Idiopathic pulmonary fibrosis (IPF) is a progressive and destructive lung disease with a poor prognosis resulting in a high mortality rate. IL-37 is an anti-inflammatory cytokine that inhibits innate and adaptive immunity by downregulating proinflammatory mediators and pathways. However, the exact role of IL-37 in lung fibrosis is unclear. In this study, we found that the IL-37 protein was expressed in alveolar epithelial cells (AECs) and alveolar macrophages in healthy controls but significantly reduced in patients with IPF. IL-37 significantly inhibited oxidative stress–induced primary mouse AEC death in a dose-dependent manner, and knockdown of IL-37 significantly potentiated human lung cancer–derived AEC (A549 cells) death. IL-37 attenuated constitutive mRNA and protein expression of fibronectin and collagen I in primary human lung fibroblasts. IL-37 inhibited TGF-β1–induced lung fibroblast proliferation and downregulated the TGF-β1 signaling pathway. Moreover, IL-37 enhanced beclin-1–dependent autophagy and autophagy modulators in IPF fibroblasts. IL-37 significantly decreased inflammation and collagen deposition in bleomycin-exposed mouse lungs, which was reversed by treatment with the autophagy inhibitor 3-methyladenine. Our findings suggested that a decrease in IL-37 may be involved in the progression of IPF and that IL-37 inhibited TGF-β1 signaling and enhancement of autophagy in IPF fibroblasts. Given its antifibrotic activity, IL-37 could be a therapeutic target in fibrotic lung diseases, including IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive, devastating lung disease characterized by alveolar epithelial cell (AEC) injury with subsequent proliferation of activated fibroblasts known as myofibroblasts. The accumulation of myofibroblasts is responsible for the excessive deposition of extracellular matrix (ECM), resulting in irreversible distortion of the lung parenchymal structure (1, 2). (Myo)fibroblasts are the major effector cells of lung fibrosis and are characterized by high-level proliferation and resistance to death (3). Many approaches have been proposed for the development of therapeutics, but few beneficial treatments for lung fibrosis are available.

Lung fibrosis is driven by the innate and adaptive immune responses to infection, cigarette smoke, dust, and other environmental stimuli. The Th1 immune response mediates the acute inflammatory response after these types of insult. Usually, the resolution of inflammation involves multiple mechanisms, including the alternation of immune cell phenotype, a switch in immunomodulatory mediators from pro- to anti-inflammatory, and activation of anti-inflammatory cytokines (4). Repeated injury due to activation of the Th2 adaptive immune response leads to sustained unresolved inflammation and damage to the alveolar epithelium, resulting in the progression of lung fibrosis (5, 6).

IL-37 is a new member of the IL-1 family that encompasses 11 structurally related members sharing a β-barrel motif. In contrast to most IL-1 family members, which have proinflammatory activities, IL-37 has emerged as a fundamental inhibitor of innate inflammation and the acquired immune response (7). Among the five splice variants of IL-37 transcripts, IL-37β is the largest cytokine and is encoded by five of the six exons spanning the IL-37 gene, of which exon 1 encodes the putative caspase-1 processing site (8, 9). The IL-37β precursor is processed by caspase-1 into a mature form, and the mature form translocates to the cell nucleus (7, 9). IL-37β–specific mRNA is expressed in various human tissues, such as lymph nodes, thymus, bone marrow, and lung (10). IL-37 has broad suppressive effects on innate inflammation and the acquired immune response (7, 11). Human IL-37–overexpressing mice are protected against endotoxin shock, colitis, hepatitis, and psoriasis (7, 1214). Recently Li et.al. reported that local treatment with IL-37 prevent bleomycin (BLM)-induced experimental lung inflammation and fibrosis. However, the exact mechanism underlying antifibrotic effect of IL-37 has not been elucidated (15). In addition, there have been no reports regarding the relevance of IL-37 in IPF.

In this study, we investigated whether IL-37 protects mice from established BLM-induced lung inflammation and fibrosis and whether IL-37 directly inhibits primary lung fibroblast proliferation and collagen synthesis, along with an examination of the possible underlying mechanisms.

The Abs used were rabbit anti–IL-37 (no. 116282; Abcam, Cambridge, U.K.), rabbit anti-collagen I (no. 34710; Abcam), mouse antifibronectin (no. 7387; Sigma-Aldrich, St. Louis, MO), rabbit anti-Akt (no. 9272; Cell Signaling Technology, Danvers, MA), rabbit anti-Akt (phospho) (no. 9271; Cell Signaling Technology), mouse anti-PI3K (no. ab86714; Abcam), rabbit anti-PI3K (phospho) (no. ab182651; Abcam), rabbit anti-mTOR (no. 2972; Cell Signaling Technology), rabbit anti-mTOR (phospho) (no. 2971; Cell Signaling Technology), rabbit anti-Smad2 (no. 5339S; Cell Signaling Technology), rabbit anti-Smad2 (phospho) (no. 3108S; Cell Signaling Technology), rabbit anti-Smad3 (no. 9523S; Cell Signaling Technology), rabbit anti-Smad3 (phospho) (no. 9520S; Cell Signaling Technology), rabbit anti–ERK 1/2 (no. 9102; Cell Signaling Technology), rabbit anti-ERK 1/2 (phospho) (no. 9101S; Cell Signaling Technology), rabbit anti-p38 MAPK (no. 9212S; Cell Signaling Technology), rabbit anti-p38 MAPK (phospho) (no. 9211S; Cell Signaling Technology), rabbit anti–L chain (LC) 3B (no. 3868S; Cell Signaling Technology), rabbit anti–autophagy-related gene 7 (ATG7) (no. 8558S; Cell Signaling Technology), rabbit anti–beclin-1 (no. 3738S; Cell Signaling Technology), rabbit anti–TGF-βRI (no. 398; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–TGF-βRI (phospho) (no. 112095; Abcam), rabbit anti–TGF-βRII (no. 61213; Abcam), rabbit anti–TGF-βRII (phospho) (no. 17007; Santa Cruz Biotechnology), rabbit anti–caspase-3 (no. 9662S; Cell Signaling Technology), mouse anti–SP-C (no. 518029; Santa Cruz Biotechnology), and mouse anti–β-actin (no. A5316; Sigma-Aldrich). SB203580, Akt-1/2 Viii, and PD98059 were purchased from Calbiochem-Novabiochem (La Jolla, CA). Reactive oxygen species (ROS)/superoxide detection assay kits (no. ab139476; Abcam) and lipid peroxidation assay kits (no. 118970; Abcam) were used. The in situ cell death detection kit (no. 11684795910; Roche, Basel, Switzerland), mouse TGF-β1 ELISA kit (no. DY1679; R&D Systems), Fluoroshield mounting medium with DAPI (no. ab104139; Abcam), BLM (Nippon Kayaku, Tokyo, Japan), and 3-methyladenine (3-MA) (no. M9281; Sigma-Aldrich) were also purchased from the sources indicated.

All human lung tissues and bronchoalveolar lavage (BAL) fluid (BALF) samples from patients with IPF (n = 20) and normal controls (n = 16) were obtained from the Biobank at Soonchunhyang University Bucheon Hospital. The diagnosis of IPF was based on an international consensus statement by the American Thoracic Society and the European Respiratory Society. Histologic diagnoses of usual interstitial pneumonia were confirmed by surgical lung biopsy specimens in all subjects. No patient with IPF had any clinical or laboratory evidence of underlying collagen vascular disease. The control samples were normal, nondiseased lung specimens obtained from patients who underwent surgery for lung cancer. Clinical data obtained for each sample included clinical information, pulmonary function test and blood gas analysis results, and smoking exposure histories (Table I). We obtained sera from 40 patients with IPF and 20 control subjects chosen from age- and sex-matched healthy volunteers without lung disease. This protocol was approved by the local ethics committee of Soonchunhyang University Bucheon Hospital (SCHBC_biobank_2012-015).

Cytocentrifuge slides containing BAL cells were air dried, fixed in methanol, and stained with Diff-Quik (American Scientific Products, Gibbstown, NJ). All subjects provided written informed consent. The protocol was approved by the local ethics committee of Soonchunhyang University Bucheon Hospital (SCHBC_IRB_2011-22).

Primary mouse AECs were isolated from wild-type mice using a previously described protocol (16) with minor modification.

Crude cell suspensions were prepared from C57BL/6 mice. The lungs were perfused with 0.9% NaCI, using a 10-ml syringe fitted with a 21-gauge needle (BD Pharmingen, San Diego, CA) through the right ventricle of the heart until they were visually free of blood. A 21-gauge i.v. catheter was inserted into the trachea and secured tightly with a suture. The lungs were filled with 1–2 ml dispase via the tracheal catheter and then allowed to collapse naturally, expelling part of the dispase. Low melting-point agarose (l%, 0.45 ml, stored in a 45°C water bath; Invitrogen, Paisley, U.K.) was infused slowly via the catheter. The lungs were immediately covered with crushed ice and incubated for 2 min. The lungs were then removed to 2 ml of dispase in 12 ml polypropylene culture tubes (Sigma-Aldrich), incubated for 45 min at room temperature, and placed on ice until the next step. The lungs were transferred into 7 ml DMEM with 0.01% DNase I in 60-mm Petri dishes. The digested tissue was carefully teased from the airways with the curved edge of curved fine-tipped forceps and gently swirled for 5–10 min. The resulting suspension was successively filtered through 100- and 40-μm Falcon cell strainers and then through 25-μm nylon mesh. The filtered suspension was centrifuged at 130 rpm × g for 8 min at 4°C and resuspended in 10 ml of 10% FBS and 1% penicillin-streptomycin in 25 mM HEPES-buffered DMEM.

