Dr. O. Distler has received consulting fees from Pfizer (more than $10,000) and from Actelion, Encysive, FibroGen, Ergonex, NicOX, Bristol-Myers Squibb, Sanofi-Aventis, United BioSource, Medac, and Biovitrium (less than $10,000 each) with regard to potential scleroderma treatments; he has received lecture honoraria from Actelion, Pfizer, Encysive, and Ergonex (less than $10,000 each).
University of Erlangen–Nuremberg, Erlangen, Germany
The transcription factor STAT-4 has recently been identified as a genetic susceptibility factor in systemic sclerosis (SSc) and other autoimmune diseases. The aim of this study was to investigate the contribution of STAT-4 in the development of a fibrotic phenotype in 2 different mouse models of experimental dermal fibrosis.
STAT-4–deficient (stat4−/−) mice and their wild-type littermates (stat4+/+) were injected with bleomycin or NaCl. Infiltrating leukocytes, T cells, B cells, and monocytes were quantified in the lesional skin of stat4−/− and stat4+/+ mice. Inflammatory and profibrotic cytokines were measured in sera and lesional skin samples from stat4−/− and stat4+/+ mice. The outcome of mice lacking STAT-4 was also investigated in the tight skin 1 (TSK-1) mouse model.
Stat4−/− mice were protected against bleomycin-induced dermal fibrosis, with a reduction in dermal thickening (mean ± SEM 65 ± 3% decrease; P = 0.03), hydroxyproline content (68 ± 5% decrease; P = 0.02), and myofibroblast counts (71 ± 6% decrease; P = 0.005). Moreover, the number of infiltrating leukocytes, especially T cells, was significantly decreased in the lesional skin of stat4−/− mice (mean ± SEM 63 ± 5% reduction in T cell count; P = 0.02). Stat4−/− mice also displayed decreased levels of inflammatory cytokines such as tumor necrosis factor α, interleukin-6 (IL-6), IL-2, and interferon-γ in lesional skin. Consistent with a primary role of STAT-4 in inflammation, STAT-4 deficiency did not ameliorate fibrosis in TSK-1 mice.
The results of this study demonstrate that the transcription factor STAT-4 exerts potent profibrotic effects by controlling T cell activation and proliferation and cytokine release. These findings confirm the results of genetics studies on the role of STAT-4 in the development of SSc.
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that affects the skin and a variety of internal organs such as the lungs, the heart, and the gastrointestinal tract. The early stages of SSc are characterized by vascular changes and the presence of inflammatory infiltrates in involved organs (1). The inflammatory infiltrates are dominated by monocytes and activated T cells. The later stages of SSc are characterized by an excessive accumulation of extracellular matrix components. SSc is caused by an interaction between susceptibility genes and environmental triggers; epidemiologic data, including family and twin studies, revealed a complex genetic component in the pathogenesis of SSc (2). Large case–control association studies and a recent unbiased genome-wide association study (GWAS) have unraveled some relevant pathogenic pathways related to immunologic processes, especially innate immunity and the type I interferon (IFN) system (3–5).
The STAT-4 transcription factor plays a key role in type I IFN receptor signaling by being activated and translocated to the nucleus after type I IFN receptor ligation (6). This transcription factor is also a central mediator in generating inflammation during protective immune responses and immune-mediated diseases. In addition, STAT-4 transmits signals from the receptors of interleukin-2 (IL-2), IL-12, and IL-23 and can therefore contribute to autoimmune responses by affecting the functions of several innate and adaptive immune cells (7). Recently, various STAT-4 variants have convincingly been established as genetic susceptibility risk factors in SSc (8–11). Various association studies have also identified STAT-4 as a susceptibility factor in different connective tissue disorders and autoimmune diseases (12, 13).
Thus, considering discrepancies between the fibrotic propensity of SSc and the specific phenotype of other connective tissue diseases characterized by the presence of a genetic variant of STAT-4, it is now crucial to determine whether identified STAT-4 contributes to the expression of the fibrotic phenotype in SSc. The aim of this study was to investigate the role of STAT-4 in fibrosis. To this end, we evaluated the outcome of mice lacking STAT-4 in different models of experimental dermal fibrosis mimicking the different stages of SSc and characterized the role of STAT-4 in fibrosis.
