Potential roles of interleukin-17A in the development of skin fibrosis in mice

Authors


Abstract

Objective

Although transforming growth factor β (TGFβ) and connective tissue growth factor (CTGF) have been considered to play central roles in the pathogenesis of systemic sclerosis (SSc), other cytokines may also be crucial for the development of SSc. The aim of this study was to examine the roles of T helper cytokines in the development of skin fibrosis.

Methods

To compare the roles of Th1, Th2, and Th17 cytokines, we examined bleomycin-induced SSc in mice deficient for interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-17A. The mechanism by which IL-17A contributes to bleomycin-induced fibrosis was investigated in vivo and in vitro. The outcome of mice lacking IL-17A was also investigated in TSK-1 mice.

Results

The loss of IL-17A significantly attenuated bleomycin-induced skin fibrosis, whereas a deficiency of IFNγ or IL-4 did not. Leukocyte infiltration and the expression of TGFβ and CTGF messenger RNA in bleomycin-injected skin were significantly reduced in IL-17A–deficient mice compared with wild-type (WT) mice. Daily bleomycin injections induced the expression of IL-17A in the skin and potent IL-17A producers in splenic CD4+ T cells from WT mice. Furthermore, a skin fibroblast cell line expressed increased TGFβ, CTGF, and collagen after the addition of recombinant IL-17A. IL-17A deficiency also attenuated skin thickness in TSK-1 mice.

Conclusion

This study demonstrates that IL-17A contributes to skin fibrosis in 2 mouse models of SSc. These findings suggest that inhibition of IL-17A represents a therapeutic target for antagonizing fibrotic skin disorders such as SSc.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by excessive extracellular matrix deposition in the skin, lungs, and other internal organs (1, 2). A growing body of evidence suggests that overproduction of extracellular matrix components by activated fibroblasts results from complex interactions between various cells, including leukocytes and fibroblasts, and via several soluble mediators, such as cytokines, chemokines, and growth factors (1, 2).

An emerging hypothesis for the pathogenesis of fibrotic disorders suggests that an imbalance between Th1 cytokines and Th2 cytokines leads to abnormal responses to tissue injury. Th2 cytokines such as interleukin-4 (IL-4), IL-6, and IL-13 stimulate the synthesis of collagen by human fibroblasts (3). In contrast, Th1 cytokines such as interferon-γ (IFNγ) and tumor necrosis factor α suppress collagen production by fibroblasts in vitro (3). Therefore, in general, a relative shift toward Th2 cytokine production rather than Th1 cytokine production can induce tissue fibrosis.

In patients with SSc, circulating T cells and T cells infiltrating the skin or lungs demonstrate a predominantly Th2 profile (4–6). Furthermore, a recent study suggested that the frequency of circulating Th17 cells is strikingly increased in patients with SSc (7). Th17 cells, which were discovered in 2007, are the third T helper cell subset that can produce IL-17A (8, 9). Th17 cells also secrete IL-17F, IL-21, and IL-22. The differentiation factors transforming growth factor β (TGFβ) plus IL-6 or IL-21, the growth and stabilization factor IL-23, and the transcription factors RORγt, RORα, and STAT-3 have been considered to be involved in the development of Th17 cells. This T helper cell subset may contribute not only to inflammation but also to fibrosis via production of IL-17A and other cytokines (10). In fact, elevated serum IL-17A levels and augmented IL-17A expression in peripheral blood lymphocytes and lesional skin have been reported in patients with SSc (7, 11, 12), although reduced plasma IL-17A levels were detected in a recent study (13).

Skin fibrosis induced by daily intradermal bleomycin injections is widely used as a representative animal model of SSc (14). Although a variety of factors have been reported to contribute to the fibrotic process, the exact mechanism remains unclear. In a previous study, administration of recombinant IFNγ, a representative Th1 cytokine, attenuated bleomycin-induced skin fibrosis (15). IFNγ-deficient mice have modest bleomycin-induced lung fibrosis (16). IL-4 signaling has been reported as critical for the development of increased skin thickness in the TSK-1 (TSK/+) mouse model, a genetic model of SSc (17, 18). In contrast, IL-4 is not required for the development of bleomycin-induced lung fibrosis (19). However, both bleomycin- and IL-1β–induced lung fibrosis are dependent on IL-17A (10). The specific roles of IFNγ, IL-4, and IL-17A in bleomycin-induced skin fibrosis have not been investigated using mice deficient for each of these cytokines.