The cells were incubated with biotinylated anti-CD32 (0.5 μg/million cells; BD Pharmingen) and biotinylated anti-CD45 (1.5 μg/million cells; BD Pharmingen) for 30 min at 37°C. Meanwhile, streptavidin-coated magnetic particles (Thermo Fisher Scientific, Waltham, MA) were washed twice in PBS (10 min each wash) in polypropylene culture tubes using a magnetic tube separator (Sigma-Aldrich). After incubation, the cells were centrifuged (130 rpm × g for 8 min at 4°C), resuspended in 7 ml of DMEM, added to the magnetic particles, and incubated with gentle rocking for 30 min at room temperature. At the end of the incubation, the tubes were attached to the magnetic tube separator with adhesive tape for 15 min. The cell suspension was aspirated from the bottom of the tube using a narrow-stemmed transfer pipet, centrifuged, and resuspended in culture media. Cell viability was determined by trypan blue staining. The purity of alveolar type II cells was assessed by pro-SPC immunofluorescence staining. As in previous studies, 4–8 × 105 cells were isolated from a single mouse. In these samples, the purity of type II pneumocytes was 90–93%. Isolated cells were maintained in 10% FBS and 1% penicillin-streptomycin in 25 mM HEPES-buffered DMEM.

Primary human lung fibroblasts were obtained from Soonchunhyang Biobank (Bucheon, South Korea) as previously described (17). Briefly, fibroblasts were derived from lung tissues obtained from IPF patients by video-assisted thoracoscopic biopsy. Lung samples were obtained by lung biopsy, usually 1 wk after hospital admission. None of the patients had been treated with corticosteroids or immunosuppressive drugs at the time of biopsy. Control fibroblasts were obtained from individuals having a lobectomy for the removal of a primary lung tumor. No morphological evidence of disease was found in the tissue samples used for the isolation of control cells. Lung fibroblasts from IPF or control specimens were isolated by mechanical dispersal and then trypsin digestion of tissues minced to 1 mm2 fragments. Fibroblast cultures were established in DMEM supplemented with 10% FCS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin and amphotericin B (0.25 μg/ml). All cells were cultured at 37°C in 95% air-5% CO2 until just before reaching confluence, which generally occurred in 1–2 wk. After passage 3, immunoblotting analyses were performed using anti-vimentin Abs on adherent cells harvested from the same culture vessels. All cells showed the morphological characteristics of fibroblasts. All experiments with IPF and control fibroblasts were performed on cells before the passage 6.

The lung tissues were dehydrated and embedded in paraffin. For histological examination, sections of samples 4-μm thick and BAL cells on slides were treated with 1.4% H2O2-methanol for 30 min to block endogenous peroxidase. Nonspecific binding was then blocked with 1.5% normal serum, and slides were incubated with rabbit anti-IL-37 polyclonal Abs (1:200; Abcam). The next day, the sections were incubated with ABC kit reagents (Vector Laboratories, Burlingame, CA). The color reaction was developed by staining with a liquid DAB+ substrate kit (Golden Bridge International, Mukilteo, WA). After immunohistochemical staining, the slides were counterstained with Harris hematoxylin for 1 min. For localization of IL-37 in the human lung, Abs against SP-C (1:100; Santa Cruz Biotechnology) were used to identify AECs. Goat anti-rabbit IgG-PE (1:100; Abcam) was used as a secondary Ab.

Proteins were extracted from lung tissues or cells in lysis buffer (no. 78510; Thermo Fisher Scientific) with proteinase and phosphatase inhibitor mixtures (no. 05892970001 and no. 04906837001; Roche) followed by centrifugation. Immunoblotting was performed as described previously (18). For each experiment, equal amounts of total protein were resolved by 10% NaDodSO4-PAGE (SDS-PAGE). The proteins were transferred onto polyvinylidene difluoride membranes (no. ISEQ00010; MilliporeSigma, Billerica, MA) and incubated with a specific primary Ab for 2 h at 37°C or for 24 h at 4°C.

After washing several times with PBS containing Tween, the membranes were incubated with anti-rabbit IgG HRP-conjugated secondary Ab (no. 7074; Cell Signaling Technology) or anti-mouse IgG HRP-conjugated secondary Ab (no. 7076), followed by chemiluminescence detection (no. 34080; Thermo Fisher Scientific and no. 1705061; Bio-Rad Laboratories, Berkeley, CA) with the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

The concentration of IL-37 was detected in the BALF by ELISA using kits from AdipoGen Life Sciences (San Diego, CA) according to the manufacturer’s instructions.

Specific pathogen-free C57BL/6 (Orient Bio, Sungnam, Gyeonggi-Do, South Korea) mice were maintained under pathogen-free conditions. All animal procedures followed a protocol approved by the Institutional Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital (SCHBC-animal-2014-009). On day 0, the mice were administered 3 U/kg BLM by intratracheal instillation (Sigma-Aldrich) dissolved in a total volume of 200 μl endotoxin-free water. On days 11–14, the mice were anesthetized with a mixture of ketamine (Yuhan, Seoul, South Korea) and xylazine (Bayer, Shawnee Mission, KS) and were administered 500 ng IL-37 (R&D Systems, Minneapolis, MN) dissolved in endotoxin-free water including 0.3% DMSO by intranasal instillation. The sham control mice were treated with PBS only. Some mice were i.p. injected with 3-MA (30 mg/kg) 2 h before IL-37 for inhibition of autophagy. On day 21, the mice were killed with an overdose of ketamine/xylazine mixture, and BAL was performed by instilling 1 ml PBS, which was gently retrieved four times as described previously (19). Cell numbers were measured using a hemocytometer, and differential cell counts were performed on slides prepared by cytocentrifugation and staining with Diff-Quik (Thermo Fisher Scientific). The BALF was centrifuged at 500 rpm × g for 10 min, and the supernatant was stored at −70°C. The Institutional Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital approved this study (SCHBC-2016-012).

A portion of each left lung was fixed in 4% (v/v) buffered paraformaldehyde and embedded in paraffin. The tissue was cut into sections 5-μm thick and stained with H&E or Masson trichrome. The right lung was snap frozen by immersion in liquid nitrogen and stored at −80°C prior to RNA and protein extraction. Lung sections were stained with H&E for histopathological analysis or with Masson trichrome for the evaluation of collagen content and distribution. The Ashcroft score for the evaluation of lung fibrosis was described previously (20).

ELISA kits were used to measure the concentrations of the active form of TGF-β1 in lung tissue (TGF-β1: no. DY1679; R&D Systems.) according to the manufacturers’ instructions.

Mouse lung tissues were homogenized in 50 mM phosphate buffer (pH 7.4) using a high-speed homogenizer. The supernatants were obtained by centrifugation at 14,000 rpm × g for 30 min at 4°C and immediately stored at −80°C until use. ROS in the supernatant of lung homogenates was determined by a method previously described (21). Briefly, 100 μl of 50 μM 2′,7′-dichlorofluorescein diacetate, 25 μl of homogenate, and 75 μl of 50 mM phosphate buffer (pH 7.4) were mixed in a total reaction volume of 200 μl. Changes in fluorescence intensity were measured at 10, 20, 30, 40, 50, and 60 min (37°C) using a SpectraMax Gemini system (Molecular Devices, Downingtown, PA) with excitation and emission wavelengths of 485 and 530 nm, respectively. ROS levels in the lungs were normalized relative to the respective protein concentration. Malondialdehyde in the supernatant of lung homogenates was assayed using a lipid peroxidation assay kit (no. 118970; Abcam) in accordance with the manufacturer’s instructions.

The sectioned mouse lung samples were placed in Bouin solution at 56°C for 1 h and stained in succession with Mayer hematoxylin solution for 5 min, Biebrich Scarlet-acid fuchsin solution for 10 min, phosphomolybdic acid–phosphotungstic acid for 15 min, and aniline blue for 2 h (staining reagents from Sigma-Aldrich). The sections were examined under a microscope.

To estimate the amount of collagen in the lung, the right lungs were used in a hydroxyproline assay (no. MAK008; Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, the lungs were weighed, homogenized in sterile water, and hydrolyzed in 12 N HCl at 120°C for 3 h. The hydrolyzed samples were incubated with 4-(dimethylamino)benzaldehyde for 90 min at 60°C, and absorbance of oxidized hydroxyproline was determined at 560 nm. The amount of collagen was expressed in micrograms per milligram lung tissue.

Human lung cancer–derived AECs (A549 cells, ATCC CCL185; American Type Culture Collection, Manassas, VA) treated with or without BLM (10 μg/ml) were seeded on six-well plates and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were grown to 80% confluence and transfected with small interfering RNA (siRNA) using Lipofectamine RNAiMAX (Invitrogen, Life Technologies, Carlsbad, CA) in serum-free Opti-MEM. Custom-designed siRNAs (OriGene Technologies, Rockville, MD) targeting human IL-37 (27-mer siRNA duplexes, each at 2 nM; SR309020) were transfected into A549 cells. A 3-μl aliquot of Lipofectamine was incubated in 500 μl Opti-MEM for 45 min at room temperature. siRNA was added to the Opti-MEM–Lipofectamine solution at a final concentration of 100 nM. The mixture was incubated for 15 min under the same conditions; Opti-MEM was added to the final volume of 1 ml, and the mixture was added to the six-well plates. The transfection solution was removed from the cells and replaced with standard medium after 6 h. The siRNA antisense sequence used was 5′-GGUAUGGAGCAUCAACCUG ACAGCT-3′. Knockdown efficacy was evaluated using an IL-37 immunoblot.

