} were also elevated after BLM instillation in T-bet–/– mice compared with wild-type controls, but IP-10 expression, which is IFN-{gamma} dependent, was generally similar to controls (Fig. 3B). Circulating concentrations of other cytokines produced by dendritic cells (IL-12 and TNF-{alpha}, keratinocyte-derived chemokine, and monocyte/macrophage attractant chemokines) also increased more in T-bet–/– mice (Fig. 3C). Several of these chemokines have been associated with fibrogenesis and Th2-mediated activation of lung epithelial cells and macrophages (20, 37).

In summary, Th2 responses to intrapulmonary administration of BLM are enhanced in T-bet–/– mice, despite the presence of high levels of IFN-{gamma}, by a non-T bet-dependent mechanism.

Increase in TGF-beta1 expression in lung tissue after BLM instillation. Th2 cytokines have a direct profibrotic effect, and they also promote expression and activation of the fibrogenic cytokine TGF-beta1 (17). Expression of TGF-beta1 and phosphorylation of Smad 3, a molecule in the TGF-beta1 signaling pathway, are important in lung fibrogenesis (29, 36). We determined lung expression of TGF-beta1 and Smad 3 by immunohistochemistry and Western blot (Figs. 4 and 5). At 7 days after BLM administration, TGF-beta1 immunostaining was positive in T-bet–/– mice in alveolar macrophages and lung epithelial cells but was negative in lungs from BALB/c wild-type mice (Fig. 4A). At 14 days after BLM instillation, the peak of the fibrotic response, TGF-beta1 staining in T-bet–/– mice was extensive in lung and airway epithelial cells and associated with interstitial connective tissue. In comparison, lungs from BALB/c mice showed only scattered positive lung epithelial cells (Fig. 4A). The immunostaining pattern for phosphorylated Smad 3 was similar (Fig. 5). Kinetic analyses of the levels of the TGF-beta1-latency-associated peptide complex (55 kDa) and the active TGF-beta1 homodimer (12.5 kDa) were performed by Western blot analysis in lung lysates of wild-type and knockout animals. Analysis at baseline showed predominant expression of the latent form, with similar levels in BALB/c and T-bet–/– mice (Fig. 4B). Instillation of BLM enhanced expression of the latent and active TGF-beta1 in T-bet–/– mice, with a pattern of progression mainly from day 7 to day 14, when fibrosis was most pronounced. The increase in the active form of TGF-beta1 was more modest in wild-type BALB/c mice and appeared late on day 14.


Figure 4
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  		Fig. 4. Expression of transforming growth factor-beta1 (TGF-beta1) increases exclusively in T-bet–/– mice after BLM injury. A: histological sections from lungs of wild-type and T-bet–/– mice 7 and 14 days after BLM. Brown stain indicates TGF-beta1; arrows, TGF-beta1-positive cells. Staining was minimal in lungs from wild-type animals but extensive in lungs from T-bet–/– animals, especially 14 days after BLM. B: Western blots showing expression of 52-kDa (Pro) and 12.5-kDa active forms of TGF-beta1 from lungs of T-bet–/– and BALB/c wild-type mice at baseline and 3, 7, and 14 days after BLM. Both forms of the protein increased after BLM, especially in T-bet–/– animals.

 

Figure 5
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  		Fig. 5. Activation of the Smad 2/3 pathway by BLM-induced lung injury only in T-bet–/– mice, as demonstrated by immunostaining for the phosphorylated form of Smad 2/3. Lungs from wild-type BALB/c and T-bet–/– mice were harvested 7 and 14 days after BLM instillation, fixed in formalin, and stained with an antibody specific for the phosphorylated form of the Smad protein, which stains brown. Increased activated Smad 3 was detected only in T-bet–/– mice, and the signal increased over the course of the BLM response.

 
Decrease in severity of BLM fibrosis and TGF-beta1 expression by in vivo depletion of CD4+, but not CD8+, cells in T-bet–/– mice. T-bet has a critical and nonredundant function in generation of type 1 immunity. However, CD8 T cell effector functions are partially independent of T-bet (16, 39, 42). Histological sections stained for CD4+ and CD8+ cells showed heavy infiltration of both cell types in lungs of BLM-treated T-bet–/– mice, whereas only small infiltrates around airways and blood vessels were observed in lungs from wild-type BALB/c mice (data not shown). To determine whether T-bet-dependent effector function in CD4+ and CD8+ T cells was required in the fibrotic response, we immunodepleted T-bet–/– mice of CD4+ or CD8+ cells and determined their response to BLM. Specific cell type depletion was confirmed by flow cytometric analysis of circulating leukocytes (Fig. 6, A and B). On day 14, histopathological analysis showed more severe inflammatory infiltrates and collagen deposition in CD8+ than in CD4+ cell-depleted animals; the response in CD4+-depleted animals was similar to that in wild-type animals (Fig. 6, C and D). We determined lung expression of the profibrotic factor TGF-beta1 in mice depleted of each of the T cell subsets. At 14 days after BLM treatment, we found similar levels of latent and active TGF-beta1 in control rat IgG-treated and in CD8-depleted T-bet–/– mice. In sharp contrast, lung expression of TGF-beta1 was markedly reduced or absent in mice lacking CD4+ T cells. Collectively, these results indicate that CD4+ T cells defective in T-bet transcription factor render the lungs susceptible to BLM-induced fibrosis (Fig. 7).