In the current study, we demonstrated that IL-17A, but not IFNγ or IL-4, has pivotal roles in the development of skin fibrosis in a murine model of bleomycin-induced skin fibrosis. Furthermore, skin thickness was attenuated by IL-17A deficiency in TSK/+ mice, another model of skin fibrosis (20).

MATERIALS AND METHODS

Mice.

IFNγ−/−, IL-4−/−, and IL-17A−/− mice (C57BL/6 genetic background) were generated as previously reported (21–23). These mice were all backcrossed at least 8 generations to C57BL/6 mice. TSK/+ mice (C57BL/6 background) and C57BL/6 mice were purchased from The Jackson Laboratory. IL-17A−/− TSK/+ mice were generated by crossing IL-17A+/− TSK/+ parents. To verify the TSK/+ genotype, polymerase chain reaction (PCR) amplification of a partially duplicated fibrillin 1 gene was carried out using genomic DNA from each mouse, as previously described (18). All mice were screened regularly for pathogens. Female mice ages 10 to 12 weeks were used for the experiments. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science approved all studies and procedures.

Intradermal bleomycin treatment.

Bleomycin was dissolved in sterile saline at a concentration of 1 mg/ml. The mice received daily intradermal injections of either bleomycin or saline (300 μl, administered using a 27-gauge needle) into their shaved backs (the para-midline, lower back region) for 4 weeks, as described previously (14).

Histologic examination of skin fibrosis.

All skin sections were obtained from the bleomycin-injected region of the lower back, as full-thickness sections extending down to the body wall musculature. The skin samples were fixed in formalin, dehydrated, embedded in paraffin, and used for immunostaining. Sections (6-μm thick) were stained with hematoxylin and eosin, Masson's trichrome, or van Gieson's reagents to identify collagen deposition in the skin. Expression of α-smooth muscle actin (α-SMA) was analyzed in paraffin-embedded specimens. After deparaffinization, samples were incubated with 3% H2O2. Positivity for α-SMA in mouse skin sections was detected by incubation with anti–α-SMA monoclonal antibodies conjugated to alkaline phosphatase (Sigma-Aldrich). Expression of the α-SMA was visualized with a New Fuchsin Substrate System (Dako). Skin thickness and the number of myofibroblasts (α-SMA–positive fibroblasts) were evaluated independently by 2 investigators (YO and MH), in a blinded manner. Fibroblasts were identified by their spindle-shaped morphology. The FreeHand tool of Adobe Photoshop Elements 3.0 was used to quantify specific van Gieson's staining in the skin sections.

Measurement of collagen content in tissue samples.

Tissue samples were embedded in paraffin, and ∼15-μm–thick sections were obtained. Sections were deparaffinized after incubation with xylol, xylol:ethanol (1:1), ethanol, water:ethanol (1:1), and water. Individual samples were placed in small test tubes and covered with 0.2 ml of a saturated solution of picric acid in distilled water that contained 0.1% fast green FCF and 0.1% sirius red F3BA. The samples were rinsed several times with distilled water until the fluid was colorless. One milliliter of 0.1N NaOH in absolute methanol (1:1 volume:volume) was added, and the eluted color was read on a spectrophotometer at 540 nm and 605 nm. The method used is based on the selective binding of sirius red F3BA and fast green FCF to collagens and noncollagenous proteins, respectively (24).

Immunohistochemical staining.