Apoptotic cells in paraffin-embedded lung tissues were labeled using a TUNEL assay kit (Roche). The numbers of TUNEL-positive (apoptotic) cells in three sections per mouse were counted under a fluorescence microscope at ×400 (Carl Zeiss Microsystems, Thornwood, NY) as described previously (22). AECs were exposed to vehicle or BLM (10 μg/ml) with IL-37 for 24 h after overnight serum starvation. The IL-37 treatment dose was increased to evaluate the dose–response relationship. The supplemented doses of IL-37 were 0, 10, 100, and 1000 ng/ml, respectively. An Annexin V FITC/propidium iodide (PI) detection kit (BD Biosciences Pharmingen, San Diego, CA) was used to determine the proportions of apoptotic and necrotic cells. Aliquots of ∼1 × 106 cells/ml were washed in PBS, surface stained, resuspended in binding buffer, incubated with FITC-conjugated annexin V and PI for 15 min in the dark at room temperature, washed, and resuspended in the binding buffer.

Primary fibroblasts obtained from patients with IPF were maintained in RPMI 1640 medium with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. To induce cell proliferation, the cells were stimulated for 48 h in medium with 0.5% FBS and TGF-β1 (5 ng/ml; R&D Systems). To evaluate the effect of IL-37, the culture medium was replaced with medium containing 0.5% FBS supplemented with TGF-β1 (5 ng/ml) and IL-37 at doses of 0, 10, 100, and 1000 ng/ml. The cells were incubated for 48 h. Control cells were maintained in medium with 0.5% FBS for 48 h. The cells were subjected to assay on day 3. The changes in cell proliferation were evaluated by phase-contrast microscopy (Carl Zeiss Microsystems).

Cell proliferation was evaluated in triplicate using water-soluble tetrazolium salt (WST)-1 (Roche) transformed into formazan by mitochondrial dehydrogenases. The cells were seeded in 96-well plates and incubated for 48 h. The cells were then stimulated with TGF-β1 (5 ng/ml) plus IL-37 (0, 10, 100, and 1000 ng/ml), and assays were performed by adding WST-1 directly to the wells and incubating them for 60 min at 37°C. The plates were then read by a scanning multiwell spectrophotometer by measuring the absorbance of the dye at a wavelength of 450 nm, with a reference wavelength of 630 nm.

RT-PCR was used to measure the expression of IL-37, collagen I, and fibronectin. Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific) and treated with DNase I (10,000 U/ml; Stratagene, La Jolla, CA) to remove any contaminating DNA. cDNA was synthesized from 3 μg total RNA using SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY) in a 20-μl reaction including 0.5 mM dNTPs, 2.5 mM MgCl2, 5 mM DTT, random hexamers (50 μg/μl), and SuperScript RT (200 U/μl) at 42°C for 50 min, followed by heat inactivation at 70°C for 15 min. The PCR conditions were as follows: denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 15 s and 60°C for 1 min. The following primers were used for amplification: IL-37 sense: 5′-CTG GCT GCC CAA AAG GAA TCA-3′; IL-37 antisense: 5′-CAC TGG GGC TCA TTT CAG CTT-3′; collagen I sense: 5′-CGG AGG AGA GTC AGG AAG G-3′; collagen I antisense: 5′-CAC AAG GAA CAG AAC AGA ACA G-3′; fibronectin sense: 5′-TCC ACA AGC GTC ATG AAG AG-3′; fibronectin antisense: 5′-CTC TGA ATC CTG GCA TTG GT-3′; GAPDH sense: 5′-AGA AGG CTG GGG CTC ATT TG-3′; and GAPDH antisense: 5′-AGG GGC CAT CCA CAG TCT TC-3′. Real-time PCR was performed using SYBR Green PCR master mix (no. 4367659; Thermo Fisher Scientific). Quantification of cDNA targets was performed using an ABI Prism 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA). Primer pairs are the same as listed above. Optimal reaction conditions for amplification of the target genes were set according to the manufacturer’s recommendations. Phosphoglycerate kinase 1 and peptidyl-prolyl isomerase A were used as an internal control. Each experiment was performed in duplicate and repeated independently at least three times.

Standard protocols for immunofluorescence microscopy were used as described previously (23). Human primary fibroblasts were plated on cover glass-bottom dishes and treated with or without the indicated agents. The cells on the dishes were washed two times, fixed in 4% paraformaldehyde for 1 h, and washed three times after fixing. The cells on the dishes were prepared and stained with the indicated primary Abs overnight at 4°C. The slides were washed twice, incubated with chrome-labeled secondary Ab (1:200) for 30 min, and washed three times after staining. Images were obtained with a confocal microscope (Zeiss LSM 510 META). The autophagosomes were identified as LC3B dots, and the autophagolysosomes were identified by coexpression of LC3B and LAMP-1.

All data are expressed as mean ± SE. The data were analyzed using the Kruskal–Wallis test, followed by the Mann–Whitney U test (with Bonferroni correction for intergroup comparison). The p values <0.05 were considered to be significant.

Lung tissue samples were obtained from patients with IPF (n = 20) and control patients (n = 16). There was no significant difference in age, sex, or smoking history between groups (Table I). However, the IPF group had significantly lower levels of the percentage of predicted forced vital capacity, DlCO, and arterial partial oxygen pressure levels compared with the control group (Table I).

Table I.
Demographic characterizations of study subjects
IPF (n = 20)NS (n = 16)
Age (y)a 63.1 (52–70) 57.6 (428–472) 
Male/Female (n15/5 12/4 
Smoking   
 SM (n
 ES (n10 
 NS (n
FVC (%)a 68.4* (55–94) 84.6 (76–101) 
FEV1 (%)a 78 (65–101) 102 (82–114) 
FEV1/FVC (%)a 80.6 (74–92) 80.9 (72–89) 
DLCO (%)a 64* (43–76) 90 (78–102) 
PaO2 (mmHg) 66* (56–94) 89 (72–102) 
BALF   
 Total cells, 105/ml 109.85* (16–142. 6) 24.4 (6.2–38.6) 
 Macrophage, % 75.3 (69.5–84.2) 88.7 (65.8–91.2) 
 Neutrophils, % 17.4 (1.8–25.4) 4.2 (0.4–7.6) 
 Eosinophils, % 1.2 (0.6–4.1) 0.4 (0–2.9) 
 Lymphocytes, % 3.8 (1.4–5.6) 3.8 (1.1–6.2) 
 Epithelial cells, % 2.6 (0–4.2) 2.5 (0–12) 
IPF (n = 20)NS (n = 16)
Age (y)a 63.1 (52–70) 57.6 (428–472) 
Male/Female (n15/5 12/4 
Smoking   
 SM (n
 ES (n10 
 NS (n
FVC (%)a 68.4* (55–94) 84.6 (76–101) 
FEV1 (%)a 78 (65–101) 102 (82–114) 
FEV1/FVC (%)a 80.6 (74–92) 80.9 (72–89) 
DLCO (%)a 64* (43–76) 90 (78–102) 
PaO2 (mmHg) 66* (56–94) 89 (72–102) 
BALF   
 Total cells, 105/ml 109.85* (16–142. 6) 24.4 (6.2–38.6) 
 Macrophage, % 75.3 (69.5–84.2) 88.7 (65.8–91.2) 
 Neutrophils, % 17.4 (1.8–25.4) 4.2 (0.4–7.6) 
 Eosinophils, % 1.2 (0.6–4.1) 0.4 (0–2.9) 
 Lymphocytes, % 3.8 (1.4–5.6) 3.8 (1.1–6.2) 
 Epithelial cells, % 2.6 (0–4.2) 2.5 (0–12) 

Data are expressed as median and 25th–75th quartile, n, or percentage except as indicated.

a

Data are presented as median (range).

*

p < 0.05 compared with the normal control.

ES, ex-smoker; NS, never smoker; SM, current smoker.

The levels of IL-37 expression in alveolar epithelium and macrophages in BALF were lower in IPF than in healthy control samples (Fig. 1A). Double-labeled immunofluorescence demonstrated that the majority of IL-37–expressing cells were type II AECs (Supplemental Fig. 1). IL-37 protein levels were significantly lower in the IPF group than in controls (Fig. 1B). IL-37 levels in BALF were significantly lower in the IPF group than in normal controls (Fig. 1C). Serum levels of IL-37 in 40 patients with IPF were significantly lower than those in 20 control subjects (Fig. 1D). In mouse lung and BALF samples, IL-37 was primarily expressed in AECs and macrophages (Fig. 2A), and the level of IL-37 expression was significantly diminished after 21 d of BLM exposure (Fig. 2B). These data demonstrate that IL-37 is constitutively expressed by alveolar macrophages and AECs. The levels of the intracellular and released forms of IL-37 in the lung were decreased in patients with IPF and BLM-treated mice.

FIGURE 1.

IL-37 protein levels were decreased in the lungs of patients with IPF. (A) Immunohistochemical staining of IL-37 in human lungs. IL-37 expression levels were examined in normal subjects (NSs) and in IPF lungs. IL-37 was strongly localized in AECs (inset) and alveolar macrophages (red arrows) in NSs. Original magnification ×40, inset; original magnification ×1000. (B) Lung IL-37 expression was measured by immunoblotting. Densitometry of IL-37 bands. Equal amounts of proteins obtained from lung lysates were subjected to immunoblotting with anti–IL-37 Ab. The blots were stripped and reprobed with anti–β-actin Ab. Lung tissues were obtained from patients with IPF and NSs who underwent thoracic surgery. *p < 0.05 versus NS. BALF (C) and serum (D) levels of IL-37, measured by ELISA. Dashed line represents the detection limit. *p < 0.05 versus NS.

FIGURE 1.

IL-37 protein levels were decreased in the lungs of patients with IPF. (A) Immunohistochemical staining of IL-37 in human lungs. IL-37 expression levels were examined in normal subjects (NSs) and in IPF lungs. IL-37 was strongly localized in AECs (inset) and alveolar macrophages (red arrows) in NSs. Original magnification ×40, inset; original magnification ×1000. (B) Lung IL-37 expression was measured by immunoblotting. Densitometry of IL-37 bands. Equal amounts of proteins obtained from lung lysates were subjected to immunoblotting with anti–IL-37 Ab. The blots were stripped and reprobed with anti–β-actin Ab. Lung tissues were obtained from patients with IPF and NSs who underwent thoracic surgery. *p < 0.05 versus NS. BALF (C) and serum (D) levels of IL-37, measured by ELISA. Dashed line represents the detection limit. *p < 0.05 versus NS.