MATERIALS AND METHODS
Bleomycin-induced dermal fibrosis in STAT-4–deficient mice.
STAT-4–deficient (stat4−/−) mice (14) on a BALB/c background were purchased from The Jackson Laboratory. Wild-type (WT) BALB/c littermates expressing STAT-4 (stat4+/+ mice) were used as controls. Skin fibrosis was induced in 6-week-old male and female mice by administering local injections of bleomycin for 3 weeks, as previously described (15). Briefly, 100 μl of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5 mg/ml was administered every other day by subcutaneous injection into defined areas of 1 cm2 on the upper back. Subcutaneous injections of 100 μl 0.9% NaCl were used as controls. Four different groups of stat4−/− mice and stat4+/+ mice (34 mice in total) were analyzed. One group of stat4−/− mice and one group of stat4+/+ mice were challenged with bleomycin, whereas the remaining 2 groups were injected with NaCl. The sex ratio was similar between groups. After 3 weeks, the mice were killed by cervical dislocation in order to analyze the dermal thickness, the hydroxyproline content, and number of myofibroblasts in lesional skin.
Inactivation of STAT-4 in tight skin (TSK) mice.
To investigate the role of STAT-4 in a model of noninflammatory SSc, STAT-4–deficient mice were crossed with TSK-1 mice to generate TSK-1 mice deficient for STAT-4 (stat4−/−/tsk1). The TSK-1 phenotype is caused by a dominant mutation in the fibrillin 1 gene (16). TSK mice are characterized by the accumulation of collagen fibers in the hypodermis, resulting in progressive hypodermal thickening. In contrast to mice with bleomycin-induced fibrosis, inflammatory infiltrates are absent in TSK-1 mice, and the aberrant activation of fibroblasts is not caused by the release of inflammation mediators from leukocytes. Similar to fibroblasts from patients with SSc, fibroblasts from TSK-1 mice are endogenously activated, with an increased release of collagen that persists for several passages in vitro. Thus, TSK-1 mice are a model for later, less inflammation-dependent stages of SSc, whereas bleomycin-induced fibrosis represents early, inflammation-dependent stages of SSc.
Genotyping of TSK-1 mice and STAT-4–deficient mice was performed by polymerase chain reaction with the following primers: for mutated fibrillin 1/TSK-1, forward 5′-GTTGGCAACTATACCTGCAT-3′, reverse 5′-CCTTTCCTGGTAACATAGGA-3′; for mutated STAT-4, forward 5′-CCTCGTCCTGCAGTTCATTC-3′, reverse 5′-TCTTTC- CAGGAGGTGTGCTC-3′. Four groups of mice were analyzed (24 mice in total). One group of TSK-1 mice expressed STAT-4 (stat4+/+), and another group of TSK-1 mice were deficient for STAT-4 (stat4−/−). The other 2 groups consisted of pa/pa (control) mice. One group of pa/pa mice expressed STAT-4 (stat4+/+), and the other group of pa/pa mice were deficient for STAT-4 (stat4−/−). Mice were killed by cervical dislocation at age 10 weeks, in order to analyze the hypodermal thickness, the hydroxyproline content, and the number of myofibroblasts in lesional skin. The local ethics committee approved all animal experiments.
Evaluation of dermal and hypodermal thickness.
Lesional skin specimens were excised, fixed in 4% formalin, and embedded in paraffin. Five-micrometer–thick sections were stained with hematoxylin and eosin. The dermal thickness was analyzed at 100-fold magnification by measuring the distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction at 4 sites in the lesional skin of each mouse (17). The hypodermal thickness in TSK-1 mice was determined by measuring the thickness of the subcutaneous connective tissue beneath the panniculus carnosus at 4 different sites on the upper back of each mouse. Two independent examiners performed the evaluation (JA and JHWD).
Assessment of the number of infiltrating leukocytes in bleomycin-treated mice.