Tissues were harvested prior to and on days 7, 14, 21, and 28 of bleomycin treatment; the numbers of infiltrating T cells, macrophages, and neutrophils were assessed by immunostaining. Deparaffinized sections were incubated with 3% H2O2. Sections were then incubated with rat monoclonal antibodies specific for CD3 (Serotec) or F4/80 (Abcam), or rabbit monoclonal antibodies specific for myeloperoxidase (MPO; NeoMarkers). Rat IgG (SouthernBiotech) was used as a control for nonspecific staining. Sections were then incubated sequentially (30 minutes at room temperature) with a biotinylated rabbit anti-rat IgG secondary antibody (BD Biosciences) or a biotinylated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology), followed by horseradish peroxidase–conjugated avidin–biotin complexes (Vectastain). Sections were washed 3 times with phosphate buffered saline (PBS) between incubations, developed with 3,3′-diaminobenzidine tetrahydrochloride dihydrate and hydrogen peroxide, and counterstained with methyl green. Each section was examined independently by 2 investigators (YO and MH), in a blinded manner, and the mean value was used for analysis.

Reverse transcriptase–PCR (RT-PCR).

Skin was harvested 7 days after intradermal bleomycin treatment. Total RNA was isolated from frozen skin specimens using RNeasy spin columns (Qiagen) and was digested with DNase I (Qiagen) to remove chromosomal DNA, in accordance with the manufacturer's protocols. Total RNA was reverse transcribed to complementary DNA using a reverse transcription system with random hexamers (Promega). Cytokine messenger RNA (mRNA) was analyzed using real-time PCR quantification, according to the manufacturer's instructions (Applied Biosystems). Sequence-specific primers and probes were designed using predeveloped TaqMan assay reagents (Applied Biosystems). Real-time PCR (40 cycles of denaturation at 92°C for 15 seconds and annealing at 60°C for 60 seconds) was performed on an ABI Prism 7000 sequence detector (Applied Biosystems). GAPDH was used to normalize mRNA. Relative expression of real-time PCR products was determined by using the ΔΔCt method (25) to compare target gene and GAPDH mRNA expression. One of the control samples was chosen as a calibrator sample.

Cytokine concentrations in tissue.

Samples of regional skin were homogenized in 600 μl of lysis buffer (10 mmoles/liter PBS, 0.1% sodium dodecyl sulfate, 1% Nonidet P40, 5 mmoles/liter EDTA containing complete protease inhibitor mixture [Roche Diagnostics]) to extract proteins. Homogenates were centrifuged at 15,000 revolutions per minute for 15 minutes at 4°C to remove debris (26). The total protein concentration in supernatants was measured with a BCA Protein Assay Kit (ThermoFisher Scientific) and was equalized in each sample. The concentration of IL-17A in supernatants was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems), according to the manufacturer's instructions.

Th17 cell induction by bleomycin treatment.

Splenocytes were harvested from wild-type (WT) mice, and CD4+ T cells were purified by negative selection using MACS technology (Miltenyi Biotec). For in vitro Th17 cell differentiation, CD4+ T cells were cultured with 20 ng/ml of recombinant IL-6 (BioLegend), 5 ng/ml of recombinant human TGFβ1 (BioLegend), 10 μg/ml of anti–IL-4 antibody (BioLegend), and 10 μg/ml of anti-IFNγ antibody (BioLegend), in combination with 3 μg/ml of anti-CD3 antibody (eBioscience) and 5 μg/ml of anti-CD28 antibody (BioLegend) for 96 hours at 37°C. To evaluate the effect of bleomycin in this process, bleomycin was added to the culture medium simultaneously.

Splenocytes were also harvested from WT mice that received intradermal injections of bleomycin for 7 days. Purified CD4+ T cells were cultured in 96-well flat-bottomed plates precoated with anti-CD3 antibody (3 μg/ml) plus anti-CD28 (5 μg/ml) for 48 hours at 37°C.

For intracellular IL-17A staining, cultured CD4+ T cells were stimulated with phorbol myristate acetate (50 ng/ml; Sigma-Aldrich), ionomycin (0.5 μg/ml; Sigma-Aldrich), and monensin (2 μM; eBioscience) for 4 hours before flow cytometric analysis. Each specimen was stimulated in triplicate wells.

