Blocking IL-17A Promotes the Resolution of Pulmonary Inflammation and Fibrosis Via TGF-β1–Dependent and –Independent Mechanisms

Tissue fibrosis is the structural basis for a variety of chronic human diseases, including idiopathic pulmonary fibrosis, cardiovascular fibrosis, liver cirrhosis, end stage kidney disease, systemic sclerosis, and autoimmune disease (1), and it is the reason why many of these diseases are incurable. Despite recent progress toward understanding the mechanisms underlying tissue fibrosis and its pathogenesis, and although many strategies have been proposed for developing therapeutic agents (2–5), there are no convincing or effective therapeutic treatments available for fibroproliferative diseases (3).

Pulmonary fibrosis is driven by both innate and adaptive immune responses to microbial inflection, physical and chemical assault, tissue injury, and other stresses (6). The innate immune system initiates a predominantly Th1-mediated acute inflammatory response after these types of insults. Nevertheless, multiple mechanisms promote the resolution of this acute inflammatory response (7), including the alternation of immune cell phenotype, the switch in immunomodulatory molecules from pro- to anti-inflammatory, and the activation of many resolving mediators. However, nonresolving chronic inflammation may result from the presence of persistent or repeated initiating stimuli or from a failure of the resolving mechanisms. The Th2-polarized adaptive immune response contributes to and sustains nonresolving inflammation in the damaged tissue that results in the progression of pulmonary fibrosis (6)

Th17 cells are a recently identified subset of effector Th cells, and IL-17A is the major cytokine released from these or other IL-17–producing cells (8). Several studies have recently suggested a possible contribution for IL-17A in the development of chronic fibroproliferative diseases (9). For instance, Yoshizaki et al. (10) found that Th1 cell cytokines were reduced, whereas Th2 and Th17 cytokines were increased during the development of bleomycin (BLM)-induced scleroderma. Baldeviano et al. (11) found IL-17A to promote the development of dilated cardiomyopathy and blockade of IL-17A to attenuate myocarditis-induced cardiac fibrosis and ameliorate ventricular function. Smith et al. (12) noted that the incidence of atherosclerosis in ApoE^−/− mice was reduced when IL-17A activity was blocked, and Wilson et al. (13) demonstrated BLM- and IL-1β–mediated pulmonary fibrosis to be IL-17A dependent. However, the mechanisms governing Th17 cells and their cytokines in the regulation of chronic inflammation and tissue repair remain poorly understood.

Using in vitro and in vivo models of pulmonary fibrosis, we have investigated whether IL-17A directly regulates the epithelial–mesenchymal transition (EMT) and collagen synthesis and whether the inhibition of IL-17A signaling protects mice from inflammation and tissue fibrosis after acute or chronic lung injury. We found that blockade of IL-17A attenuates tissue injury, inflammation, fibrosis, and dysfunction in the acutely and chronically injured lungs by reversing the immunosuppressive environment and restoring the autophagic activity in damaged lung tissues. Our studies suggest that using an immune stimulator of autophagic activity, such as IL-17A antagonism, rather than immunosuppressive agents may be a promising therapeutic strategy for the treatment of devastating fibroproliferative diseases such as idiopathic pulmonary fibrosis.