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FIGURE 2.

IL-37 is expressed in the AECs and macrophages in the mouse lung and are decreased by BLM. (A) Immunohistochemical staining of IL-37 in mouse lungs. IL-37 was strongly localized in AECs (inset) and alveolar macrophages in the control lung. Original magnification ×100, inset, original magnification ×400. (B) IL-37 expression was quantified by immunoblotting and densitometric analysis (n = 6 per group). *p < 0.05 versus control. Equal amounts of proteins obtained from lung lysates were subjected to immunoblotting with anti–IL-37 Ab. The blots were stripped and reprobed with anti–β-actin Ab.

FIGURE 2.

IL-37 is expressed in the AECs and macrophages in the mouse lung and are decreased by BLM. (A) Immunohistochemical staining of IL-37 in mouse lungs. IL-37 was strongly localized in AECs (inset) and alveolar macrophages in the control lung. Original magnification ×100, inset, original magnification ×400. (B) IL-37 expression was quantified by immunoblotting and densitometric analysis (n = 6 per group). *p < 0.05 versus control. Equal amounts of proteins obtained from lung lysates were subjected to immunoblotting with anti–IL-37 Ab. The blots were stripped and reprobed with anti–β-actin Ab.

Close modal

Lung cell apoptosis is critical in the pathogenesis of lung fibrosis (24). To evaluate the effect of IL-37 on cell death, BLM-exposed primary mouse AECs or A549 cells were used. Primary mouse AECs were cultured in the presence of BLM (10 μg/ml) with increasing concentrations of IL-37 for 24 h. IL-37 siRNA-treated A549 cells were also treated with BLM (10 μg/ml), and the proportions of apoptosis and necrotic cells were assessed by flow cytometry. The IL-37 treatment significantly decreased apoptosis and necrosis in a dose-dependent manner (Fig. 3A). As the maximal beneficial effect was similar at 100 and 1000 ng/ml of IL-37, we decided to use 100 ng/ml of IL-37 in subsequent experiments. When we performed siRNA-mediated silencing of IL-37 in A549 cells, the numbers of apoptotic and necrotic cells were dramatically augmented (Fig. 3B). These data indicate that IL-37 plays a protective role against oxidative stress–induced AEC death.

FIGURE 3.

Antiapoptotic effect of IL-37 on BLM-treated AECs. (A) Mouse primary AECs were exposed to BLM (10 μg/ml) and/or human IL-37 (10, 100, and 1000 ng/ml) for 24 h in serum-free medium. *p < 0.05 versus apoptosis or necrosis of the BLM+/IL-37–treated groups. Apoptosis was defined as Annexin V+/both PI+ and PI. Necrosis was defined as PI+/both Annexin V+ and Annexin V. (B) IL-37 siRNA and scrambled siRNA were transfected into A549 cells. BLM (10 μg/ml) was added 48 h post-siRNA transfection, and the samples were analyzed after 24 h. The cells were double stained with Annexin V/PI and analyzed by flow cytometry. *p < 0.05 versus apoptosis or necrosis of the scramble/BLM+-treated groups.

FIGURE 3.

Antiapoptotic effect of IL-37 on BLM-treated AECs. (A) Mouse primary AECs were exposed to BLM (10 μg/ml) and/or human IL-37 (10, 100, and 1000 ng/ml) for 24 h in serum-free medium. *p < 0.05 versus apoptosis or necrosis of the BLM+/IL-37–treated groups. Apoptosis was defined as Annexin V+/both PI+ and PI. Necrosis was defined as PI+/both Annexin V+ and Annexin V. (B) IL-37 siRNA and scrambled siRNA were transfected into A549 cells. BLM (10 μg/ml) was added 48 h post-siRNA transfection, and the samples were analyzed after 24 h. The cells were double stained with Annexin V/PI and analyzed by flow cytometry. *p < 0.05 versus apoptosis or necrosis of the scramble/BLM+-treated groups.

Close modal

We examined the effect of IL-37 on primary lung fibroblasts isolated from patients with IPF. The cells were treated with IL-37 (100 ng/ml) for up to 48 h. IL-37 reduced basal levels of the transcripts of fibronectin and collagen I, which are major ECM components, as determined by real-time PCR (Fig. 4A). These changes in protein paralleled the changes in mRNA levels determined by immunoblotting (Fig. 4B). We measured PI3K/AKT and p38 MAPK–ERK levels in fibroblasts to examine the possible underlying mechanisms. Both PI3K/AKT and p38 MAPK–ERK pathways are known signaling pathways regulating the activation of ECM molecules in fibroblasts (25, 26). IL-37 significantly inhibited phosphorylation from PI3K/AKT, ERK 1/2, and p38 MAPK in fibroblasts (Fig. 4C). After treatment with the specific inhibitors of Akt (Akt-1/2 Viii; 10 μM), ERK (PD98059; 20 μM), and p38 MAPK (SB203580; 10 μM), collagen I expression was reduced in the fibroblasts (Fig. 4D). These results suggest that IL-37 reduced fibrogenic activities of fibroblasts by decreasing the levels of constitutive main ECM protein expression, which was at least partly associated with the downregulation of PI3K/AKT and p38 MAPK–ERK signaling.

FIGURE 4.

IL-37 attenuated constitutive expression of the ECM molecules fibronectin and collagen, but not α-SMA, in primary IPF fibroblasts. IL-37 also inhibited the expression of PI3K/AKT, ERK, and MAPK signaling molecules in IPF fibroblasts. IPF fibroblasts were treated with or without IL-37 (100 ng/ml) for 48 h. (A) The cells were lysed, and RNA was extracted. RT-PCR was performed to assess mRNA levels of fibronectin, collagen I, and α-SMA shown as gel images. Each mRNA was quantified by real-time PCR, and the results are shown as graphs. (B) Fibronectin and collagen I expression were quantified by immunoblotting and densitometric analysis (n = 4 per group). (C) AKT-PI3K-mTOR and ERK/MAPK pathway proteins were quantified by immunoblotting and densitometric analysis. (D) IPF fibroblasts were treated with AKT, ERK, and MAPK inhibitors for 48 h. Expression of collagen was quantified by immunoblotting and densitometric analysis. Error bars represent mean ± SEM (n = 4). *p < 0.05 versus vehicle treatment.

FIGURE 4.

IL-37 attenuated constitutive expression of the ECM molecules fibronectin and collagen, but not α-SMA, in primary IPF fibroblasts. IL-37 also inhibited the expression of PI3K/AKT, ERK, and MAPK signaling molecules in IPF fibroblasts. IPF fibroblasts were treated with or without IL-37 (100 ng/ml) for 48 h. (A) The cells were lysed, and RNA was extracted. RT-PCR was performed to assess mRNA levels of fibronectin, collagen I, and α-SMA shown as gel images. Each mRNA was quantified by real-time PCR, and the results are shown as graphs. (B) Fibronectin and collagen I expression were quantified by immunoblotting and densitometric analysis (n = 4 per group). (C) AKT-PI3K-mTOR and ERK/MAPK pathway proteins were quantified by immunoblotting and densitometric analysis. (D) IPF fibroblasts were treated with AKT, ERK, and MAPK inhibitors for 48 h. Expression of collagen was quantified by immunoblotting and densitometric analysis. Error bars represent mean ± SEM (n = 4). *p < 0.05 versus vehicle treatment.

Close modal

Primary lung fibroblasts obtained from patients with IPF were treated with various doses of IL-37 (10–1000 ng/ml) and TGF-β1 (5 ng/ml) for 48 h. IL-37 significantly inhibited TGF-β1–induced increased cell proliferation in a dose-dependent manner, as determined by the WST-1 assay (Fig. 5A). We tested whether IL-37 inhibited TGF-β1–induced myofibroblast differentiation and the production of ECM proteins in IPF fibroblasts to further explore the mechanisms of IL-37–mediated inhibition of TGF-β1–induced fibroblast proliferation. The TGF-β1–induced increases in collagen I and fibronectin expression were inhibited by IL-37 in a dose-dependent manner (Fig. 5B). However, expression of the myofibroblast differentiation marker gene, α-smooth muscle actin (α-SMA), was not diminished by IL-37 (Fig. 5B). These data suggest that IL-37 suppressed TGF-β1–induced cell proliferation but not myofibroblast differentiation. Next, we determined whether IL-37 interfered with the TGF-β1 signaling pathway. We examined Smad-dependent and non-Smad signaling pathways. We observed that IL-37 (100 ng/ml) inhibited the TGF-β1–induced increase in Smad2 and Smad3 phosphorylation in fibroblasts (Fig. 5C). TGF-β1 also induced non-Smad responses, including ERK- and p38 MAPK-mediated signaling (27). IL-37 also reduced phosphorylation of ERK and p38 MAPK induced by TGF-β1 (Fig. 5D). IL-37 appeared to downregulate Smad-dependent and non-Smad signaling pathways induced by TGF-β1. TGF-β1 signaling is initiated by the binding of TGF-β1 to TβR I and TβR II on the cell membrane (28). Increased tyrosine phosphorylation of TβR II was downregulated by IL-37 after treatment with TGF-β1 (Fig. 5E). These results suggest that IL-37 mediates an inhibitory effect on TβR II phosphorylation in IPF fibroblasts.

FIGURE 5.