Figure 6
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  		Fig. 6. Decrease in severity of BLM-induced injury in BALB/c T-bet–/– mice depleted of CD4+, but not CD8+, T cells. T-bet–/– animals were immunodepleted of CD4+ or CD8+ cells before instillation of BLM. A and B: fluorescence-activated cell sorting analysis of peripheral white blood cells demonstrating specific depletion of CD4+ (A) and CD8+ (B) cell types. C and D: histological analysis of sections from lungs of animals killed after 14 days. Hematoxylin and eosin-stained sections show decreased cellular infiltrates and fibrosis in a CD4+-depleted animal similar to the wild-type response, whereas CD8+ depletion did not alter the BLM response.

 

Figure 7
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  		Fig. 7. Western blot analysis for TGF-beta1 in lungs from T-bet–/– mice treated with a control immune serum or immunodepleted of CD4+ or CD8+ cells and treated with BLM and harvested 14 days later. Animals treated with anti-CD8 or isotype control serum expressed similar levels of TGF-beta1. In animals in which CD4+ cells were depleted, TGF-beta1 levels were very low or not detectable.

 
Circulating concentrations of IFN-{gamma} 14 days after BLM instillation are shown for the T cell subset-immunodepleted animals and isotype-treated controls in Fig. 8. The principal cell source of the increase in IFN-{gamma} after BLM instillation in T-bet–/– animals shown in Fig. 3B appears to be CD8+, rather than CD4+, cells, because CD8+ depletion significantly reduced the IFN-{gamma} response, whereas CD4+ depletion had no effect. In contrast, levels of the Th2 cytokine IL-10 were significantly lower only in CD4-depleted T-bet–/– mice.


Figure 8
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  		Fig. 8. Serum concentrations of IFN-{gamma} 14 days after BLM administration in T-bet–/– mice treated with a control immune serum or immunodepleted of CD4+ (Dep-CD4+) or CD8+ (Dep-CD8+) T cells. CD8+ cell depletion significantly decreased the IFN-{gamma} response, but CD4+ depletion had no effect. *Significantly different (P < 0.05).

 

   DISCUSSION
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 ABSTRACT
 METHODS
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 DISCUSSION
GRANTS
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A balance between Th1 and Th2 cytokines has an apparent role in the development of fibrosis in the lungs (46, 47). In particular, the balance between IFN-{gamma}/IL-13 and TGF-beta1 appears to regulate inflammatory and fibrotic responses. We demonstrate here that the Th1 transcription factor T-bet in CD4+ T cells determines the susceptibility to BLM-induced pulmonary fibrosis in BALB/c mice, which are naturally resistant to BLM. We found Th2-biased immune responses, enhanced expression of TGF-beta1, and lung fibrosis in BLM-treated T-bet–/– mice. Immunodepletion of CD4+, but not CD8+, T cells restored the wild-type response.

Clarification of the immunology of BLM-induced lung fibrosis in mice may contribute to the understanding of the pathogenesis of the human disease. Recent evidence indicates that patients with IPF have an immune environment that is polarized toward a type 2 response (32, 46, 47). It is possible that recurrent or continuous lung injury in susceptible individuals exhausts their capacity to mount an appropriate type 1 response (IFN-{gamma}), shifting the immune environment toward a type 2 response (IL-4, IL-13, and IL-5) with consequent upregulation of profibrotic genes.