Male C57BL/6J mice (17 ± 1 g, 6–8 wk) were obtained from Vital River Laboratory Animal Technology (Beijing, China). The mouse IL-17A–F proteins were purchased from Peprotech and eBioscience (San Diego, CA). Cycloheximide (CHX), 3-methylamphetamine (3-MA), and rapamycin were obtained from Sigma (St. Louis, MO). TUNEL assay kits were obtained from Roche (Basel, Switzerland). Alexa Fluor 488, 594, 633 and 647 and DQ collagen-coupled FITC Abs were obtained from Invitrogen (San Diego, CA). Anti-goat IgG and anti-mouse α-SMA, E-cadherin, Snail, Slug, ZO-1, IL-17R, retinoic acid-related orphan receptor γt (RORγt), p–NF-κB, NF-κB, p-smad3, smad3, p-stat3, stat3, L chain 3 (LC3) II/I, Beclin-1, lysosome-associated membrane protein 1 (LAMP-1), collagen I, Vps34, p62, p-mTOR, mTOR, β-tublin, and β-actin Abs were obtained from Cell Signaling Technology (Danvers, MA). FITC-conjugated, PE-conjugated, or PE–Cy5–conjugated anti-mouse CD4, IL-13, IL-17A, IFN-γ, CD11c, MHC class II, CD40, CD80, CD86, and Foxp3 Abs were purchased from eBioscience. The neutralizing mouse IL-17A mAb was purchased from R&D Systems (Minneapolis, MN). The Sircol collagen assay kits were obtained from Biocolor (Carrickfergus, U.K.). The ELISA kits for TGF-β1, IL-6, IL-23, and IL-17A were purchased from eBioscience. BLM was purchased from Nippon Kayaku (Tokyo, Japan). Endotoxin levels in the BLM and neutralizing Ab solutions were <0.01 ng/ml, as tested by the Limulus amebocyte lysate assay from BioWhittaker (Basel, Switzerland). Other materials were purchased from commercial sources.

The mouse type II alveolar epithelial cells (MLE-12) were cultured in DMEM–Ham's Nutrient Mixture F12 (1:1; Hyclone) supplemented with 2% FBS and l-glutamine (full-nutrient medium). The cells were passaged every 2 d.

The expression of collagen I mRNA was analyzed as described previously (14). In brief, total RNA was isolated from MLE-12 cells using the TRIzol kit (Invitrogen) according to the manufacturer’s instructions. Next, the RNA was reverse-transcribed and amplified. PCR was performed using a Mycycler thermal cycler, and the amplified products were analyzed by agarose gel. The following specific primer sequences were used: 5′-CCAGAGTGGAACAGCGATTAC-3′ and 5′-GCAGGCGAGATGGCTTATTT-3′ (mouse collagen I); and 5′-TAACCAACTGGGACGATATG-3′ and 5′-AAACAGGGACAGCACAGCCT-3′ (mouse β-actin). All values obtained were normalized to the values obtained for β-actin.

Male C57BL/6J mice (17 ± 1 g, 6–8 wk) were obtained from Vital River Laboratory Animal Technology. The mice were anesthetized with 50 mg/kg pentobarbital i.p. (Merck). Using an insulin syringe, 50 μl of LPS-free saline, clinical grade BLM (3.0 U/kg), or silica (0.2 g/kg) was injected directly into the trachea as previously described (15). The IL-17A–neutralizing or isotype-matched control Ab (2 μg/mouse) was injected i.v. in 200 μl saline on days 14, 17, and 21 after BLM instillation or on days 60, 63, 70, 77, and 84 after silica instillation. Mice were sacrificed by excessive doses of anesthesia for the collection of single-cell suspensions, bronchoalveolar lavage fluid (BALF), and lungs on days 28 after BLM instillation or day 90 after silica instillation. The lungs were excised and were fixed or frozen for morphological evaluation or measurement of hydroxyproline content.

All animal protocols conformed to the Guidelines for the Care and Use of Laboratory Animals prepared and approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.

The lungs were rapidly excised, fixed with 4% paraformaldehyde, and embedded in paraffin for histopathological examination as previously described (15). Lung tissue sections (5 μm thick) were prepared and stained with Sirius, H&E, or Masson’s trichrome. The grade of pulmonary inflammation and fibrosis was blindly assessed by a professional pathologist. The average integrated OD (IOD) of collagen deposition from 10 randomly chosen regions per tissue sample at a magnification of ×200 was determined using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD).

Single-cell suspensions from lung tissue were prepared as previously described (15) with minor modifications. Briefly, the lungs were inflated with dispase II, were allowed to collapse, and were then placed in 1 ml dispase II with gentle agitation at room temperature for 45 min. The lungs were minced to 1-mm pieces and were resuspended in 2 ml dispase II containing collagenase IV (2 μg/ml) and DNase (50 μg/ml). The digested lung tissues were resuspended in DMEM supplemented with 10% FBS and were sequentially filtered through 200-μm filters. The expression of various surface molecules and the numbers of immune cells, such as marrow-derived dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), M1-type macrophages (M1 cells), and regulatory T cells (Tregs) were analyzed from the lung single-cell suspensions.