Flow cytometric analysis.

For 2-color or 3-color immunofluorescence analyses, cultured T cells were stained with anti-CD4 antibody (BD Biosciences) and anti–IL-17A antibody (BD Biosciences). Cells with the forward and side light-scatter properties of lymphocytes were analyzed on a FACScan flow cytometer (BD Biosciences). Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), according to the manufacturer's instructions, and stained with phycoerythrin-conjugated mouse anti–IL-17A monoclonal antibody. CD4+ T cells from IL-17A−/− mice served as negative controls to demonstrate specificity and to establish background IL-17A staining levels.

Fibroblast culture.

Mouse NIH3T6 fibroblasts were obtained from ATCC. Fibroblast cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Gibco BRL), 1% vitamin solutions, and 2 mM L-glutamine. For the experiments, cultures were placed in fresh, serum-free medium containing 0.1% bovine serum albumin for 24 hours prior to the addition of cytokines.

Fibroblast stimulation by IL-17A.

Cultured fibroblasts were stimulated for 24 hours with the indicated final concentrations of recombinant IL-17A or TGFβ1. The concentration of soluble collagen in cultured fibroblast supernatants was measured using a Sircol soluble collagen assay kit (Biocolor), which is a dye-binding method for the analysis of acid- and pepsin-soluble collagens. Cultured fibroblasts were used for quantitative RT-PCR analysis of TGFβ1 and connective tissue growth factor (CTGF).

Statistical analysis.

A Mann-Whitney U test was used for determining the significance of differences in sample means, and a Bonferroni test was used for multiple comparisons.

RESULTS

Amelioration of bleomycin-induced skin fibrosis by loss of IL-17A, but not IFNγ or IL-4.

IFNγ, IL-4, and IL-17A are frequently used representatives of Th1, Th2, and Th17 cytokines, respectively. To determine which T helper cytokines are critical for bleomycin-induced skin fibrosis, we assessed the results of daily intradermal administration of bleomycin in IFNγ−/−, IL-4−/−, IL-17A−/−, and WT mice. Dermal thickness, defined as the distance between the top of the granular layer and the dermal–adipose layer junction, was determined in 5 random grids on 3 different sections from each mouse (Figures 1A and B).

Figure 1.

Skin fibrosis induced by intradermal bleomycin injections. Interferon-γ–deficient (IFNγ−/−) mice, interleukin-4–deficient (IL-4−/−) mice, IL-17A−/− mice, and wild-type (WT) mice were treated with daily intradermal injections of bleomycin (BLM) for 28 days. A, Dermal thickness. B, Representative images of skin tissue stained with Masson's trichrome. Arrows indicate skin thickness. Original magnification × 40. C, Histologic evaluation of skin fibrosis. Left, Size of fibrotic areas, as evaluated by van Gieson's staining. Middle, Number of α-smooth muscle actin (α-SMA)–positive fibroblastic cells. Right, Collagen content in lesional skin tissue sections. Bars show the mean ± SEM (n = 10 mice in each group). ∗ = P < 0.05. PBS = phosphate buffered saline.

IL-17A deficiency caused a significant reduction in the mean dermal thickness, as evaluated by hematoxylin and eosin staining or Masson's trichrome staining. In contrast, the loss of IFNγ or IL-4 did not affect dermal thickness. The size of fibrotic areas, as evaluated by van Gieson's staining, was consistent with the findings for dermal thickness (Figure 1C). Likewise, the frequency of α-SMA–positive myofibroblasts and the local skin collagen content, which were evaluated in 3 different sections from each mouse, were significantly reduced in IL-17A–deficient mice (Figure 1C). The loss of IFNγ or IL-4 did not significantly affect the size of fibrotic areas, the frequency of myofibroblasts, or the skin collagen content. Thus, loss of IL-17A, but not IFNγ or IL-4, ameliorated skin fibrosis induced by daily intradermal bleomycin treatment.

Skin inflammation induced by daily intradermal bleomycin injections.

