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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Feb 16;104(8):2827–2830. doi: 10.1073/pnas.0700021104

Transcription factor T-bet regulates skin sclerosis through its function in innate immunity and via IL-13

Antonios O Aliprantis *,†, Jingsong Wang *,†, John W Fathman *, Raphael Lemaire ‡, David M Dorfman §, Robert Lafyatis ‡, Laurie H Glimcher *,†,
PMCID: PMC1815266  PMID: 17307869

Abstract

Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-β and IL-4, more than type 1 cytokines, like IFN-γ, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2−/− mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet−/− mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.

Keywords: scleroderma, fibrosis, cytokine

It has been estimated that diseases characterized by tissue fibrosis cause 45% of deaths in the United States (1). Fibrosis commonly occurs secondary to chronic inflammation, such as liver cirrhosis from viral or parasitic infections, or after organ damage, such as the left ventricular scarring that ensues after myocardial infarction. The primary fibrosing disorders, such as pulmonary fibrosis and systemic sclerosis (SSc) (scleroderma), are rare and less well understood, but offer insight into the dysregulation of extracellular matrix production. SSc is an autoimmune disease characterized by the excessive deposition of collagen, fibronectin, and proteoglycans by activated fibroblasts in the skin, internal organs, and blood vessels, resulting in fibrosis and a proliferative small vessel vasculopathy (2). Few therapeutic options are available for this disabling disease.

Soluble mediators from a differentiated type 2 immune response have been proposed to drive the fibrotic process in SSc (3). Accordingly, increased levels of IL-4, IL-13, and TGF-β can be demonstrated in the blood or affected tissues of SSc patients (4, 5). Animal models of human SSc support this hypothesis. A duplication in the fibrillin-1 gene in mice causes a tight skin phenotype (Tsk/+) with pathological changes similar to SSc. Tsk/+ mice deficient in IL-4, IL-4 receptor, or STAT6 do not develop disease (6). Humans treated with the antineoplastic agent, bleomycin, can develop pulmonary fibrosis or frank SSc (7). Intrapulmonary or intradermal administration of bleomycin in mice consequently results in lung or skin fibrosis, respectively (7). Interestingly, agonists of type I immunity, like IFN-γ, IL-12, or CpG, are protective in these murine models of SSc (7, 8). Thus, restoring a balance between type I and type II cytokines may be an attractive strategy for the treatment of SSc and other fibrosing disorders.

Given the pleiotropic role that transcription factors play in shaping immune responses, we studied the function of the transcription factor T-box expressed in T cells (T-bet) in murine bleomycin-induced scleroderma. T-bet, the master regulator of type 1 T helper (Th1) cell differentiation, activates and represses type I and type II cytokines, respectively. Thus, T-bet-deficient mice display impaired IFN-γ production and overproduction of TGF-β, IL-4, and IL-10, resulting in spontaneous asthma, the paradigmatic Th2 disease (9, 10). Here, we show that mice deficient in T-bet develop exacerbated bleomycin-induced scleroderma. Unexpectedly, despite its well-characterized role in T cells, T-bet exerts its antifibrotic effect within the innate immune system likely by repressing the profibrotic cytokine, IL-13.

Results

Development of Bleomycin-Induced Scleroderma Does Not Require an Intact Adaptive Immune System.

Pulmonary fibrosis is a well-known side effect of the antineoplastic agent, bleomycin. Daily s.c. injections of bleomycin into mice for 3–4 weeks results in the deposition of homogeneous collagen bundles and cellular infiltrates that histologically resembles human scleroderma (11). Moreover, a single, intratracheal instillation of bleomycin results in lung injury and fibrosis and is a model for the human disease, idiopathic pulmonary fibrosis (12). The relative role of the adaptive and innate immune system in bleomycin-induced tissue fibrosis is controversial. Both nude mice, and mice immunodepleted of CD4 and CD8 T cells with antibodies, display attenuated bleomycin-mediated pulmonary disease (13, 14). Conversely, other investigators have reported that SCID mice develop both bleomycin-induced pulmonary fibrosis and dermal sclerosis comparable with that of wild-type mice (15, 16). The SCID mutation, which resides in a gene encoding a DNA-dependent protein kinase, is “leaky” with ≈15% of mice developing functional lymphocytes (17). Therefore, to definitively test the contribution of the adaptive immune system to bleomycin-induced dermal sclerosis, we performed this model in RAG2−/− knockout mice (RAG2 KO), which are deficient in the recombinase gene necessary for antigen receptor rearrangement and therefore lack mature T and B cells (18). After 4 weeks of bleomycin treatment, RAG2−/− and WT mice developed comparable increases in skin hydroxyproline content (Fig. 1), a reliable maker of collagen deposition and skin sclerosis (11). We conclude that the development of dermal sclerosis in this model is not mediated by the adaptive immune system and must involve innate immune cells.