BALF was collected and harvested as previously described (16). In brief, the trachea was exposed by a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter. Bilateral BALF was collected after instilling two 0.5-ml aliquots of sterile saline. Approximately 0.9–1.0 ml BALF was retrieved per mouse. The cellular viabilities were >99%, as assessed by 0.4% trypan blue exclusion. The cells were then washed, resuspended, and analyzed by a hematology analyzer or stained with PE–Cy5–conjugated, allophycocyanin-conjugated, or FITC-conjugated Abs for flow cytometry. The supernatant from the BALF was frozen and analyzed by ELISA or Sircol collagen assay.

Surface molecule expression on lung cells was analyzed using multicolor flow cytometry as described previously (15). In brief, lung cells were harvested, washed, and suspended in cold PBS containing 3% FBS and 0.02% NaN₃. The cells were then incubated with a mixture of rat and mouse IgG (1:1) to reduce nonspecific binding, which was followed by serial incubations with saturating concentrations of FITC-conjugated, PE-conjugated, and/or PE–Cy5–conjugated mAb for 1 h at 4°C. Isotype-matched mAbs were used in control samples. At least 20,000 stained cells were analyzed using CellQuest software (BD Biosciences).

The concentrations of IL-17A, TGF-β1, IL-6, and IL-23 in BALF were detected by ELISA using kits from eBioscience according to the manufacturer’s instructions.

The collagen contents of BALF and cell culture supernatants were measured using Sircol collagen assay kits from Biocolor according to the manufacturer’s instructions.

Collagen deposition was determined by measuring the total hydroxyproline content of the lungs according to the revised Reddy GK method (15). In brief, the lungs were hydrolyzed with 2.5 N NaOH at 120°C, 0.1 kPa for 40 min. After neutralization with hydrochloric acid, the hydrolyzation products were diluted with distilled water. The hydroxyproline content of the hydrolyzation products was assessed calorimetrically at 550 nm with p-dimethylaminobenzaldehyde. The results were represented as micrograms per lung.

MLE-12 cells in the logarithmic phase were cultured with serum-free media and treated with collagen–FITC for 1 h as previously described (17). The cells were then rinsed three times with PBS, given fresh DMEM–Ham's Nutrient Mixture F12 media, and treated with the indicated agents for 2 h. After this incubation, the cells were washed, collected, and suspended in cold PBS containing 3% FBS and 0.02% NaN₃. Twenty thousand stained cells were analyzed using CellQuest software (BD Biosciences).

Standard protocols for immunofluorescence microscopy were used as described previously (18). MLE-12 cells were planted on the coverglass-bottom dishes and treated with or without indicated agents. The cells on the dishes were washed two times, fixed by 4% paraformaldehyde for 1 h, and washed three times after fixing. The cells on the dishes and lung sections (5 μm thick) were prepared and stained with indicated primary Abs overnight at 4°C. The sections were washed twice, incubated with fluorochrome-labeled secondary Abs (1:200) for 30 min, and washed three times after staining. Images were obtained with a Leica SP2 confocal microscope (Leica Microsystems, Exton, PA) and analyzed with Leica confocal software. The autophagosomes were identified by LC3 dots, and the autophagolysosomes were identified by the coexpression of LC3 and LAMP-1. Autophagic cell death was identified by the combination of LC3 and TUNEL staining (19), and autophagic collagen degradation was identified by costaining for LC3 and collagen I.

Total lung protein was extracted and separated (20 μg/lane) on 12% SDS-PAGE gels containing 1 mg/ml gelatin under nonreducing conditions. After electrophoresis, the gels were soaked in renaturing buffer and equilibrated in developing buffer. The gels were incubated in fresh developing buffer overnight at 37°C. Subsequently, the gels were stained with 0.5% Coomassie blue R250 and destained in a 25% methanol, 20% acetic acid solution (13).