IL-37 diminish TGF-β1–induced fibroblast proliferation and the downregulation of TGF-β1 signaling pathway. Primary cultured IPF fibroblasts were starved of serum overnight and cotreated with or without TGF-β1 (5 ng/ml) and IL-37 (10, 100, and 1000 ng/ml) for 48 h. (A) Proliferation of fibroblasts was estimated by WST-1 assay. Error bars represent mean ± SEM of four different primary fibroblast cultures, each assayed in quadruplicate. (B) Collagen I, fibronectin, and α-SMA expression were examined by immunoblotting. (CE) The fibroblasts were treated with TGF-β1 (5 ng/ml) with or without IL-37 (100 ng/ml) for 24 h. TGF-β1–induced Smad (C) and non-Smad (D) signaling and TGF-β1R signaling (E) were evaluated by immunoblotting. *p < 0.05 versus TGF-β1+/vehicle+ group.

FIGURE 5.

IL-37 diminish TGF-β1–induced fibroblast proliferation and the downregulation of TGF-β1 signaling pathway. Primary cultured IPF fibroblasts were starved of serum overnight and cotreated with or without TGF-β1 (5 ng/ml) and IL-37 (10, 100, and 1000 ng/ml) for 48 h. (A) Proliferation of fibroblasts was estimated by WST-1 assay. Error bars represent mean ± SEM of four different primary fibroblast cultures, each assayed in quadruplicate. (B) Collagen I, fibronectin, and α-SMA expression were examined by immunoblotting. (CE) The fibroblasts were treated with TGF-β1 (5 ng/ml) with or without IL-37 (100 ng/ml) for 24 h. TGF-β1–induced Smad (C) and non-Smad (D) signaling and TGF-β1R signaling (E) were evaluated by immunoblotting. *p < 0.05 versus TGF-β1+/vehicle+ group.

Close modal

Lung fibroblasts are the principal components of the interstitium, which are the main producers of the ECM, and proliferation of (myo)fibroblasts is the main pathological finding in IPF (29). Recent reports have implicated a defective fibroblast autophagic process in the pathogenesis of IPF (30, 31). We investigated whether IL-37 regulated autophagic flux in fibroblasts. First, we observed an effect of IL-37 (100 ng/ml) that resulted in the formation of cytoplasmic vacuoles after 6 h of treatment, suggesting the possible involvement of autophagy (Fig. 6A). Conversion of LC3-I to LC3-II is recognized as a hallmark of autophagy (32). We evaluated LC3B I/II expression in IPF fibroblasts by immunofluorescence and immunoblotting. IL-37 increased LC3B-expressing vacuoles of fibroblasts, according to results from immunofluorescence microscopy (Fig. 6B). Immunoblotting revealed an increase in the LC3B-II/I ratio in fibroblasts (Fig. 6C). ATG7 and beclin-1 are key autophagy proteins in the canonical autophagy pathway (33). IL-37 enhanced ATG7 and beclin-1 expression in lung fibroblasts (Fig. 6C). These data suggest that IL-37 activates autophagy via a canonical pathway.

FIGURE 6.

IL-37 enhanced autophagy in IPF fibroblasts. (A) IL-37 induced vacuolar changes in fibroblasts. IPF fibroblasts were treated with IL-37 (100 ng/ml) for 6 h, and light microscopy was performed. Original magnification ×400, inset; original magnification ×1000. Scale bar, 50 μm. (B) Laser confocal microscopy was performed to detect LC3B and LAMP-1 by indirect immunofluorescence. Scale bar, 10 μm. (C) LC3B, ATG7, and beclin-1 expression were evaluated by immunoblotting. Densitometric analysis of independent experiments was performed. Error bars represent mean ± SEM (n = 4). *p < 0.05 versus IL-37 group.

FIGURE 6.

IL-37 enhanced autophagy in IPF fibroblasts. (A) IL-37 induced vacuolar changes in fibroblasts. IPF fibroblasts were treated with IL-37 (100 ng/ml) for 6 h, and light microscopy was performed. Original magnification ×400, inset; original magnification ×1000. Scale bar, 50 μm. (B) Laser confocal microscopy was performed to detect LC3B and LAMP-1 by indirect immunofluorescence. Scale bar, 10 μm. (C) LC3B, ATG7, and beclin-1 expression were evaluated by immunoblotting. Densitometric analysis of independent experiments was performed. Error bars represent mean ± SEM (n = 4). *p < 0.05 versus IL-37 group.

Close modal

BLM, which is a potent fibrosis inducer, causes oxidative lung damage. To determine whether IL-37 has an antioxidative effect, the ROS levels in the mouse lung were measured. As expected, ROS levels were significantly elevated in the BLM-treated mice, and IL-37 markedly suppressed ROS generation (Supplemental Fig. 2A). To quantify the oxidative injury in the lung, we measured lung lipid peroxidation. BLM treatment led to a significant increase in lipid peroxidation in the control mice. However, IL-37 treatment showed no increase in lung lipid peroxidation in response to BLM (Supplemental Fig. 2B). Next, we examined whether IL-37 diminished BLM-induced lung cell apoptosis. Apoptosis of lung cells was assessed by TUNEL assay, which also evaluated cleaved caspase-3 expression. The increases in the number of TUNEL-positive cells and the expression of cleaved caspase-3 by BLM were significantly decreased by IL-37 (Supplemental Fig. 3A, 3B). These observations indicated that IL-37 protected against BLM-induced oxidative damage and cell death in the mouse lung.

Next, we determined whether IL-37 had antifibrotic activity in the BLM-induced lung fibrosis model. First, we confirmed the development of lung fibrosis after BLM treatment on day 10 (data not shown). IL-37 (500 ng/ml) was administered intranasally on days 11–13, and lung samples were evaluated on day 21. H&E staining showed that IL-37 efficiently reversed BLM-induced lung structural distortion (Fig. 7A). In line with the H&E staining results, BALF analysis showed that IL-37 significantly attenuated lung inflammation and reduced the total numbers of macrophages, neutrophils, and lymphocytes (Fig. 7B). Furthermore, IL-37 treatment clearly reduced lung fibrosis caused by collagen deposition, as shown by Masson trichrome staining and Ashcroft scoring (Fig. 7C, 7D). The hydroxyproline assay further confirmed the antifibrotic activity of IL-37 during the BLM treatment (Fig. 7E). Active form of TGF-β1 increased significantly in the lung following exposure to BLM but decreased significantly after IL-37 treatment (Fig. 7F).

FIGURE 7.

IL-37 attenuated BLM-induced lung inflammation and fibrosis in a mouse model. (A) Photographs of H&E staining in control and BLM-treated mouse lungs treated with IL-37 (500 ng/mouse/day) intranasally on days 11–13. Lung samples were collected on day 21. Original magnification ×100. (B) Cell counts in BALF, collected on day 21. The total number of cells was counted using a hemocytometer. Differential cell counts in BALF were analyzed from 500 cells stained with Diff-Quik (n = 8 per group). (C) Masson trichrome staining. Original magnification ×200. (D) Quantification of lung fibrosis by the Ashcroft score (n = 8 per group). Data are expressed as mean ± SEM. (E) Collagen measurement from the hydroxyproline assays of control and BLM-treated mouse lungs, with and without IL-37 treatment (n = 8 per group). (F) Active TGF-β1 levels in control and BLM-treated mouse lungs, with and without IL-37 treatment (n = 8 per group). TGF-β1 levels in lung lysates were measured by ELISA. *p < 0.05 versus BLM-treated.

FIGURE 7.

IL-37 attenuated BLM-induced lung inflammation and fibrosis in a mouse model. (A) Photographs of H&E staining in control and BLM-treated mouse lungs treated with IL-37 (500 ng/mouse/day) intranasally on days 11–13. Lung samples were collected on day 21. Original magnification ×100. (B) Cell counts in BALF, collected on day 21. The total number of cells was counted using a hemocytometer. Differential cell counts in BALF were analyzed from 500 cells stained with Diff-Quik (n = 8 per group). (C) Masson trichrome staining. Original magnification ×200. (D) Quantification of lung fibrosis by the Ashcroft score (n = 8 per group). Data are expressed as mean ± SEM. (E) Collagen measurement from the hydroxyproline assays of control and BLM-treated mouse lungs, with and without IL-37 treatment (n = 8 per group). (F) Active TGF-β1 levels in control and BLM-treated mouse lungs, with and without IL-37 treatment (n = 8 per group). TGF-β1 levels in lung lysates were measured by ELISA. *p < 0.05 versus BLM-treated.

Close modal

As IL-37 diminished lung fibrosis and activated autophagy in vitro, we evaluated whether autophagy inhibition reversed the therapeutic effect of IL-37 in BLM-induced lung fibrosis. The autophagy inhibitor 3-MA was administered 2 h before the IL-37 treatment in BLM-treated mice. First, the effect of 3-MA on BLM treatment was evaluated in the lungs of mice. Treatment with 3-MA tended to increase inflammation and lung destruction compared with mice treated with BLM alone (Supplemental Fig. 4A, 4B). 3-MA inhibited the IL-37–mediated decrease in lung destruction and reduction of collagen deposition by BLM (Fig. 8A–D). The LC3 I/II ratio, a characteristic marker of autophagy activation, increased significantly in response to IL-37 in the lung (Fig. 8E). IL-37 enhanced expression of ATG7 and beclin-1; however, p-mTOR expression decreased (Fig. 8E). 3-MA inhibited the expression of autophagy-related proteins in the lung (Fig. 8E). These findings demonstrate that IL-37 plays a role in antifibrotic activity and that this effect is related to the activation of autophagy in the fibrotic lung.

FIGURE 8.