Infiltrating leukocytes in the lesional skin of stat4−/− mice and stat4+/+ mice were quantified in hematoxylin and eosin–stained sections. Eight different high-power fields from different tissue sites for each mouse were evaluated for mononuclear/inflammatory cells at 400-fold magnification by 2 examiners blinded to the treatment (JA and JHWD). All images were captured with a Nikon Eclipse 80i microscope equipped with a Sony DSP 3CCD camera (18).
The collagen content in lesional skin samples was evaluated by hydroxyproline assay (19). After digestion of punch biopsy specimens (3 mm diameter) in 6M HCl for 3 hours at 120°C, the pH of the samples was adjusted to 7 with 6M NaOH. Afterward, samples were mixed with 0.06M chloramine T and incubated for 20 minutes at room temperature. Next, 3.15M perchloric acid and 20% p-dimethylaminobenzaldehyde were added, and samples were incubated for an additional 20 minutes at 60°C. The absorbance was determined at 557 nm with a SpectraMax 190 microplate spectrophotometer (Molecular Devices). For direct visualization of collagen fibers, trichrome staining was performed using the Trichrome (Masson) Stain Kit (Sigma-Aldrich).
Immunohistochemistry for α-smooth muscle actin (α-SMA), CD3, CD4, CD8, CD22, and CD68.
The expression of α-SMA, T cells, and macrophages was quantified in paraffin-embedded sections of lesional skin from stat4−/− mice and stat4+/+ mice. Myofibroblasts were identified by staining for α-SMA, as previously described (20, 21). After deparaffinization and blocking with 5% horse serum and 3% H2O2, skin sections were incubated with anti–α-SMA antibodies (clone 1A4; Sigma-Aldrich). Polyclonal rabbit anti-mouse antibodies labeled with horseradish peroxidase (Dako) were used as secondary antibodies.
To quantify the numbers of infiltrating T cells, B cells, and monocytes, skin sections were stained for CD3, CD22, and CD68, respectively. CD4 and CD8 T cell subsets were also quantified after staining for CD4 and CD8. After deparaffinization, antigen retrieval with Tris–EDTA–Tween and blocking with 10% goat serum and 0.3% H2O2, sections were incubated with polyclonal rabbit anti-human antibodies against CD3 or CD22 (Abcam), monoclonal rabbit anti-human antibodies against CD8 (Novus Biologicals), or monoclonal mouse anti-human antibodies against CD4 or CD68 (Abcam and Novus Biologicals, respectively). Polyclonal horseradish peroxidase–labeled goat anti-rabbit or rabbit anti-mouse immunoglobulins (Dako) were used as secondary antibodies.
Irrelevant isotype–matched antibodies were used as controls. Staining was visualized with diaminobenzidine–peroxidase substrate solution (Sigma-Aldrich). Sections stained with α-SMA were counterstained with hematoxylin. The number of myofibroblasts was determined at 200-fold magnification in 4 different sections for each mouse. T cells, CD4 and CD8 T cell subsets, B cells, and monocytes were counted in 8 different sections of lesional skin for each mouse, at 400-fold magnification. Counting was performed in a blinded manner by 2 experienced examiners (JA and JHWD).
Assessment of infiltrating T cell proliferation.
To determine the proliferation of infiltrative T cells, double staining for CD3 and the proliferation marker Ki-67 was performed. Skin sections were first stained by immunohistochemistry for CD3, as described above, and were then incubated with polyclonal rabbit anti-human antibodies against Ki-67 (Abcam). Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulins (Santa Cruz Biotechnology) were used as secondary antibodies. The slides were then viewed by microscopy using appropriate fluorescence filters. The number of double-labeled Ki-67–positive and CD3-positive cells, in relation to the total number of CD3-positive cells, was evaluated in a blinded manner.
Inflammatory and profibrotic cytokine measurement in serum or lesional skin samples from bleomycin-treated mice.