Proteins were extracted from lung tissue using a Qproteome plasma nuclei protein kit (Qiagen, Toronto, ON, Canada). Protein concentrations were determined using a Coomassie Plus reagent. SDS-PAGE and Western blotting were conducted as described previously (14).

Data are represented as the mean ± SE. Statistical analyses were performed using one-way ANOVA, for which the p value was set at 0.05, and using Tukey–Kramer's or Dunnett’s post hoc multiple comparison tests. The survival rates were analyzed using the Kaplan–Meier method. All statistics were analyzed using SPSS 17.0 software.

The activation of the EMT is important for the pathogenesis of tissue fibrosis, as well as for cancer metastasis and embryonic development (20). To determine the role of IL-17A in the regulation of the EMT, the expression of the positive EMT markers Snail, Slug, and α-SMA and the negative EMT markers E-cadherin and ZO-1 was evaluated in the cultured mouse alveolar epithelial (MLE-12) cells. We found that IL-17A stimulated the expression of Snail, Slug, and α-SMA but suppressed the expression of E-cadherin and ZO-1 in a concentration-dependent manner (Fig. 1A). IL-17RA (IL-17R) is the only receptor for IL-17A in rodents (21). Therefore, we examined how IL-17A regulated the expression of these EMT markers by blocking the IL-17R with an anti–IL-17R Ab. We found that IL-17A could neither activate the expression of positive markers of EMT nor attenuate the expression of the negative markers of the EMT in the cells pretreated with the anti–IL-17R Ab (Fig. 1B), indicating that IL-17A promoted the EMT progression by activating IL-17R in the mouse alveolar epithelial cells. TGF-β1 is a pivotal enhancer of EMT (22). We wondered if the IL-17A–induced EMT depended on TGF-β1. We found that both IL-17A and TGF-β1 inhibited the expression of E-cadherin and enhanced the expression of α-SMA (Fig. 1C). However, the pro-EMT role of IL-17A could be attenuated by TGF-β1 antagonism, but the pro-EMT role of TGF-β1 could not be blocked by IL-17A antagonism (Fig. 1C). These data indicated that IL-17A stimulates EMT progress in a TGF-β1–dependent manner.

The accumulated collagen in fibrotic tissue is a critical component in the formation of fibrotic scars (6). We therefore examined whether IL-17A could directly regulate the secretion of collagen by these lung epithelial cells. We found that IL-17A stimulated a concentration- and time-dependent secretion of collagen by MLE-12 cells (Fig. 2A, 2B). However, only IL-17A, but not other members of the IL-17 family, could stimulate the secretion of collagen in these cells (Fig. 2C). The IL-17A–induced secretion of collagen required de novo protein synthesis because the IL-17A–induced expression of collagen I mRNA was completely abrogated in the presence of the protein synthesis inhibitor CHX (Fig. 2D). As TGF-β1 is a positive regulator of collagen, we examined whether IL-17A–stimulated collagen production depended on the presence of TGF-β1. Indeed, blocking TGF-β1 with a TGF-β1–neutralizing Ab completely inhibited the IL-17A–stimulated collagen secretion (Fig. 2E). Furthermore, IL-17A induced the expression of TGF-β1 in these cells in concentration- and time-dependent manners (Fig. 2F, 2G). Taken together, these results indicate that IL-17A can induce the production of collagen in the mouse alveolar epithelial cells in a TGF-β1–dependent manner.