Inhibition of autophagy reversed the therapeutic effects of IL-37 in experimental lung fibrosis. Mice were treated with IL-37 (500 ng/mouse/day) intranasally on days 11–13 after BLM exposure. Some mice were given 3-methyadenine (3-MA; 30 mg/kg/day, i.p.) 2 h before the IL-37 treatment. The mice were killed on day 21 after BLM administration. (A) H&E-stained image; original magnification ×100. (B) Masson trichrome staining. Original magnification ×200. (C) Quantification of lung fibrosis by the Ashcroft score. Data are expressed as mean ± SEM (n = 6 per group). (D) Collagen levels from lung lysates, measured with a hydroxyproline assay kit. (E) The expression of autophagy-related proteins from lung lysates, including LC3 I/II, ATG7, beclin-1, mTOR, and p-mTOR, detected by immunoblotting. Densitometric analysis of the experiments (n = 6 per group). Error bars represent mean ± SEM. *p < 0.05 versus BLM+ or BLM+/IL-37+/3-MA+.

FIGURE 8.

Inhibition of autophagy reversed the therapeutic effects of IL-37 in experimental lung fibrosis. Mice were treated with IL-37 (500 ng/mouse/day) intranasally on days 11–13 after BLM exposure. Some mice were given 3-methyadenine (3-MA; 30 mg/kg/day, i.p.) 2 h before the IL-37 treatment. The mice were killed on day 21 after BLM administration. (A) H&E-stained image; original magnification ×100. (B) Masson trichrome staining. Original magnification ×200. (C) Quantification of lung fibrosis by the Ashcroft score. Data are expressed as mean ± SEM (n = 6 per group). (D) Collagen levels from lung lysates, measured with a hydroxyproline assay kit. (E) The expression of autophagy-related proteins from lung lysates, including LC3 I/II, ATG7, beclin-1, mTOR, and p-mTOR, detected by immunoblotting. Densitometric analysis of the experiments (n = 6 per group). Error bars represent mean ± SEM. *p < 0.05 versus BLM+ or BLM+/IL-37+/3-MA+.

Close modal

In this study, we showed that IL-37 levels decreased substantially in the lungs of patients with IPF and in the lungs of BLM-exposed mice. Treatment with IL-37 inhibited BLM-induced increased oxidative stress and apoptotic cell death, which are parts of the main pathophysiology of IPF. Our findings indicate that IL-37 protected against BLM-induced lung inflammation and fibrosis. These beneficial effects were associated with suppressed upregulation of neutrophilic inflammation and antiapoptotic activity of IL-37 that attenuate AEC death in the BLM-exposed lung.

In addition, IL-37 had antifibrotic activities by 1) suppressing TGF-β1 receptor signaling, 2) inhibiting fibronectin and collagen I production, and 3) inducing canonical autophagy in cultured primary lung fibroblasts and in vivo. This study is the first, to our knowledge, to report on the role of IL-37 in IPF lung and the therapeutic effect of IL-37 on BLM-mediated experimental lung fibrosis and to elucidate the possible mechanism.

TGF-β1 signaling is essential to fibrosis and involves multiple organs, including the lungs (34). In this study, the IL-37 treatment attenuated TGF-β1–induced Smad2 and Smad3 phosphorylation in primary lung fibroblasts. Moreover, IL-37 inhibited non-Smad signaling, particularly the ERK and MAPK pathways. As Smad-dependent and non-Smad pathways were inhibited by IL-37, we evaluated whether IL-37 could modulate TGF-β receptor expression. The TGF-β receptor is a heterodimer of TβRI and TβRII receptors; the ligand binds the TβRII, which activates the TβRI serine-threonine kinase and, in turn, phosphorylates serine residues in receptor-activated SMADs (35). Recent reports have demonstrated that phosphorylation of the TβRII tail tyrosine residues regulates the activation of receptors and their downstream signaling (36, 37). Rojas et al. (38) reported that TGF-β receptor levels regulate the specificity of signaling pathway activation and the biological effects of TGF-β1 and, in particular, that TβRII expression levels are correlated with Smad signaling and MAPK–ERK signaling. We showed that IL-37 inhibits TβRII phosphorylation induced by TGF-β1. The exact molecular mechanism by which IL-37 modulates TβRII dephosphorylation remains unknown.

We also observed that IL-37 suppressed mRNA and protein expression of the ECM components fibronectin and collagen I in lung fibroblasts (Fig. 4A, 4B). This activity appears to be independent of TGF-β1 signaling based on downregulated constitutive expression of ECM proteins. In our study, IL-37 inhibited the PI3K/AKT and ERK and MAPK signaling pathway, which regulates cellular proliferation and metabolism (Fig. 4C). Inhibition of the PI3K pathway is known to reduce the basal expression of fibronectin and collagen I in fibroblasts (39). ERK and MAPK signaling also has been shown to control cellular processes associated with fibrosis, including cell growth, proliferation, migration, protection from apoptosis, and myofibroblast transformation (40, 41). Interestingly, we also found that IL-37 did not downregulate α-SMA transcripts, key molecules involved in myofibroblast differentiation (Fig. 4A). These data suggest that IL-37 primarily inhibits the proliferative, but not the differentiation, signaling pathway in IPF fibroblasts. The PI3K/AKT/mTOR signaling pathway regulates cell growth, apoptosis, and other important cellular signaling activities (42, 43). Mercer et al. (44) reported that the PI3K pathway was activated in fibroblastic foci, which are cardinal lesions in IPF, and that a PI3Kinase/mTOR inhibitor decreased profibrotic responses to TGF-β1 in primary human lung fibroblasts, suggesting that the regulation of the PI3K/mTOR pathway by IL-37 may be a promising therapeutic target in IPF.

Autophagy is a fundamental homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. Autophagy is activated in response to intracellular or extracellular factors, such as hypoxia, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection (45, 46). Autophagy is an intrinsic cellular defense mechanism in the innate and adaptive immune systems (47, 48). As cytokines are crucial for innate and adaptive immune responses, examination of interactions between cytokines and autophagy could elucidate a novel mechanism for control of the immune response (49, 50). However, how IL-37 regulates autophagy, especially in lung fibrosis, is not known. In the current study, IL-37 enhanced the formation of autophagosomes by activating the expression of key autophagic molecules, such as LC3-II, beclin-1, and ATG7, in IPF fibroblasts and BLM-induced fibrotic lung tissues (Figs. 6, 8E). We found that IL-37 suppressed mTOR, which is a downstream mediator of PI3K/AKT signaling that inhibits macroautophagy (51) and is part of an ATG7-dependent canonical pathway (Fig. 8E) (33). One study demonstrated impaired autophagy in lung tissues from patients with IPF, measured by p62 accumulation, as well as decreased LC-II expression (31). However, whether defective autophagy is a causal factor or an epiphenomenon that accompanies the aging process in patients with IPF has not been elucidated clearly (52, 53). Decreased clearance of ECM proteins by autophagy leads to their accumulation, contributing to the progression of fibrosis. Thus, the enhancement of autophagy may help to resolve fibrosis.

The inhibition of autophagy with 3-MA reversed the antifibrotic effects of IL-37 (Fig. 8A–D). These data indicate that increased autophagy contributed mainly to the antifibrotic effect on IL-37 in the BLM-treated lung. In addition to autophagy, the blockage of TGF-β1 signaling and the inhibition of PI3K/AKT signaling contributed to the antifibrotic effect of IL-37.

One study demonstrated that IL-37 suppresses the production of proinflammatory cytokines, such as IL-1β, TNF-α, and IL-6, from LPS-stimulated monocytes (54). IL-37 also dampened Th1, Th2, and Th17 effector responses in animal models of asthma and rheumatoid arthritis (55, 56). IL-37 exerts anti-inflammatory effects under intracellular and extracellular conditions. After synthesis, one third of the IL-37 precursor is cleaved intracellularly and translocated to the nucleus (7). IL-37 interacts with Smad3 during this process, suggesting that Smad3 mediates the inhibition of inflammatory cytokine production (7, 57). Extracellular IL-37 binds to IL-18Rα and recruits the orphan decoy.

IL-1R8 leads to augmentation of the anti-inflammatory pathway instead of activation of the IL-18 pathway (58, 59). That is, IL-37 binds to the IL-18 binding protein, which is a natural antagonist of IL-18 (60). Therefore, we speculate that IL-37 may related to the downregulation of PI3K/AKT signaling via IL-18 inactivation by binding as IL-18 is a known activator of PI3K/AKT signaling (61). Two studies have shown that the anti-inflammatory activity of IL-37 requires the IL-18Ra and TIR-8 receptors in allergic airway disease and sepsis models (55, 62). Kitasato et al. (63) reported that IL-18 and IL-18Ra are strongly expressed in bronchoalveolar epithelium and alveolar macrophages of patients with IPF, whereas IL-18Ra is strongly expressed in interstitial cells, especially in fibroblastic foci. However, whether IL-18 has profibrotic activity in patients with IPF is unclear. Further studies using IL-18Ra and TIR8 knockout mice are needed to evaluate the contribution of this interaction in lung fibrosis.

Some limitations of this study should be discussed. We revealed two different mechanisms for the regulation of the fibrotic response: TGF-β1 receptor signaling and the recovery of impaired autophagic activity by IL-37. However, we did not elucidate whether these mechanisms interact or occur independently. Moreover, distinguishing the main mechanism for the antifibrotic effect of IL-37 is difficult.

In summary, the current study demonstrated that IL-37 expression was decreased in IPF lungs and that treatment with IL-37 protected against BLM-induced experimental lung injury/fibrosis. The mechanisms of the IL-37 antifibrotic activity were the inhibition of TGF-β1 signaling and enhancement of autophagy. IL-37 may represent a novel therapeutic strategy for fibrotic lung diseases, including IPF.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by Ministry of Science, Information and Communication Technologies and Future Planning NRF-2016R1E1A1A01943481 and NRF 2019R1A2C1006351.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AEC

alveolar epithelial cell

ATG7

autophagy-related gene 7

BAL

bronchoalveolar lavage

BALF

BAL fluid

BLM

bleomycin

ECM

extracellular matrix

IPF

idiopathic pulmonary fibrosis

LC

L chain

3-MA

3-methyladenine

PI

propidium iodide

ROS

reactive oxygen species

siRNA

small interfering RNA

α-SMA

α-smooth muscle actin

WST

water-soluble tetrazolium salt.