Cytokine levels were measured in the serum and skin of 24 stat4−/− and stat4+/+ mice that received bleomycin or NaCl injections (6 mice per group). Mouse skin tissue lysate was prepared by homogenization in modified radioimmunoprecipitation assay buffer (50 mM Tris/HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, and complete EDTA-free protease inhibitor cocktail [Roche]) with a Precellys 24 homogenizer/grinder (Peqlab). Tissue and cell debris was removed by centrifugation. The protein concentration was determined using the amido black method (22). Serum and skin lysates were assayed by multiplex bead array technology (Bender MedSystems) for the following cytokines: tumor necrosis factor α (TNFα), IL-6, IFNγ, IL-2, IL-4, IL-5, IL-10, and transforming growth factor β (TGFβ).
Data were expressed as the mean ± SEM. Student's t-test was used for statistical analyses. P values less than 0.05 were considered significant.
STAT-4 deficiency and protection against bleomycin-induced dermal fibrosis.
To evaluate the role of STAT-4 in fibrosis, stat4−/− mice and their stat4+/+ littermates were challenged with bleomycin. Skin architecture and dermal thickness did not differ between stat4−/− mice and stat4+/+ mice injected with NaCl, suggesting that under physiologic conditions, the skin phenotype in stat4−/− mice with nonfibrotic conditions is not altered (Figures 1A and B). Following injection of bleomycin, dermal thickness increased in both stat4−/− and stat4+/+ mice (Figures 1A and B). However, stat4−/− mice were protected against bleomycin-induced fibrosis. In stat4−/− mice, the mean ± SEM increase in dermal thickness was 17 ± 3%, compared with 55 ± 5% in stat4+/+ mice (P = 0.04). Following bleomycin challenge, dermal thickness was reduced by 65 ± 3% in stat4−/− mice compared to stat4+/+ mice (P = 0.03) (Figures 1A and B).
Consistent with decreased dermal thickening, reduced accumulation of collagen following bleomycin challenge was observed on trichrome-stained skin sections from stat4−/− mice (Figure 1C). In addition, the hydroxyproline content in the lesional skin of stat4−/− mice was significantly lower than that in the skin of stat4+/+ mice (mean ± SEM decrease 68 ± 5%; P = 0.02) (Figure 1D). Bleomycin challenge also significantly reduced the number of myofibroblasts in the lesional skin of stat4−/− mice compared to stat4+/+ mice (mean ± SEM decrease 71 ± 6%; P = 0.005) (Figures 1E and F).
STAT-4–regulated T cell proliferation into lesional skin.
Inflammatory infiltrates are characteristic features of the early stages of SSc that are mimicked in the mouse model of bleomycin-induced fibrosis. Infiltrating leukocytes contain mostly T cells, with a perivascular distribution, and stimulate fibroblast activation and collagen synthesis via the release profibrotic factors (1, 23). To analyze whether STAT-4 influences the outcome of bleomycin-induced fibrosis by regulating leukocyte infiltration or proliferation, we next quantified the number of leukocytes in lesional skin. Following bleomycin treatment, inflammation was significantly reduced in stat4−/− mice compared to stat4+/+ mice (mean ± SEM reduction 62 ± 4%; P = 0.02) (Figures 2A and B).
To investigate which leukocyte populations are affected, we first quantified the number of T cells in fibrotic skin. Following bleomycin treatment, T cell counts were significantly lower in stat4−/− mice compared to stat4+/+ mice (mean ± SEM decrease 63 ± 5%; P = 0.02) (Figure 3A). Both CD4 and CD8 T cell subsets were significantly reduced in the skin of bleomycin-treated stat4−/− mice compared with stat4+/+ mice (Figures 3B and C). The number of CD4- and CD8-positive T cells was reduced in stat4−/− mice, by 51 ± 12% (P = 0.01) and 40 ± 8% (P = 0.02), respectively. In contrast to T cells, the number of B cells and monocytes did not significantly differ between stat4−/− and stat4+/+ mice following bleomycin challenge (Figures 3D and E).
STAT-4 has been suggested to regulate the proliferation of T cells (24, 25). To determine whether the decrease in T cell infiltration in the lesional skin of stat4−/− mice might be caused by reduced T cell proliferation, T cells were stained for the proliferation marker Ki-67. Following bleomycin challenge, the number of Ki-67–positive T cells was significantly reduced in stat4−/− mice. Staining for Ki-67 was detected in a mean ± SEM of 85 ± 3% of T cells in skin sections from stat4+/+ mice and in 47 ± 8% of T cells in skin sections from stat4−/− mice (P = 0.03) (Figure 3F).