Given the observed roles for IL-17A in the promotion of EMT, collagen production, and TGF-β1 expression, we further examined the expression of IL-17A, IL-17A–related regulatory cytokines, and components of IL-17A signaling pathway in fibrotic lung tissue. We found that IL-17A and its related partners IL-6, TGF-β1, and IL-23 (Fig. 3A) were upregulated in the BALF from the BLM-challenged mice. RORγt is an essential transcription factor for Th17 cells, and it has an intimate relationship with the secretion of IL-17A (23). We found that expression of RORγt and the IL-17R was also increased in fibrotic lung tissues (Fig. 3B). These studies indicate that IL-17A has a crucial role in the pathogenesis of pulmonary fibrosis and that targeting IL-17A might be of therapeutic potential for this devastating disease. Indeed, blocking IL-17A activity reversed BLM-established pulmonary fibrosis, as demonstrated by the reduced deposition of collagen (Fig. 3D, 3E), the hydroxyproline content (Fig. 3F), the levels of collagen in the BALF (Fig. 3G), the expression of α-SMA (Fig. 3H), and the activation of MMP2 (Fig. 3I). These factors also contributed to the increased survival of BLM-challenged mice (Fig. 3J).

Because nonresolving inflammation is known to drive pulmonary fibrosis (7), we examined whether the targeting of IL-17A could impact the resolution of inflammation in fibrotic lung tissue. IL-17A antagonism suppressed the recruitment and infiltration of inflammatory cells (Fig. 3C, Table I) as well as the expression of the inflammatory cytokines IL-17A, TGF-β1, IL-6, and IL-23 (Fig. 4A) in the BLM-challenged lung tissue. Next, we examined the effects of blocking IL-17A on fibrotic tissue-infiltrating inflammatory cells, as the balance between the Th1 and Th2 responses can determine the development and progression of pulmonary fibrosis (6). We found that treatment with anti–IL-17A Ab shifted the Th2-polarized response toward a Th1-polarized response in the injured lung tissue, as indicated by the reduction of lung-infiltrating Th2 cells, Th17 cells, Tregs, pDCs, and M2-type macrophages (M2 cells) and the increase in Th1 cells, M1 cells, and mDCs (Fig. 4B). Moreover, anti–IL-17A treatment attenuated the phosphorylation of the transcription factors NFκB-p65, smad3, and stat3 (Fig. 4C), each of which have been shown to promote pulmonary fibrosis (15). These data indicate that targeting IL-17A can attenuate BLM-induced pulmonary fibrosis by promoting the resolution of the BLM-induced inflammatory response.

As the BLM-induced lung fibrotic response is the result of acute injury/inflammation, we further asked whether IL-17A antagonism could show therapeutic efficacy for chronic pulmonary fibrosis. We generated a mouse model of silica-induced pulmonary fibrosis by administering silica dust into the lungs for 2 mo, and these mice were treated with anti–IL-17A for an additional 30 d. This blockade of IL-17A activity attenuated silica-induced pulmonary fibrosis and decreased the recruitment and infiltration of inflammatory cells (Fig. 5A), the deposition of collagen (Fig. 5B, 5C), the hydroxyproline content (Fig. 5D), the secretion of collagen in the BALF (Fig. 5E), and the expression of α-SMA (Fig. 5F). These findings indicate that IL-17A has a critical role in the pathogenesis of acute and chronic pulmonary fibrosis and that targeting IL-17A may have general therapeutic efficacy for pulmonary fibrosis that is independent of fibrotic origin.