1
Raghu
,
G.
,
H. R.
Collard
,
J. J.
Egan
,
F. J.
Martinez
,
J.
Behr
,
K. K.
Brown
,
T. V.
Colby
,
J. F.
Cordier
,
K. R.
Flaherty
,
J. A.
Lasky
, et al
ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis
.
2011
.
An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.
Am. J. Respir. Crit. Care Med.
183
:
788
824
.
2
Wolters
,
P. J.
,
H. R.
Collard
,
K. D.
Jones
.
2014
.
Pathogenesis of idiopathic pulmonary fibrosis.
Annu. Rev. Pathol.
9
:
157
179
.
3
Wynn
,
T. A.
,
T. R.
Ramalingam
.
2012
.
Mechanisms of fibrosis: therapeutic translation for fibrotic disease.
Nat. Med.
18
:
1028
1040
.
4
Keane
,
M. P.
,
R. M.
Strieter
.
2002
.
The importance of balanced pro-inflammatory and anti-inflammatory mechanisms in diffuse lung disease.
Respir. Res.
3
:
5
.
5
Belperio
,
J. A.
,
M.
Dy
,
L.
Murray
,
M. D.
Burdick
,
Y. Y.
Xue
,
R. M.
Strieter
,
M. P.
Keane
.
2004
.
The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis.
J. Immunol.
173
:
4692
4698
.
6
Keane
,
M. P.
2008
.
The role of chemokines and cytokines in lung fibrosis.
Eur. Respir. Rev.
17
:
151
156
.
7
Nold
,
M. F.
,
C. A.
Nold-Petry
,
J. A.
Zepp
,
B. E.
Palmer
,
P.
Bufler
,
C. A.
Dinarello
.
2010
.
IL-37 is a fundamental inhibitor of innate immunity.
Nat. Immunol.
11
:
1014
1022
.
8
Kumar
,
S.
,
C. R.
Hanning
,
M. R.
Brigham-Burke
,
D. J.
Rieman
,
R.
Lehr
,
S.
Khandekar
,
R. B.
Kirkpatrick
,
G. F.
Scott
,
J. C.
Lee
,
F. J.
Lynch
, et al
.
2002
.
Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production.
Cytokine
18
:
61
71
.
9
Sharma
,
S.
,
N.
Kulk
,
M. F.
Nold
,
R.
Gräf
,
S. H.
Kim
,
D.
Reinhardt
,
C. A.
Dinarello
,
P.
Bufler
.
2008
.
The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines.
J. Immunol.
180
:
5477
5482
.
10
Boraschi
,
D.
,
D.
Lucchesi
,
S.
Hainzl
,
M.
Leitner
,
E.
Maier
,
D.
Mangelberger
,
G. J.
Oostingh
,
T.
Pfaller
,
C.
Pixner
,
G.
Posselt
, et al
.
2011
.
IL-37: a new anti-inflammatory cytokine of the IL-1 family.
Eur. Cytokine Netw.
22
:
127
147
.
11
Luo
,
Y.
,
X.
Cai
,
S.
Liu
,
S.
Wang
,
C. A.
Nold-Petry
,
M. F.
Nold
,
P.
Bufler
,
D.
Norris
,
C. A.
Dinarello
,
M.
Fujita
.
2014
.
Suppression of antigen-specific adaptive immunity by IL-37 via induction of tolerogenic dendritic cells.
Proc. Natl. Acad. Sci. USA
111
:
15178
15183
.
12
Bulau
,
A. M.
,
M.
Fink
,
C.
Maucksch
,
R.
Kappler
,
D.
Mayr
,
K.
Wagner
,
P.
Bufler
.
2011
.
In vivo expression of interleukin-37 reduces local and systemic inflammation in concanavalin A-induced hepatitis.
ScientificWorldJournal
11
:
2480
2490
.
13
McNamee
,
E. N.
,
J. C.
Masterson
,
P.
Jedlicka
,
M.
McManus
,
A.
Grenz
,
C. B.
Collins
,
M. F.
Nold
,
C.
Nold-Petry
,
P.
Bufler
,
C. A.
Dinarello
,
J.
Rivera-Nieves
.
2011
.
Interleukin 37 expression protects mice from colitis.
Proc. Natl. Acad. Sci. USA
108
:
16711
16716
.
14
Teng
,
X.
,
Z.
Hu
,
X.
Wei
,
Z.
Wang
,
T.
Guan
,
N.
Liu
,
X.
Liu
,
N.
Ye
,
G.
Deng
,
C.
Luo
, et al
.
2014
.
IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production.
J. Immunol.
192
:
1815
1823
15
Jia
,
H.
,
J.
Liu
,
B.
Han
.
2018
.
Reviews of interleukin-37: functions, receptors, and roles in diseases.
Biomed Res. Int.
2018
: 3058640.
16
Corti
,
M.
,
A. R.
Brody
,
J. H.
Harrison
.
1996
.
Isolation and primary culture of murine alveolar type II cells.
Am. J. Respir. Cell Mol. Biol.
14
:
309
315
.
17
Uhal
,
B. D.
,
I.
Joshi
,
A. L.
True
,
S.
Mundle
,
A.
Raza
,
A.
Pardo
,
M.
Selman
.
1995
.
Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.
Am. J. Physiol.
269
:
L819
L828
.
18
Baek
,
A. R.
,
J. M.
Lee
,
H. J.
Seo
,
J. S.
Park
,
J. H.
Lee
,
S. W.
Park
,
A. S.
Jang
,
J.
Kim
,
E. S.
Koh
,
S. T.
Uh
, et al
.
2016
.
Apolipoprotein A1 inhibits TGF-β1-induced epithelial-to-mesenchymal transition of alveolar epithelial cells.
Tuberc. Respir. Dis. (Seoul)
79
:
143
152
.
19
Kim
,
T. H.
,
Y. H.
Lee
,
K. H.
Kim
,
S. H.
Lee
,
J. Y.
Cha
,
E. K.
Shin
,
S.
Jung
,
A. S.
Jang
,
S. W.
Park
,
S. T.
Uh
, et al
.
2010
.
Role of lung apolipoprotein A-I in idiopathic pulmonary fibrosis: antiinflammatory and antifibrotic effect on experimental lung injury and fibrosis.
Am. J. Respir. Crit. Care Med.
182
:
633
642
.
20
Hübner
,
R. H.
,
W.
Gitter
,
N. E.
El Mokhtari
,
M.
Mathiak
,
M.
Both
,
H.
Bolte
,
S.
Freitag-Wolf
,
B.
Bewig
.
2008
.
Standardized quantification of pulmonary fibrosis in histological samples.
Biotechniques
44
:
507
511, 514–517
.
21
Paraidathathu
,
T.
,
H.
de Groot
,
J. P.
Kehrer
.
1992
.
Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.
Free Radic. Biol. Med.
13
:
289
297
.
22
Lee
,
E.
,
E. J.
Lee
,
H.
Kim
,
A.
Jang
,
E.
Koh
,
S. T.
Uh
,
Y.
Kim
,
S. W.
Park
,
C. S.
Park
.
2013
.
Overexpression of apolipoprotein A1 in the lung abrogates fibrosis in experimental silicosis.
PLoS One
8
: e55827.
23
Mi
,
S.
,
Z.
Li
,
H.-Z.
Yang
,
H.
Liu
,
J.-P.
Wang
,
Y.-G.
Ma
,
X.-X.
Wang
,
H.-Z.
Liu
,
W.
Sun
,
Z.-W.
Hu
.
2011
.
Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-β1-dependent and -independent mechanisms.
J. Immunol.
187
:
3003
3014
.
24
Uhal
,
B. D.
2008
.
The role of apoptosis in pulmonary fibrosis.
Eur. Respir. Rev.
17
:
138
144
.
25
Somanath
,
P. R.
,
E. S.
Kandel
,
N.
Hay
,
T. V.
Byzova
.
2007
.
Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly.
J. Biol. Chem.
282
:
22964
22976
.
26
Lim
,
I. J.
,
T. T.
Phan
,
E. K.
Tan
,
T. T.
Nguyen
,
E.
Tran
,
M. T.
Longaker
,
C.
Song
,
S. T.
Lee
,
H. T.
Huynh
.
2003
.
Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids.
J. Biol. Chem.
278
:
40851
40858
.
27
Mu
,
Y.
,
S. K.
Gudey
,
M.
Landström
.
2012
.
Non-Smad signaling pathways.
Cell Tissue Res.
347
:
11
20
.
28
Pomeraniec
,
L.
,
M.
Hector-Greene
,
M.
Ehrlich
,
G. C.
Blobe
,
Y. I.
Henis
.
2015
.
Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.
Mol. Biol. Cell
26
:
3117
3127
.
29
Bjoraker
,
J. A.
,
J. H.
Ryu
,
M. K.
Edwin
,
J. L.
Myers
,
H. D.
Tazelaar
,
D. R.
Schroeder
,
K. P.
Offord
.
1998
.
Prognostic significance of histopathologic subsets in idiopathic pulmonary fibrosis.
Am. J. Respir. Crit. Care Med.
157
:
199
203
.
30
Araya
,
J.
,
J.
Kojima
,
N.
Takasaka
,
S.
Ito
,
S.
Fujii
,
H.
Hara
,
H.
Yanagisawa
,
K.
Kobayashi
,
C.
Tsurushige
,
M.
Kawaishi
, et al
.
2013
.
Insufficient autophagy in idiopathic pulmonary fibrosis.
Am. J. Physiol. Lung Cell. Mol. Physiol.
304
:
L56
L69
.
31
Patel
,
A. S.
,
L.
Lin
,
A.
Geyer
,
J. A.
Haspel
,
C. H.
An
,
J.
Cao