Because we observed that only IL-17A loss affected skin fibrosis, subsequent experiments were performed solely in IL-17A−/− mice. The numbers of CD3+ T cells, MPO-positive neutrophils, and F4/80-positive macrophages were assessed in 5 random grids on 3 different sections of bleomycin-injected skin tissue specimens under 400× magnification. When compared with skin tissue from WT mice, skin tissue from IL-17A−/− mice exhibited significantly reduced numbers of CD3+ T cells and F4/80-positive macrophages on day 7 but not on day 14 (Figures 2A and B). Conversely, the numbers of MPO-positive neutrophils were significantly reduced in IL-17A−/− mice on day 14 relative to WT controls (Figure 2A); the numbers of MPO-positive neutrophils in the 2 groups were not significantly different on day 7. Thus, inflammatory cell infiltration was generally modest in IL-17A−/− mice and was associated with reduced skin fibrosis.

Figure 2.

Inflammatory cell infiltration in the skin of IL-17A−/− mice and WT mice during intradermal bleomycin treatment. A, Numbers of CD3+ cells, myeloperoxidase (MPO)–positive neutrophils, and F4/80+ macrophages on day 7 and day 14. Bars show the mean ± SEM (n = 10 mice in each group). ∗ = P < 0.05. B, Representative images showing CD3 and F4/80 staining in tissue sections from IL-17A−/− mice and WT mice. Original magnification × 400. See Figure 1 for other definitions.

Effect of bleomycin on T cell cytokines.

Next, we assessed mRNA levels of cytokines in CD4+ T cells isolated from the spleens of WT mice that received intradermal bleomycin injections. Overall, bleomycin treatment nonspecifically increased the expression of several cytokines, including IFNγ, IL-6, IL-17A, and TGFβ1 (Figure 3A). The induction of IL-17A was particularly prominent, with an ∼5-fold increase compared with PBS-treated mice. For this reason, the concentration of IL-17A in skin lysates was measured by ELISA before and during bleomycin treatment. Interestingly, bleomycin injections significantly increased the local IL-17A concentration, with a peak on day 7 (Figure 3B).

Figure 3.

IL-17A expression in splenocytes and lesional skin from bleomycin-treated WT mice. A, Expression of IFNγ, IL-4, IL-6, IL-17A, and transforming growth factor β1 (TGFβ1) mRNA in CD4+ T cells isolated from the spleens of WT mice treated with bleomycin or PBS, as measured by quantitative reverse transcriptase–polymerase chain reaction on day 7. B, Concentrations of IL-17A in supernatants of skin homogenates, as measured by enzyme-linked immunosorbent assay. The total protein concentration in supernatants was equalized in each sample. C, Frequency of T cells that have potential to produce IL-17A. Splenocytes were harvested from WT mice that had been treated with bleomycin for 7 days. Purified CD4+ T cells were cultured in 96-well flat-bottomed plates precoated with anti-CD3 antibody plus anti-CD28 for 48 hours at 37°C. Cultured T cells were used for flow cytometric analysis of intracellular IL-17A. Each specimen was stimulated in triplicate wells. Bars show the mean ± SEM (n = 5 mice in each group). ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for other definitions.

We also evaluated whether bleomycin could induce Th17 cell differentiation. Initially, we examined whether bleomycin itself can directly affect in vitro Th17 cell differentiation, by culturing splenic T cells from WT mice in Th17 cell–inducing culture conditions. Although significant numbers of Th17 cells were induced during the in vitro Th17 cell differentiation assay (mean ± SEM 13.9 ± 3.4%), the frequency of Th17 cells was not significantly changed by the addition of bleomycin (10.5 ± 3.3%) (results not shown).

Next, we investigated whether daily intradermal bleomycin injections can induce Th17 cell induction in vivo. Bleomycin treatment significantly increased the frequency of T cells that have potential to produce IL-17A, although the population was small (Figure 3C). Thus, daily bleomycin injections can induce expression of cytokines, including IL-17A, in vivo. These findings indicated that bleomycin does not directly affect Th17 cell differentiation, but that daily bleomycin injections may indirectly enhance IL-17 production in the skin and splenic T cells.