We have recently shown the activation of autophagy and autophagy-related cell death to be essential for the resolution of inflammation and fibrosis after acute tissue injury (24) (H.-Z. Yang et al., unpublished observations). Thus, we sought to determine whether IL-17A had a role in the regulation of autophagy and whether the antifibrotic effects of IL-17A antagonism were related to IL-17A–regulated autophagy in the fibrotic lung tissue. The microtubule-associated protein LC3 (or Atg8) is a unique molecular marker for autophagosomes. The conversion of LC3-I (cytosolic form) to LC3-II (membrane-bound lipidated form) is widely recognized as an early mark of autophagy activation (25). We found the instillation of BLM to increase significantly autophagy activation, as indicated by the increased ratio of LC3-II/LC3-I in fibrotic tissues (Fig. 6A). Moreover, blocking IL-17A further enhanced the ratio of LC3-II/LC3-I in fibrotic tissues (Fig. 6A), which indicates that IL-17A promotes tissue fibrosis by regulating the activation of autophagy. We subsequently examined the expression of Beclin-1, Vps34, and the ratio of p–Bcl-2/Bcl-2 because this core protein complex regulates autophagy and is required for autophagosome formation in the autophagy process (26). The expression of Beclin-1 was increased, but the expression of Vps34 and the ratio of p–Bcl-2/Bcl-2 were decreased in the fibrotic lung tissues from the untreated mice. However, the expression of Beclin-1 and Vps34, as well as the ratio of p–Bcl-2/Bcl-2, was increased in the fibrotic lung tissues from the anti–IL-17A–treated animals (Fig. 6A). Importantly, the “cargo” protein p62, which is involved in the trafficking of intracellular aggregates or unfolding proteins to the degradation pathway and is recognized as a marker of autophagy flux (27), had significantly enhanced expression in the lung tissues of untreated or IgG-treated mice compared with that in the lung tissues of sham and IL-17A Ab-treated mice (Fig. 6A). This indicated that there was impaired autophagy in the lungs of untreated and IgG-treated mice but that IL-17A antagonism reversed this impairment. We also detected the expression of a negative regulator of autophagy, mTOR, which is independent of the Beclin-1 core complex. We found that phosphorylation of mTOR was increased in the untreated and IgG-treated fibrotic lungs but was decreased in the IL-17A Ab-treated lungs (Fig. 6A). Moreover, blocking IL-17A significantly enhanced the colocalization of LC3 and TUNEL in the fibrotic tissue (Fig. 6B). This indicated that blockade of IL-17A promoted the autophagy-associated cell death, which is a reliable marker for the autophagy activation (28). Indeed, blocking IL-17A enhanced the coexpression of LAMP-1 and the autolysosomal maker LC3 (29) in fibrotic lung tissues (Fig. 6C), which indicated that blocking IL-17A promoted the formation of autolysosomes. Taken together, these data indicate that autophagy is impaired in BLM-challenged lung tissue and that blocking IL-17A can induce an effectual autophagy and induce an autophagy-associated cell death in the fibrotic lung tissue.

To determine whether the activation of autophagy was responsible for the efficiency of IL-17A antagonism in pulmonary fibrosis, we examined whether IL-17A could directly regulate autophagy in cultured lung epithelial cells. We found that IL-17A significantly inhibited the starvation-induced increase of LC3 foci (Fig. 7A) in MLE-12 cells. IL-17A also inhibited the formation of the autophagosome, reduced the ratio of LC3-II/LC3-I, and reduced the expression of Beclin-1, p62, Vps34, p–Bcl-2/Bcl-2, and p-mTOR/mTOR in these cells (Fig. 7B). These data indicate that IL-17A can inhibit autophagy. However, IL-17A–inhibited autophagy did not depend on TGF-β1 because TGF-β1 neither changed autophagic activity nor attenuated the IL-17A–suppressed autophagy in these cells (Supplemental Fig. 1).

Because we found IL-17A to stimulate collagen production and attenuate autophagy in MLE-12 cells and IL-17A blockade to attenuate acute and chronic pulmonary fibrosis via autophagy activation, we therefore asked whether autophagy participated directly in the regulation of collagen production by epithelial cells. Rapamycin, a classical autophagy agonist (30), was used to determine the role of IL-17A in the regulation of collagen production. Activation of autophagy by rapamycin reduced the IL-17A–induced increase in the secretion of collagen by MLE-12 cells (Fig. 7C). We further examined whether autophagy participated in the regulation of collagen degradation in these cells. MLE-12 cells were incubated with DQ collagen-coupled FITC for 1 h, after which the medium was removed, and the cells were washed intensively. The cells were then treated with or without IL-17A and autophagy-regulating agents for 2 h. Because FITC annihilation is accompanied by collagen degradation, the surplus of FITC in the cells can be used to measure the efficacy of collagen degradation (17). We found that the activation of autophagy by rapamycin promoted collagen degradation, whereas the inhibition of autophagy by 3-MA and IL-17A attenuated collagen degradation (Fig. 7D). These data indicate that IL-17A attenuates the degradation of collagen by inhibiting autophagy.