Fig. 1.

Fig. 1.

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Bleomycin-induced skin sclerosis does not require the adaptive immune system. BALB/c (WT) or RAG2−/− mice on BALB/c background (RAG2 KO) received s.c. injections of PBS (□) or bleomycin (■) for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay. Statistically significant increases were observed in the hydroxyproline content in the skin of bleomycin-treated WT and RAG2−/− mice compared with PBS-treated control mice (P = 0.020 and 0.019, respectively). No statistical difference was observed in the hydroxyproline content in the skin of bleomycin-treated WT or RAG2−/− mice (∗, P > 0.05).

Increased Severity of Bleomycin-Induced Sclerosis in the Absence of T-bet.

The transcription factor T-bet governs the differentiation of CD4+ T cells into Th1 cells characterized by the production of the cytokine, IFN-γ. Recent evidence, however, has uncovered an important role for T-bet in innate immunity. T-bet is required for optimal production of IFN-α and IFN-γ from plasmacytoid and classical CD11chigh dendritic cells, respectively (19). Moreover, dendritic cells deficient in T-bet are unable to mediate inflammatory arthritis (20). We decided to investigate the role of T-bet in the bleomycin model of scleroderma for the following reasons: (i) the innate immune system mediates bleomycin-induced skin sclerosis (Fig. 1), (ii) skin sclerosis in the bleomycin model can be attenuated by exogenous administration of IFN-γ (7), and (iii) T-bet is important for the production of IFN-γ by innate immune cells (19). Accordingly, WT and T-bet−/− mice were subjected to daily injections of bleomycin for 4 weeks. Histopathological analysis of the skin from bleomycin-treated mice revealed the hallmark of dermal sclerosis: thickening of the dermis caused by the deposition of homogenous collagen bundles. Both conventional H&E staining (Fig. 2 A–D) and trichrome staining (Fig. 2 E–H), which highlights collagen bundles, revealed exacerbated dermal sclerosis in the T-bet−/− mice compared with WT mice. Lastly, skin hydroxyproline content (Fig. 3A), a quantitative marker of collagen content, was significantly higher in T-bet−/− mice compared with WT mice after 4 weeks of bleomycin treatment. Two approaches were taken to investigate whether T-bet was acting within innate or adaptive immune cells to suppress dermal sclerosis in response to bleomycin. First, the T-bet null allele was crossed onto the RAG2−/− background to generate T-bet−/−/RAG2−/− double-knockout mice (T-bet/RAG2 DKO). Interestingly, T-bet KO and T-bet/RAG2 DKO mice developed similarly increased levels of skin hydroxyproline after 4 weeks of bleomycin treatment compared with WT mice (Fig. 3A). Our second approach was to test the bleomycin-induced dermal sclerosis model in transgenic mice that overexpress T-bet in T cells under the control of the CD2 promoter (21). Despite increased production of IFN-γ from T cells of this mouse (21), no difference was observed in the accumulation of hydroxyproline in the skin after 4 weeks of s.c. bleomycin administration (Fig. 3B). Together, these data indicate that T-bet functions within cells of the innate immune system to attenuate bleomycin-induced dermal sclerosis.

Fig. 2.

Fig. 2.

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Histopathological analysis of bleomycin-treated T-bet KO mice reveals increased dermal sclerosis compared with WT mice. Skin sections from BALB/c (WT) and T-bet−/− (Tbet KO) mice injected daily for 4 weeks with bleomycin or PBS were stained with H&E (A–D) or Masson trichrome (E–H). Note the increased skin thickness and more robust deposition of collagen observed in bleomycin-treated TbetKO mice compared with similarly treated WT mice.

Fig. 3.

Fig. 3.

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The transcription factor T-bet functions within the innate immune system to augment bleomycin-induced skin sclerosis. (A) BALB/c (WT), T-bet−/− mice (Tbet KO), and RAG2−/− × T-bet−/− DKO mice (Tbet/RAG2 DKO) received s.c. injections of PBS (□) or bleomycin (■) for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay (∗, P < 0.002; ∗∗, P < 0.05). The results are representative of four independent experiments. (B) BALB/c (WT) and transgenic mice that overexpress T-bet under the control of the CD2 promoter were treated with bleomycin and analyzed as in (A) (∗, P > 0.05). The data represent the average values of each group of mice (n = 3–6) with standard deviation. Asterisks denote statistical significance. The results are representative of two independent experiments.

IL-13 Is a Functional Target of T-bet and Profibrotic Cytokine in Bleomycin-Induced Skin Fibrosis.