Association of IL-17A loss with decreased mRNA expression of profibrogenic growth factors and intercellular adhesion molecule 1 (ICAM-1) in the skin.

Fibrosis is influenced by a variety of cytokines and chemokines. We assessed whether IL-17A deficiency affects the expression of TGFβ and CTGF, 2 well-known fibrogenic growth factors, in the skin of mice receiving daily bleomycin injections. We observed that mRNA levels of both TGFβ and CTGF were markedly reduced on both day 7 and day 14 in mice deficient for IL-17A (Figure 4A). We also examined adhesion molecules and chemokines and observed that mRNA levels of ICAM-1 were significantly lower in IL-17A−/− mice than in WT controls on day 7 (Figure 4B). Messenger RNA levels of CXCL2, CCL2, and CCL3 were not significantly different between IL-17A−/− mice and WT mice on day 7. Thus, the local expression of TGFβ, CTGF, and ICAM-1 was decreased in the skin of bleomycin-treated IL-17A−/− mice.

Figure 4.

Messenger RNA levels of fibrogenic factors in IL-17A−/− mice. A, Expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in bleomycin-treated WT mice and IL-17A−/− mice on days 7 and 14, as determined by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). B, Expression of intracellular adhesion molecule 1 (ICAM-1), CXCL2, CCL2, and CCL3 mRNA in bleomycin-treated WT and IL-17A−/− mice, as determined by quantitative RT-PCR on day 7. Bars show the mean ± SEM (n = 5 mice in each group). ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for other definitions.

Effect of recombinant IL-17A on collagen production from fibroblasts.

To confirm the direct effect of IL-17A on fibrosis, we cultured a mouse fibroblast cell line with or without recombinant IL-17A. Consistent with our in vivo data, the addition of IL-17A resulted in a dose-dependent increase in the production of soluble collagen (Figure 5A). Furthermore, TGFβ and CTGF mRNA expression was increased in cultured fibroblasts following the addition of IL-17A (Figure 5B). These findings indicate that IL-17A can stimulate collagen synthesis from skin fibroblasts, directly and/or indirectly, via fibroblast production of cytokines such as TGFβ and CTGF.

Figure 5.

Effect of the addition of recombinant interleukin-17A (IL-17A) to a skin fibroblast cell line. A, IL-17A–induced dose-dependent increase in soluble collagen production, as determined using a Sircol soluble collagen assay kit. B, IL-17A–induced increase in transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA expression. For comparison, recombinant TGFβ1 was also added to some cultures. Bars show the mean ± SEM results of 4 experiments. ∗ = P < 0.05; ∗∗ = P < 0.01. CTL = control.

Reduced skin thickness in IL-17A–deficient TSK mice.

We further analyzed the fibrotic potential of IL-17A in the TSK/+ mouse model, a genetic mouse model of SSc that resembles the later stages of SSc and is characterized by endogenous activation of fibroblasts without inflammatory infiltration (20, 27). In TSK/+ mice, skin fibrosis is established by age 10 weeks and is attributable to marked hypodermal thickness. Hypodermal thickness, which was defined as the thickness of a subcutaneous loose connective tissue layer (i.e., the hypodermis or superficial fascia) beneath the panniculus carnosus, was measured in 5 grids on 3 different sections of each mouse. Hypodermal thickness was significantly reduced in IL-17A−/− TSK/+ mice compared with control TSK/+ mice (Figure 6).

Figure 6.

Atttenuation of hypodermal thickness by IL-17A deficiency in TSK/+ mice. A, Representative hematoxylin and eosin–stained skin tissue sections from WT, TSK/+, and IL-17A−/− TSK/+ mice. Arrows indicate the thickness of the hypodermis beneath the panniculus carnosus. Original magnification × 40. B, Hypodermal thickness in WT mice, TSK/+ mice, and IL-17A−/− TSK/+ mice. Bars show the mean ± SEM (n = 5 mice in each group). ∗ = P < 0.05. See Figure 1 for definitions.