Because the blockade of IL-17A was found to attenuate pulmonary fibrosis by restoring IL-17A–suppressed autophagy in fibrotic lung tissue, we examined whether inhibition of autophagy with the antagonist 3-MA prior to anti–IL-17A treatment could reverse the therapeutic effect of IL-17A–neutralizing Ab on pulmonary fibrosis. 3-MA prevented the IL-17A blockade-mediated reduction in collagen deposition (Fig. 8A), secretion of collagen in the BALF (Fig. 8B), and expression of α-SMA (Fig. 8C) in fibrotic lung tissue. 3-MA treatment also significantly reduced animal survival, which was increased after anti–IL-17A treatment (Fig. 8D). Because increased deposition of collagen is at the origin of tissue fibrosis (3), and because blocking IL-17A–reduced production of collagen can be reversed by autophagy inhibition in vitro (Fig. 7D), we examined whether altering autophagic activity could regulate the in vivo deposition of collagen in fibrotic lung tissue. We found LC3 and collagen to colocalize significantly in areas of fibrotic tissue, and blocking IL-17A reduced the deposition of collagen in fibrotic tissues. However, this effect was reversed by treatment with the autophagy antagonist 3-MA (Fig. 8E). 3-MA also reduced the number of autophagolysosomes (Fig. 8F) and inhibited the expression of autophagy-related proteins (Fig. 8G). These findings support an antifibrotic role for IL-17A antagonism that results in the activation of autophagy in the fibrotic tissue (Fig. 9).

In this study, we have demonstrated that IL-17A, a cytokine released from Th17 and other IL-17–producing cells, plays a crucial role in the development and progression of both acute and chronic inflammation-induced pulmonary fibrosis. We observed that IL-17A, but not other members of the IL-17 family, not only stimulates collagen production and EMT activation but also attenuates the autophagy-regulated degradation of collagen in cultured lung epithelial cells. Moreover, in fibrotic lung tissues, we found increased expression of IL-17A and IL-17A–related molecules as well as components of the IL-17A signaling pathway. Blocking IL-17A activity promoted the resolution of acute and chronic inflammation, resulted in a shift from the suppressive immune response in the fibrotic lung toward a Th1 response, and effectively induced the activation of autophagy, which prompted the degradation of collagen. Thus, IL-17A antagonism showed considerable efficacy against BLM-induced acute pulmonary fibrosis and silica-induced chronic pulmonary fibrosis. Moreover, the antifibrotic effects of IL-17A antagonism were due to the restoration of IL-17A–suppressed autophagic activity and autophagy-associated cell death, which is essential for the resolution of inflammation and tissue fibrosis after acute or chronic tissue injury (Fig. 9). Indeed, the inhibition of autophagy with 3-MA reversed the antifibrotic effects of IL-17A antagonism. These observations indicate that reversal of immunosuppressive tissue environment and restoration of IL-17A–suppressed autophagy in fibrotic lung tissue are responsible for the antifibrotic effect of IL-17A antagonism.