IL-13 is a type II cytokine that promotes tissue fibrosis. In mice, overexpression of IL-13 in the lung results in airway fibrosis (1). Blocking IL-13 activity with either antibodies or an IL-13 receptor fusion protein can reduce collagen deposition in the livers of mice challenged with Schistosoma mansoni, or in the lungs of mice exposed to bleomycin or Aspergillus (1). Moreover, airway remodeling and fibrosis observed in T-bet-deficient mice can be suppressed by neutralizing antibodies to IL-13 (10). Data from human studies suggest that IL-13 is a pathogenic cytokine in scleroderma. IL-13 levels are increased in the serum of patients with scleroderma and are associated with nailfold capillary abnormalities, a clinically relevant window into the vasculopathy that afflicts these patients (4, 5, 22). Therefore, the role of IL-13 in bleomycin-induced scleroderma was investigated by using mice genetically deficient in this profibrotic cytokine. Compared with WT mice, IL-13−/− (IL-13 KO) failed to develop an increase in skin collagen content after 4 weeks of treatment with bleomycin (Fig. 4). To determine whether IL-13 was also responsible for the augmented bleomycin-induced skin sclerosis observed in T-bet-deficient mice, IL-13−/− T-bet−/− double-knockout mice (IL-13/T-bet DKO) were generated. The phenotype of the IL-13/T-bet DKO was identical to that of the IL-13 single-knockout mice, with greatly attenuated bleomycin-induced collagen deposition compared with WT mice (Fig. 4). These data clearly situate IL-13 downstream of T-bet as a profibrotic mediator of skin sclerosis in response to bleomycin.

Fig. 4.

Fig. 4.

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T-bet regulate dermal sclerosis through IL-13. Seven- to 8-week-old BALB/c (WT), IL-13−/− mice on BALB/c background (IL-13 KO), and T-bet−/− × IL-13−/− mice (Tbet/IL-13 DKO) received s.c. bleomycin injections for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay (∗, P < 0.05; ∗∗, P < 0.05). The data represent the average values of each group of mice (n = 3–5) with standard deviation. Asterisks denote statistical significance. The results shown are representative of three separate experiments.

Discussion

In recent years, an important role for innate immune cells in the development of various autoimmune diseases, such as lupus and rheumatoid arthritis, has become apparent. The presence of T cells in the fibrotic lesions of scleroderma and accompanying characteristic serum autoantibodies has focused research mainly on the adaptive immune system. In contrast, relatively little has been done to evaluate the contribution of the innate immune system in the development of this disease. Here, we have shown that bleomycin-induced skin sclerosis is driven by IL-13 (Fig. 4) derived from the innate immune system and that the transcription factor T-bet is an endogenous suppressor of this response (Figs. 2–4). Although the mechanism of action of T-bet in this model remains unresolved, we believe T-bet functions within the innate immune system. First, the adaptive immune system is dispensable for bleomycin-induced dermal fibrosis (Fig. 1). Second, we have never been able to demonstrate T-bet expression in cells of mesenchymal origin (data not shown). Third, recently, our laboratory has shown that T-bet is critical for IFN-γ production by dendritic cells after stimulation with either IL-12 or CpG oligodeoxynucleotides, a Toll-like receptor 9 (TLR9) agonist (19). Interestingly, prior studies have shown that exogenous administration of IFN-γ or CpG oligodeoxynucleotides can ameliorate dermal fibrosis in bleomycin and Tsk/+ disease models, respectively (7, 8). The attenuation of dermal fibrosis by IFN-γ may result from inhibition of TGF-β-mediated collagen synthesis in fibroblasts (23). Therefore, the absence of T-bet may result in reduced tissue levels of IFN-γ, promoting IL-13-driven fibrosis. Alternatively, T-bet may directly regulate IL-13 production by the innate immune system. Further experimentation is needed to resolve the precise cellular role of T-bet in this model.

The data presented in Figs. 2 and 3 indicate that T-bet is a suppressor of skin fibrosis and agree with the previous observation that T-bet deficiency promotes lung remodeling and airway fibrosis (24). Distinct from our results in the skin, however, T-bet appears to function within adaptive immune cells (T cells) to attenuate fibrosis in the lung. The loss of T-bet leads to spontaneous Th2 skewing and the overproduction of type II cytokines, such as IL-4, IL-13, and TGF-β in bronchoalveolar lavage fluid. Moreover, T-bet-deficient CD4+ T cells are sufficient to transfer pulmonary disease to unaffected SCID recipients (24). Lastly, in contrast to our results, T-bet deficiency appears to promote bleomycin-induced lung fibrosis, in resistant BALB/c mice, through CD4+ T cells (25). Together, these prior investigations, and the findings presented here, highlight T-bet as a negative regulator of profibrotic tissue responses, which acts in a site-specific manner within the innate (skin) and adaptive (lung) immune systems.