DISCUSSION

By using cytokine-deficient mice, we determined that IL-17A, but not IFNγ or IL-4, is necessary for maximal bleomycin-induced skin fibrosis. Among mice deficient for IFNγ, IL-4, or IL-17A, only IL-17A–deficient mice demonstrated reduced skin fibrosis following bleomycin treatment. Daily intradermal bleomycin injections triggered IL-17A production in the lesional skin. Additionally, IL-17A was critical for the induction of TGFβ and CTGF in fibrotic skin during bleomycin treatment. The addition of recombinant IL-17A to a skin fibroblast cell line increased the production of collagen, TGFβ, and CTGF. Furthermore, the loss of IL-17A also significantly reduced skin thickness in another model of SSc, TSK/+ mice, suggesting the critical roles of IL-17A in skin fibrosis.

To our knowledge, this study is the first to identify specific roles for IL-17A in the bleomycin-induced skin fibrosis model, via the use of transgenic mice or blocking antibodies. Recently, IL-17A has been implicated in the development of tissue fibrosis and SSc. For example, IL-17A was shown to be critically involved in the development of bleomycin-, silica-, or IL-1β–induced lung fibrosis (10, 28, 29), and serum IL-17A levels were increased in mice treated with daily intradermal bleomycin injections (30). Elevated serum IL-17A levels and augmented IL-17A expression in peripheral blood lymphocytes and lesional skin have also been reported in patients with SSc (11, 12). Another study demonstrated that patients with SSc had markedly increased numbers of circulating Th17 cells (7, 31). In addition, a crucial role for IL-17A in sclerodermatous chronic graft-versus-host disease has been reported (32). Our findings support these observations and extend them, by providing a potential and possibly important function for IL-17A in promoting skin fibrosis.

To clarify the mechanism behind IL-17A regulation of fibrosis, we first determined whether bleomycin could induce IL-17A production. Splenic T cells from WT mice treated intradermally with bleomycin had augmented IFNγ, IL-6, IL-17A, and TGFβ1 mRNA expression; the increase in IL-17A expression was most striking. In addition, daily intradermal bleomycin injections markedly increased the IL-17A concentration in lesional skin, with a peak on day 7. These findings are similar to those of a recent study that showed elevated IL-17A expression in the bronchoalveolar lavage fluid and lung tissue of mice with bleomycin-induced lung fibrosis (10). In our study, daily bleomycin injections increased the potential source of Th17 cells in splenocytes. These findings suggest that the augmented IL-17A production induced by bleomycin treatment cooperated with other proinflammatory or profibrogenic cytokines to promote the development of skin fibrosis.

Although IL-17A has been shown to promote fibroblast proliferation, the direct effect of IL-17A on collagen synthesis in humans has not been reported (11). Therefore, IL-17A may be contributing to the development of skin fibrosis indirectly via induction of other molecules critical for fibrosis or inflammation. To examine this possibility, we examined mRNA expression of profibrogenic molecules in the bleomycin-induced fibrotic skin of IL-17A−/− mice, using real-time RT-PCR. The expression of TGFβ, CTGF, and ICAM-1 mRNA was dramatically reduced by the loss of IL-17A. Because TGFβ and CTGF are central players in the fibrotic process (1), induction of these cytokines by IL-17A is likely important for the development of skin fibrosis in vivo.

ICAM-1 is also considered to be important for skin fibrosis and/or skin inflammation in both bleomycin-induced skin fibrosis and in TSK/+ mice (30, 33). Our results are consistent with those from a prior study that revealed roles for IL-17A in ICAM-1 induction in human endothelial cells (11). Furthermore, human IL-17A has been shown to induce cytokines such as IL-6, IL-8, and granulocyte colony-stimulating factor from fibroblasts (34). Our data agree with these previous findings and indicate that IL-17A may be contributing indirectly to bleomycin-induced skin fibrosis via induction of adhesion molecules and various profibrogenic and proinflammatory cytokines.