,
I. O.
Rosas
,
D.
Morse
.
2012
.
Autophagy in idiopathic pulmonary fibrosis.
PLoS One
7
: e41394.
32
Kabeya
,
Y.
,
N.
Mizushima
,
T.
Ueno
,
A.
Yamamoto
,
T.
Kirisako
,
T.
Noda
,
E.
Kominami
,
Y.
Ohsumi
,
T.
Yoshimori
.
2000
.
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
EMBO J.
19
:
5720
5728
.
33
Codogno
,
P.
,
M.
Mehrpour
,
T.
Proikas-Cezanne
.
2011
.
Canonical and non-canonical autophagy: variations on a common theme of self-eating?
Nat. Rev. Mol. Cell Biol.
13
:
7
12
.
34
Fernandez
,
I. E.
,
O.
Eickelberg
.
2012
.
The impact of TGF-β on lung fibrosis: from targeting to biomarkers.
Proc. Am. Thorac. Soc.
9
:
111
116
.
35
Kamato
,
D.
,
M. L.
Burch
,
T. J.
Piva
,
H. B.
Rezaei
,
M. A.
Rostam
,
S.
Xu
,
W.
Zheng
,
P. J.
Little
,
N.
Osman
.
2013
.
Transforming growth factor-β signalling: role and consequences of Smad linker region phosphorylation.
Cell. Signal.
25
:
2017
2024
.
36
Chen
,
X.
,
H.
Wang
,
H. J.
Liao
,
W.
Hu
,
L.
Gewin
,
G.
Mernaugh
,
S.
Zhang
,
Z. Y.
Zhang
,
L.
Vega-Montoto
,
R. M.
Vanacore
, et al
.
2014
.
Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling.
J. Clin. Invest.
124
:
3295
3310
.
37
Allison
,
S. J.
2014
.
Fibrosis: regulation of fibrotic signalling by TGF-β receptor tyrosine phosphorylation.
Nat. Rev. Nephrol.
10
:
484
.
38
Rojas
,
A.
,
M.
Padidam
,
D.
Cress
,
W. M.
Grady
.
2009
.
TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta.
Biochim. Biophys. Acta
1793
:
1165
1173
.
39
Fuentes-Calvo
,
I.
,
A. M.
Blázquez-Medela
,
N.
Eleno
,
E.
Santos
,
J. M.
López-Novoa
,
C.
Martínez-Salgado
.
2012
.
H-Ras isoform modulates extracellular matrix synthesis, proliferation, and migration in fibroblasts.
Am. J. Physiol. Cell Physiol.
302
:
C686
C697
.
40
Xu
,
S. W.
,
S. L.
Howat
,
E. A.
Renzoni
,
A.
Holmes
,
J. D.
Pearson
,
M. R.
Dashwood
,
G.
Bou-Gharios
,
C. P.
Denton
,
R. M.
du Bois
,
C. M.
Black
, et al
.
2004
.
Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK.
J. Biol. Chem.
279
:
23098
23103
.
41
Madala
,
S. K.
,
S.
Schmidt
,
C.
Davidson
,
M.
Ikegami
,
S.
Wert
,
W. D.
Hardie
.
2012
.
MEK-ERK pathway modulation ameliorates pulmonary fibrosis associated with epidermal growth factor receptor activation.
Am. J. Respir. Cell Mol. Biol.
46
:
380
388
.
42
Franke
,
T. F.
,
C. P.
Hornik
,
L.
Segev
,
G. A.
Shostak
,
C.
Sugimoto
.
2003
.
PI3K/Akt and apoptosis: size matters.
Oncogene
22
:
8983
8998
.
43
DeBerardinis
,
R. J.
,
J. J.
Lum
,
G.
Hatzivassiliou
,
C. B.
Thompson
.
2008
.
The biology of cancer: metabolic reprogramming fuels cell growth and proliferation.
Cell Metab.
7
:
11
20
.
44
Mercer
,
P. F.
,
H. V.
Woodcock
,
J. D.
Eley
,
M.
Platé
,
M. G.
Sulikowski
,
P. F.
Durrenberger
,
L.
Franklin
,
C. B.
Nanthakumar
,
Y.
Man
,
F.
Genovese
, et al
.
2016
.
Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF.
Thorax
71
:
701
711
.
45
Levine
,
B.
,
G.
Kroemer
.
2008
.
Autophagy in the pathogenesis of disease.
Cell
132
:
27
42
.
46
Glick
,
D.
,
S.
Barth
,
K. F.
Macleod
.
2010
.
Autophagy: cellular and molecular mechanisms.
J. Pathol.
221
:
3
12
.
47
Schmid
,
D.
,
C.
Münz
.
2007
.
Innate and adaptive immunity through autophagy.
Immunity
27
:
11
21
.
48
Xu
,
Y.
,
N. T.
Eissa
.
2010
.
Autophagy in innate and adaptive immunity.
Proc. Am. Thorac. Soc.
7
:
22
28
.
49
Harris
,
J.
2011
.
Autophagy and cytokines.
Cytokine
56
:
140
144
.
50
Wu
,
T. T.
,
W. M.
Li
,
Y. M.
Yao
.
2016
.
Interactions between autophagy and inhibitory cytokines.
Int. J. Biol. Sci.
12
:
884
897
.
51
Codogno
,
P.
,
A. J.
Meijer
.
2005
.
Autophagy and signaling: their role in cell survival and cell death.
Cell Death Differ.
12
(
Suppl. 2
):
1509
1518
.
52
Rubinsztein
,
D. C.
,
G.
Mariño
,
G.
Kroemer
.
2011
.
Autophagy and aging.
Cell
146
:
682
695
.
53
Tashiro
,
K.
,
M.
Shishido
,
K.
Fujimoto
,
Y.
Hirota
,
K.
Yo
,
T.
Gomi
,
Y.
Tanaka
.
2014
.
Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components.
Biochem. Biophys. Res. Commun.
443
:
167
172
.
54
Quirk
,
S.
,
D. K.
Agrawal
.
2014
.
Immunobiology of IL-37: mechanism of action and clinical perspectives.
Expert Rev. Clin. Immunol.
10
:
1703
1709
.
55
Lunding
,
L.
,
S.
Webering
,
C.
Vock
,
A.
Schröder
,
D.
Raedler
,
B.
Schaub
,
H.
Fehrenbach
,
M.
Wegmann
.
2015
.
IL-37 requires IL-18Rα and SIGIRR/IL-1R8 to diminish allergic airway inflammation in mice.
Allergy
70
:
366
373
.
56
Ye
,
L.
,
B.
Jiang
,
J.
Deng
,
J.
Du
,
W.
Xiong
,
Y.
Guan
,
Z.
Wen
,
K.
Huang
,
Z.
Huang
.
2015
.
IL-37 alleviates rheumatoid arthritis by suppressing IL-17 and IL-17-triggering cytokine production and limiting Th17 cell proliferation.
J. Immunol.
194
:
5110
5119
.
57
Grimsby
,
S.
,
H.
Jaensson
,
A.
Dubrovska
,
M.
Lomnytska
,
U.
Hellman
,
S.
Souchelnytskyi
.
2004
.
Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity.
FEBS Lett.
577
:
93
100
.
58
Wang
,
W. Q.
,
K.
Dong
,
L.
Zhou
,
G. H.
Jiao
,
C. Z.
Zhu
,
W. W.
Li
,
G.
Yu
,
W. T.
Wu
,
S.
Chen
,
Z. N.
Sun
, et al
.
2015
.
IL-37b gene transfer enhances the therapeutic efficacy of mesenchumal stromal cells in DSS-induced colitis mice.
Acta Pharmacol. Sin.
36
:
1377
1387
.
59
Dinarello
,
C. A.
,
C.
Nold-Petry
,
M.
Nold
,
M.
Fujita
,
S.
Li
,
S.
Kim
,
P.
Bufler
.
2016
.
Suppression of innate inflammation and immunity by interleukin-37.
Eur. J. Immunol.
46
:
1067
1081
.
60
Novick
,
D.
,
S. H.
Kim
,
G.
Fantuzzi
,
L. L.
Reznikov
,
C. A.
Dinarello
,
M.
Rubinstein
.
1999
.
Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response.
Immunity
10
:
127
136
.
61
Chandrasekar
,
B.
,
S.
Mummidi
,
W. C.
Claycomb
,
R.
Mestril
,
M.
Nemer
.
2005
.
Interleukin-18 is a pro-hypertrophic cytokine that acts through a phosphatidylinositol 3-kinase-phosphoinositide-dependent kinase-1-Akt-GATA4 signaling pathway in cardiomyocytes.
J. Biol. Chem.
280
:
4553
4567
.
62
Nold-Petry
,
C. A.
,
C. Y.
Lo
,
I.
Rudloff
,
K. D.
Elgass
,
S.
Li
,
M. P.
Gantier
,
A. S.
Lotz-Havla
,
S. W.
Gersting
,
S. X.
Cho
,
J. C.
Lao
, et al
.
2015
.
IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.
Nat. Immunol.
16
:
354
365
.
63
Kitasato
,
Y.
,
T.
Hoshino
,
M.
Okamoto
,
S.
Kato
,
Y.
Koda
,
N.
Nagata
,
M.
Kinoshita
,
H.
Koga
,
D. Y.
Yoon
,
H.
Asao
, et al
.
2004
.
Enhanced expression of interleukin-18 and its receptor in idiopathic pulmonary fibrosis.
Am. J. Respir. Cell Mol. Biol.
31
:
619
625
.

The authors have no financial conflicts of interest.

Supplementary data