Our results also indicate that IL-13 promotes bleomycin-induced dermal fibrosis through innate immunity. In addition to activated Th2 cells, innate immune cells, including mast cells and dendritic cells, produce IL-13, particularly after TLR2 stimulation (26, 27). A recent study showed that bleomycin, a product derived from the bacterium streptomyces, activates TLR2 (28). These data and ours suggest that bleomycin promotes tissue fibrosis via activation of TLR2 on innate immune cells resulting in the production of IL-13. IL-13 may, in turn, promote collagen production by dermal fibroblasts either directly or via the activation of TGF-β pathways (1).

Scleroderma and other primary fibrosing disorders are recalcitrant illnesses with no effective therapeutic options. Furthermore, many common disorders like vascular heart disease, graft rejection, and liver disease, although initially treatable, have a prominent, and often intractable, terminal fibrotic stage. Our data surprisingly indicate that the innate immune system is capable of executing a sclerotic tissue response and support the established profibrotic properties of IL-13. Moreover, the data presented here implicate T-bet as a potent suppressor of dermal sclerosis and negative regulator of IL-13-mediated fibrosis. We suggest that elucidation of the T-bet target genes that negatively regulate tissue fibrosis within the innate immune system may provide novel therapeutic targets for fibrosing disorders.

Materials and Methods

Mice.

The generation of T-bet−/− mice has been described. T-bet−/− mice were backcrossed eight generations onto the BALB/c (H-2d) background. IL-13−/− mice (29) were obtained from Joan Stein-Streilein (Brigham and Women's Hospital, Boston, MA). RAG2−/− mice (BALB/c background) were obtained from The Jackson Laboratory (Bar Harbor, ME). T-bet−/−RAG2−/− and IL-13−/−RAG2−/− mice were generated by crossing T-bet−/−, IL-13−/−, and RAG2−/− mice on a BALB/c background. All mice were housed in a pathogen-free facility at the Harvard School of Public Health, and all animal studies were performed according to institutional and National Institutes of Health guidelines for animal use and care. The drinking water of RAG2−/− mice was supplemented with sulfatrim to prevent bacterial infection.

Skin Sclerosis Induction.

Groups of 7- to 8-week-old mice were injected with 100 μg of bleomycin sulfate (Sigma, St. Louis, MO) dissolved in 100 μl of sterilized PBS s.c. into the shaved back daily for 4 weeks (11). Control groups were injected with PBS in the same fashion. Skin samples were obtained from the injection sites by punch biopsy for further examination.

Histopathology and Immunohistochemistry Studies on the Mouse Skin.

On the day after the final injection, the skin at the injection site was removed by using a dermal biopsy punch (Miltex, York, PA), fixed in 10% formalin solution, and embedded in paraffin. The general histological appearance of tissue was examined by routine H&E and Masson trichrome stain (11).

Measurement of Hydroxyproline.

Full-thickness punch-biopsy specimens of 5 mm diameter were obtained from the bleomycin injection site and stored at –80°C. Collagen deposition was determined by measuring the content of hydroxyproline in the skin (30). Briefly, the skin pieces were hydrolyzed with 2 M sodium hydroxide at 120°C for 20 min. The hydrolyzate was then oxidated with chloramine-T (Sigma) for 25 min at room temperature. Ehrlich's aldehyde reagent (Sigma) was added to each sample, and the chromophore was developed by incubating the samples at 65°C for 20 min. Absorbance of each sample was read at 550 nm by using a spectrophometer. Results were expressed as hydroxyproline score, which represents value of OD550 per 5-mm skin piece dissolved in a total of 1 ml of mixture of the solutions described above. For all hydroxyproline assays, a standard curve was performed by using a known quantity of hydroxyproline. The reported hydroxyproline scores fell on the linear portion of this standard curve.

Statistical Analysis.

The unpaired Student t test was used for all comparisons of continuous variables, with a value of P < 0.05 considered statistically significant.

Acknowledgments

We thank Dorothy Zhang for help with preparation and analysis of histological sections; Lisa de Elizalde and Ryan Ghan for expert manuscript preparation; and Aimee Angle-Zahn, Landy Kangaloo, Betty Tang, and Jacobo Ramirez for technical assistance and animal care. This work was supported by a grant from the Scleroderma Research Foundation (to L.H.G.); a Scleroderma Research Foundation Fellowship grant (to J.W.); and National Institutes of Health Grants AR46983 (to R. Lafyatis), AI56296 (to L.H.G.), and K08 AI57434 (to J.W.). A.O.A. is the recipient of the Abbott Scholar Award in Rheumatology Research.

Abbreviations

SSc

systemic sclerosis

T-bet

T-box expressed in T cells

Th

T helper

TLR

Toll-like receptor.

Footnotes

Conflict of interest statement: L.H.G. has equity in and is on the corporate board of Bristol–Myers Squibb Company.

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