Reduced levels of proinflammatory cytokines in the serum and skin lysates of bleomycin-treated STAT-4–deficient mice.
To further characterize how STAT-4 regulates leukocyte infiltration and secondary fibroblast activation in lesional skin, the levels of different cytokines were determined in serum (Figure 4A) and lesional skin (Figure 4B). Compared with stat4+/+ mice treated with bleomycin, stat4−/− mice treated with bleomycin had reduced levels of several cytokines that have been implicated in inflammation and fibrosis, such as IFNγ (mean ± SEM reduction 58 ± 3%; P = 0.02), TNFα (71 ± 5% reduction; P = 0.01), IL-6 (50 ± 4% reduction; P = 0.02), and IL-2 (63 ± 4% reduction; P = 0.03), in lesional skin. No differences in the levels of IL-4, IL-5, IL-10, and TGFβ were observed between stat4−/− and stat4+/+ mice treated with bleomycin. Similar results were observed in serum. These data suggest that STAT-4 is associated with a proinflammatory profile, promoting tissue infiltration and proliferation of T cells and the release of selective proinflammatory and profibrotic cytokines, which may contribute to collagen synthesis and finally to the development of fibrosis.
Role of STAT-4 deficiency in fibrosis in the TSK-1 mouse model.
Based on the results in the model of bleomycin-induced dermal fibrosis, we concluded that STAT-4 regulates fibroblast activation and fibrosis indirectly by controlling T cell activation and cytokine release (26). To confirm this hypothesis, we analyzed the effects of STAT-4 in the TSK-1 mouse, a model of fibrosis that is less dependent on inflammation compared with bleomycin-induced fibrosis. This model is characterized by the absence of inflammatory infiltrates and by endogenous activation of fibroblasts (16, 27). Consistent with our hypothesis, STAT-4 deficiency did not significantly ameliorate fibrosis in the TSK-1 mouse model. No differences in hypodermal thickness were observed between stat4−/−/tsk1 mice and their stat4+/+/tsk1 littermates (Figures 5A–D). The hydroxyproline content and myofibroblast counts did not differ between stat4−/−/tsk1 mice and stat4+/+/tsk1 mice. Consistent with these observations, the release of collagen was not altered in fibroblasts isolated from stat4−/− mice (data not shown).
Over the past several years, hypothesis-driven candidate gene and GWAS approaches have enabled the identification of several genetic susceptibility genes for SSc that are linked to the immune system (3–5, 8). Among these, STAT4 has been identified in several independent studies (8–11). However, STAT4 variants also contribute to other autoimmune diseases, and it is therefore critical to determine the specific role of STAT4 in the fibrotic component of connective tissue disease (20, 28–31). In the current study, we present the first translational data and evidence that STAT-4 might regulate fibroblast activation and collagen release indirectly by orchestrating leukocyte infiltration and regulating proinflammatory cytokine production in the inflammatory stages of SSc.
The early stages of SSc are characterized by the infiltration of inflammatory cells into involved skin (1, 2). The infiltrating leukocytes release inflammatory and profibrotic cytokines that stimulate collagen synthesis in resident fibroblasts (2, 32, 33). T cells represent a major component of this infiltrate (32, 33). Moreover, activated T cells have been suggested to play a key role in the induction of the hyperactive and altered functional phenotype of SSc fibroblasts (32). As in human SSc, T cells are present in increased numbers and in an activated state in the lesional skin of bleomycin-challenged mice (33). Our results demonstrate that knockdown of STAT-4 significantly reduced the number and the proliferation of infiltrating T cells in bleomycin-treated mice. In contrast, the release of collagen was not altered in fibroblasts from STAT-4–deficient mice, and deficiency of STAT-4 did not significantly ameliorate fibrosis in TSK-1 mice, which represent a less inflammation-dependent model of fibrosis. The role of immune cells, and especially T cells, in the development of fibrosis in TSK-1 mice remains controversial.