Although a number of studies have highlighted the differential regulatory roles for Th1 and Th2 responses in the pathogenesis of tissue fibrosis (15, 31), it is unclear how the Th1/2 balance affects the progression of tissue fibrosis. Recent studies have indicated that autophagy acts as an immune effector mechanism against intracellular pathogens and functions to heal injured tissue and remove cellular debris. For example, the Th1 cytokine IFN-γ induces autophagy in macrophages to eliminate Mycobacterium tuberculosis, whereas the Th2 cytokines IL-4 and IL-13 inhibit autophagic control of intracellular M. tuberculosis (32). These findings establish a mechanism by which the status of Th1/Th2 polarization differentially affects the immune response to intracellular pathogens. Furthermore, they suggest that autophagy can be positively or negatively affected by Th1 or Th2 cytokines for its participation in the pathogenesis of tissue fibrosis. In contrast, few studies on this topic have generated significant insight on the role of other T cell subsets, such as Th17 cells, in this process. Our current study has indicated that IL-17A directly inhibits the formation of autophagosomes by attenuating the expression of several key autophagic molecules, such as Beclin-1, Vps34, and the ratio of p–Bcl-2/Bcl-2. Antagonism of IL-17A not only restored the suppressive activity of these autophagic molecules but also caused a shift in the immunosuppressive tissue environment toward a Th1 environment, which may have generated a suitable tissue environment for an effective autophagic response in the fibrotic lung tissue that led to antifibrotic activity. Indeed, an immunosuppressive tissue environment associated with the impaired autophagy was observed in fibrotic lung tissue from untreated or IgG-treated mice, despite the activation of earlier autophagic signals. We recently found that autophagy functions as an effector mechanism after TLR4 activation to promote the resolution of chronic inflammation and to attenuate pulmonary and myocardial fibrosis after acute lung or heart injury (H.-Z. Yang et al. unpublished observations). Our studies demonstrate that an active immune response is crucial for the resolution of chronic inflammation and tissue fibrosis after acute or chronic tissue injury. Therefore, our studies provide novel insights into the pathogenesis of tissue fibrosis and indicate that the Th1/Th2 paradigm for the pathogenesis of fibroproliferative lung diseases requires further clarification to achieve greater prevention and treatment of pulmonary fibrosis.

Our work indicates that Th17 cells and the cytokine IL-17A, similarly to Th2 cells and Th2 cytokines, contribute to the pathogenesis of pulmonary fibrosis and participate in the pathogenesis of fibrosis in multiple organs, such as for UUO-induced renal fibrosis and BDL-induced hepatic fibrosis (S. Mi, Z. Li, H. Liu, H.-Z. Yang, and Z.-W. Hu, unpublished observations). Therefore, a general mechanism may be responsible for the profibrotic role of IL-17A in these different organs (33). Indeed, our studies demonstrated that the IL-17A–stimulated production of collagen requires de novo protein expression and depends on the presence of TGF-β1, a pivotal profibrotic factor, which is supported by the finding of Wilson et al. (13) in that TGF-β1 is required for IL-17A–mediated pulmonary fibrosis. However, although the IL-17A–induced secretion of collagen depends on TGF-β1 expression, the IL-17A antagonism-mediated restoration of autophagic activity in fibrotic lung tissue was not dependent on the expression of TGF-β1. Indeed, TGF-β1 has been found to stimulate autophagy in human hepatocellular carcinoma cells (34) and renal tubules (35), whereas our study demonstrated that TGF-β1 does not regulate the autophagy in lung epithelial cells (Supplemental Fig. 1). Thus, these studies indicate that IL-17A can promote pulmonary fibrosis through both TGF-β1–dependent and –independent mechanisms (Fig. 9).

Not only do our findings reveal a novel role for the IL-17A signaling pathway in the pathogenesis of pulmonary fibrosis after acute or chronic lung injury, but they also suggest that targeting the components of IL-17A signaling pathway is a novel strategy for the development of therapeutic agents against fibroproliferative diseases that are resistant to the current treatments. For instance, Th1 cytokine IFN-γ has been tested in a clinical trial to treat pulmonary fibrosis but with a controversial therapeutic efficacy (3). Recent studies indicate that IFN-γ can augment the TGF-β1–induced EMT in A549 cells by upregulating TbetaR-I (36). The innate immune component of kidney IRI requires dual activation of the IL-12/IFN-γ and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17 is upstream of IL-12/IFN-γ (37), and IFN-γ and IL-17 cooperatively contribute to inflammation in proteoglycan-induced arthritis (38). Thus, IFN-γ promotes EMT and inflammation by activating IL-17A and/or TGF-β1 signaling pathways, which sustains chronic inflammation and tissue fibrosis to counteract, at least partly, the IFN-γ–induced antifibrotic effect. Blocking IL-17A signaling may attenuate the IFN-γ–mediated profibrotic effects because IL-17A antagonism blocks both IL-17A and TGF-β1 signaling pathways.

The authors have no financial conflicts of interest.