Mice lacking T-bet have defective production of IFN-{gamma} in CD4, natural killer (NK), dendritic, and effector memory CD8+ T cells (8, 42, 50), although IFN-{gamma} production is unaffected in CD8+ T cells after polyclonal stimulation. Normal levels of IFN-{gamma} have been reported in T cells from T-bet–/– mice in an oxalone-induced colitis model and in herpes simplex virus type 2-specific CD8 T cells, among others (26, 40). We found increased circulating levels of IFN-{gamma} in BLM-treated T-bet–/– mice. This response was significantly attenuated with CD8+, but not CD4+, T cell depletion, indicating that CD8+ cells were likely the principal source of the cytokine. T-bet also controls IFN-{gamma} expression in NK cells, regulating innate immune responses (31, 44, 53). The capacity of pulmonary NK cells to produce IFN-{gamma} seems to be a key determinant of resolution or progression of pulmonary fibrosis, as was determined by adoptive transfer of NK cells in CXCR3-deficient mice (15). In T-bet–/– mice, the inability of NK cells to produce IFN-{gamma} could contribute to Th2 polarization, favoring fibrosis, instead of resolution. Dendritic cells from T-bet–/– mice can also contribute to fibrosis by defective Th1 polarization and production of significantly higher amounts of the Th2-specific thymus- and activation-related chemokine, which selectively recruits Th2 cells to the site of the inflammation (48).

CD8+ and CD4+ T cells play a role in BLM-induced fibrosis, because depletion of individual T cell subsets attenuates lung fibrosis, and fibrosis is completely prevented by simultaneous depletion of both T cell subsets (21). Our results specifically implicate Th2-biased CD4+ T cells in the increased susceptibility to BLM-induced lung fibrosis in T-bet–/– mice. Th2 cytokines have different roles in the regulation of tissue remodeling and fibrosis. We found increased levels of IL-13 and IL-5 in BLM-challenged T-bet–/– mice. IL-13 shares many functional activities with IL-4, because both cytokines use the same IL-4R{alpha} chain and STAT6 pathway. IL-13 has been demonstrated to be a dominant effector of fibrosis in several models, including the BLM model, and IL-5 preferentially facilitates the production of profibrotic cytokines, including TGF-beta1 and IL-13 (3, 12). We found that depletion of CD4 T cells in T-bet–/– mice was associated with diminution of the profibrotic TGF-beta and Th2 cytokines. The low levels of TGF-beta1 are potentially the result of the depletion of TGF-beta-producing CD4 T cells plus decreased fibroblast TGF-beta production. Recent reports indicate an autocrine activation of lung fibroblasts from T-bet–/– mice stimulated by the release of Th2 cytokines from CD4 T cells (7)

We found marked increases in expression of TGF-beta1 and its signaling molecule Smad 3 in the lungs of T-bet–/– mice after BLM administration that were specific to the T-bet–/– phenotype. A reciprocal relation between TGF-beta1 and T-bet has been reported. TGF-beta1 suppresses T-bet expression in activated T cells, and regulatory CD62L– CD4+ T cells in T-bet–/– mice overexpress TGF-beta1 (26). Enhanced expression of TGF-beta1 is associated with an increase in Smad 3 and a decrease in the TGF-beta1 inhibitor Smad 7. Binding of Smad 3 to the TGF-beta1 receptor is followed by importin-1beta- and RanGTPase-mediated import of Smad 3 into the nucleus, where it controls expression of TGF-beta1 target genes (45). The presence of TGF-beta1 during priming of CD4 T cells suppresses IFN-{gamma} expression as well as development of Th1 effector cells expressing IFN-{gamma} at recall stimulation by a direct effect on the CD4+ cell. Overexpression studies indicate that TGF-beta1 inhibits IFN-{gamma} expression in effector Th1 cells through inhibition of T-bet expression (19). Consistent with these data, our studies show strong expression of TGF-beta1 in T-bet–/– mice after BLM treatment.

In conclusion, our data suggest that a deficiency of the Th1 transcription factor T-bet in CD4+ T lymphocytes results in a dominant Th2-biased response to BLM and renders an otherwise BLM-resistant mouse strain susceptible to lung fibrosis. These data support the theory that the immune system has an important role in lung fibrogenesis and, specifically, implicate T-bet as one of the regulators of this process. Although inhibition of immune responses has been shown to attenuate fibrosis in animal models but is less successful in humans, this information may provide rationales for developing new interventions that modulate the fibrotic response.


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This work was supported by National Heart, Lung, and Blood Institute Grants 5 P01 HL-0669496-02 and K01 HL-073154, University Research Committee (URC) Emory University Grant 2003100, and the McKelvey Lung Transplantation Center at Emory University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Rojas, Div. of Pulmonary, Allergy, and Critical Care Medicine, Center for Translational Research of the Lung, Emory Univ. School of Medicine, Atlanta, GA 30322 (e-mail: mrojas{at}emory.edu)

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

* A. L. Mora and J. Xu contributed equally to this work. Back


   REFERENCES
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