Consistent with these in vivo data, we observed that recombinant IL-17A enhanced the expression of both TGFβ and CTGF in a cultured mouse skin fibroblast cell line. Furthermore, the addition of IL-17A increased collagen synthesis in these fibroblasts. In a recent study, mRNA levels of Colα2(I) and TGFβ were shown to be increased in cultured fibroblasts derived from mouse skin (30). Although our current results are similar to those of the previous study, we also detected increased CTGF expression in cultured fibroblasts following the addition of IL-17A. However, it is unclear whether the augmented collagen synthesis was directly caused by IL-17A. Another previous study showed that intratracheal administration of IL-17A induced lung fibrosis that was TGFβ dependent (10). Furthermore, an analysis of T helper cell subsets demonstrated that Th17 cells were the main producers of TGFβ1 both in vivo and in vitro (35). Therefore, our results for fibroblasts may also be reflecting the effects of increased TGFβ production. Further studies will be needed to determine whether IL-17A is directly affecting CTGF production by fibroblasts, and whether CTGF is contributing to IL-17A–induced collagen synthesis.

IL-17A and Th17 cells have been considered to be critical mediators of various autoimmune diseases and inflammatory bowel or skin diseases, in addition to their roles in host defense (8, 9). However, fibrotic changes are uncommon in these disorders. This may be due to different functions and/or expression of IL-17A or Th17 cells that is dependent on each disease, organ, phase, and environment (expression of other cytokines). In fact, recent studies showed significant plasticity of Th17 cells and suggested the possibility that the pathogenetic function of these cells may be mediated through a Th17-to-Th1 phenotype (IFNγ producer) transition in other autoimmune conditions such as autoimmune colitis and diabetes (36, 37). In contrast, Th17 cells require TGFβ for sustained expression of IL-17A (37). Therefore, in a TGFβ-rich environment such as fibrotic disorders (including SSc), Th17-to-Th1 conversions may be infrequent compared with the conversions that occur in other autoimmune or inflammatory disorders. Furthermore, TGFβ is highly expressed by Th17 cells and acts in an autocrine manner to maintain Th17 cells (35). Therefore, the function of IL-17A and Th17 may be different depending on the profile of cytokines and Th cell subsets that contribute to the development of each disease.

Recently, a list of criteria to use in selecting the most promising molecular targets for SSc trials was proposed (38). According to those criteria, the antifibrotic effects of specific molecules should be confirmed in at least 2 complementary animal models of SSc. Although in the current study we mainly analyzed the roles of IL-17A in a model of bleomycin-induced SSc, similar findings were also observed in TSK/+ mice. Bleomycin-induced skin fibrosis is characterized by dense inflammatory cell infiltration in lesional skin. Inflammatory cells have been considered to contribute to the development of skin fibrosis by stimulating profibrogenic cytokine production from fibroblasts. In contrast, TSK/+ mice are characterized by the absence of inflammation and by endogenous activation of fibroblasts. Thereby, the bleomycin-induced model and the TSK/+ mouse model mimic the early progressive stage and the late stage of SSc, respectively (27). Thus, our data indicate that IL-17A has fibrotic effects in 2 mouse models of SSc with different pathologic mechanisms.

We believe that further studies that include the administration of blocking antibody will be needed to clarify the roles of IL-17A in the development of SSc. Nonetheless, our findings suggest that inhibition of IL-17A represents a promising therapeutic target for antagonizing fibrotic skin disorders such as SSc. Because anti-human monoclonal antibodies against IL-17A or IL-17 receptor have been used for clinical trials in other autoimmune and inflammatory disorders (39–41), this strategy could be rapidly advanced into clinical trial testing in patients with SSc.

AUTHOR CONTRIBUTIONS

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. Hasegawa 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. Okamoto, Hasegawa, Fujimoto.

Acquisition of data. Okamoto.

Analysis and interpretation of data. Okamoto, Hasegawa, Matsushita, Hamaguchi, Huu, Iwakura, Fujimoto, Takehara.

Acknowledgements

We thank Dr. W. Ishida, for technical advice and Ms M. Matsubara and Y. Yamada for technical assistance.

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