Although a role for CD4+ T cells and B cells in the activation of collagen synthesis has been suggested (34, 35), bone marrow transplantation experiments have challenged the contribution of immune cells. The transfer of enriched B cells or T cells increased autoantibody production but did not cause skin fibrosis, and transfer of T and B lymphocytes led to only mild fibrotic lesions compared to the massive fibrosis observed in TSK-1 mice (36). In line with this finding, the Fbn1 mutation also induced fibrosis in RAG-2 mice that lack mature B cells and T cells, and TSK-1 mice homozygous for a mutation at the Prkdcscid locus, thus lacking mature T and B lymphocytes, developed a fibrotic phenotype in the absence of a functional immune system (37, 38). The role of T cells might also depend on the mouse strains used, and differences in the mouse strains used might influence the outcome in the TSK-1 mouse model. For example, strain-specific differences between BALB/c and C57BL/6 mice in T cell response, cytokine production, and hypodermal thickness have been previously reported (35).
These findings tend to indicate that STAT-4 exerts profibrotic effects indirectly in the early inflammatory stages of SSc by orchestrating the activation and proliferation of T cells into lesional skin rather than by direct effects on the collagen synthesis of fibroblasts. STAT-4 is also a critical factor involved in the regulation of cytokine balance. Inactivation of STAT-4 leads to decreased secretion of selective cytokines in the lesional skin of bleomycin-treated mice. STAT-4 interacts directly with DNA sequences in the IFNγ promoter to increase gene transcription. As expected, we observed decreased levels of IFNγ in the serum and lesional skin of stat4−/− mice treated with bleomycin. However, the role of IFNγ in the promotion of fibrosis remains unclear, because this cytokine displays both profibrotic and antifibrotic properties (39–41).
In this study, we demonstrated that STAT-4 promotes the release of IL-6 and TNFα from infiltrating leukocytes. The levels of these cytokines in STAT- 4–deficient mice treated with bleomycin were lower than those in WT mice. These cytokines have been implicated in the activation of fibroblasts, collagen synthesis, and subsequent fibrosis. IL-6 is overexpressed by endothelium and fibroblasts in the involved skin of patients with SSc (42). Dermal fibroblasts from patients with SSc are reported to constitutively produce up to 30-fold higher levels of IL-6 than those from healthy control subjects (43). IL-6 promotes fibrosis by enhancing inflammation and is a potent inducer of excessive collagen production and proliferation of SSc fibroblasts, notably by exerting autocrine regulation of fibroblasts (40). Moreover, blocking of the IL-6 response using anti–IL-6 antibodies results in a significant reduction of type I procollagen in cultured SSc fibroblasts (44). Based on these results, a clinical trial to evaluate the efficacy of tocilizumab in SSc is currently in preparation.
Expression of TNFα is observed during the early stages of SSc (45). Preclinical studies indicate that inhibition of TNFα might exert antifibrotic effects in the early inflammatory stages of SSc. Inhibition of TNFα in bleomycin-induced dermal fibrosis resulted in a significant reduction in dermal thickness, collagen accumulation, and the number of infiltrating myofibroblasts (46). Similar results were also observed for pulmonary fibrosis (47, 48). Our results in the mouse model of bleomycin-induced dermal fibrosis might be directly transferable to patients with SSc. Although the molecular basis for a functional role of the different STAT-4 variants has not yet been identified, an inflammatory expression profile with increased plasma levels of IL-6 and TNFα was observed in patients carrying the identified STAT-4 polymorphism (10).
In summary, we have demonstrated that STAT-4 regulates the activation of fibroblasts by promoting the infiltration of leukocytes into lesional skin and by stimulating the release of cytokines involved in both inflammatory and fibrotic processes. Inactivation of STAT-4 decreased leukocyte activation and significantly ameliorated inflammation-driven fibrosis. These findings confirm the results of genetics studies on the role of STAT-4 in the development of the fibrotic phenotype of SSc.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Avouac had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Avouac, O. Distler, Schett, Allanore, J. H. W. Distler.