Background
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal form of fibrosing interstitial pneumonia of unknown cause. The typical clinical course includes dyspnea, decreased exercise capacity, dry cough, and death at 2.5–5 years after diagnosis [
1]. Although recent studies have provided insights into the pathophysiology of IPF, treatment options for this disease remain limited [
1]. A prerequisite for the development of potential therapeutic targets is a better understanding of the pathogenesis of IPF.
IPF is characterized by heterogeneous pulmonary lesions at different stages of evolution, with foci of proliferative fibroblasts and myofibroblasts, abnormal lung epithelial cells, and an overwhelming matrix accumulation in the lung interstitium [
2]. The origins of the invasive lung myofibroblasts and their activation are unknown but are probably multiple, including activation of lung resident fibroblasts, recruitment of circulating fibrocyte blood mesenchymal precursors, and mesenchymal transformations of alveolar type II epithelial cells (ATII), endothelial cells, pericytes, and mesothelial cells [
3].
Transforming growth factor beta 1 (TGF-β1) is a well-studied, profibrotic growth factor that plays a key role in IPF by driving lung fibroblast activation and promoting mesenchymal transformations of different cell types [
2]. However, other important fibrogenic mediators are also elevated in diseased lung tissue and participate in the pathogenesis of IPF [
2,
3]. Therefore, an effective treatment for IPF must address its multiple mechanisms.
Tyrosine kinases are a complex, heterogeneous group of cell signal transducers that regulate a wide variety of physiological cell processes including metabolism, growth, differentiation, and apoptosis [
4]. Deregulated tyrosine kinase activity can promote the development and progression of neoplastic, cardiovascular, and fibrotic diseases. Currently, the only two drugs approved for the treatment of IPF are pirfenidone and nintedanib [
1]. Nintedanib, a multi-tyrosine kinase inhibitor, ameliorates IPF progression and symptoms by blocking the tyrosine kinases coupled to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) receptors [
5,
6]. It is therefore likely that specific tyrosine kinase inhibition can block the signal transduction of multiple key mediators of fibrosis.
Janus kinases (JAKs) are receptor-associated tyrosine kinases with central roles in cytokine and growth factor signaling. Like other receptor-associated tyrosine kinases, cytokine binding induces autophosphorylation and activation of JAK kinases [
7]. In turn, JAK kinases recruit and phosphorylate signal transducer and activator of transcription (STAT) proteins. Upon activation, STATs dimerize and translocate to the nucleus, where they activate the transcription of several target genes [
7]. Alterations in JAK2 signaling cause profound changes in the cellular response to cytokine stimulation. TGF-β1 signaling induces phosphorylation and activation of JAK2, which then interacts and phosphorylates STAT3 to induce fibrotic responses [
8]. In addition, JAK2 can be activated by other profibrotic mediators, including PDGF, VEGF, interleukin (IL)-6, IL-13, angiotensin II (ANGII), serotonin (5-HT), and endothelin (ET-1) [
9,
10]. STAT3 phosphorylation has been detected in fibrotic lung tissue from IPF patients and participates in both the fibroblast to myofibroblast transition and lung epithelial cell damage; therefore, it is an attractive therapeutic target in IPF [
11‐
13]. By contrast, the role of JAK2 in IPF has not been studied. Several JAK2 and STAT3 inhibitors are currently being evaluated in clinical trials involving various malignancies and inflammatory diseases. Thus, demonstration of anti-fibrotic effects in experimental models of fibrosis may have direct translational implications. In this study, we established different in vitro and in vivo models relevant to IPF to analyze the participation of JAK2 in IPF and the dependent and independent relationships of this protein with STAT3.
Methods
See the Online Supplement for more detailed descriptions of these methods.
Patients
Human lung tissue was obtained from IPF patients who underwent surgery for organ transplantation (n = 12). Healthy lung explant control samples were obtained from the organ transplant program of the University General Consortium Hospital of Valencia, Spain. The protocol was approved by the local research and independent ethics committee of the University General Consortium Hospital of Valencia (CEIC21/2013). Informed written consent was obtained from each patient.
Isolation and culture of human ATII cells and lung fibroblasts and in vitro experimental conditions
Primary ATII cells were obtained from the lung parenchyma of IPF patients as previously outlined [
14]. The cells were suspended in Dulbecco’s Modified Eagle’s Medium plus 10% fetal calf serum, 2 mM
l-glutamine, 100 U penicillin/mL, and 100 g streptomycin/mL. Primary human lung fibroblasts were obtained from the lung parenchyma of IPF patients with macroscopically fibrotic areas of disease, as previously described [
15]. The A549 human alveolar type II cell line and MRC5 normal lung fibroblasts were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in supplemented Roswell Park Memorial Institute 1640 medium as outlined [
16]. For the in vitro studies, ATII/A549 or primary lung fibroblast/MRC5 were stimulated with recombinant TGF-β1 (5 ng/mL; Sigma Aldrich, St. Louis, MO, USA) or IL-6 (50 ng/mL; Sigma) together with IL-13 (50 ng/mL; Sigma) for the indicated times, replacing the culture medium and stimulus every 24 h. TGF-β1 (5 ng/mL), IL-6 (50 ng/mL), and IL-13 (50 ng/mL) have been shown to induce cell phenotypic changes, including epithelial to mesenchymal transition, at the concentrations used in this study [
14,
17,
18]. JSI-124 (Sigma) is a selective JAK2/STAT3 inhibitor that at a concentration of 1 μM suppresses JAK2/STAT3 activation in A549 cells [
19]; NSC-33994 (Sigma) is a selective JAK2 inhibitor that at 1 μM completely inhibits JAK2 activity without affecting other tyrosine kinases [
20]); 5, 15-DPP (Sigma) is a selective STAT3 inhibitor that at 1 μM completely inhibits STAT3 activity without affecting other STATs [
21]. These inhibitors were added 30 min before the stimulus and left in the medium together with the stimulus until the effects were evaluated. Trypan blue staining of the cells was > 95%, which showed that none of the drugs altered viability.
Western blotting
Changes in the expression levels of proteins in human and rat lung tissues, in ATII/A549, and in lung fibroblast/MRC5 were examined by western blotting. The bands shown on the films were analyzed by densitometry using Image J 1.42q software (available at
http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). Target protein levels are expressed as the percentage of the densitometry values of the endogenous control (β-actin).
Small interfering RNA experiments
Total RNA was isolated from cells/lung tissue using TriPure® isolation reagent (Roche, Indianapolis, IN, USA) as previously described [
22]. Small interfering RNAs (siRNAs), including the scrambled siRNA control and JAK2 and STAT3 gene-targeted siRNAs, were designed by Ambion (Huntingdon, Cambridge, UK). A549 and MRC5 cells were transfected with siRNA (50 nM) in serum and antibiotic-free medium as previously reported [
23].
Histological and immunohistochemical studies
Lung histology and immunohistochemistry were conducted as previously reported [
24]. Tissue blocks (4 μm thickness) were stained with hematoxylin & eosin to assess fibrotic injury and pulmonary artery remodeling, and with Masson’s trichrome (Sigma-Aldrich, Madrid, Spain) to detect collagen deposition. The severity of lung fibrosis was scored on a scale from 0 (normal lung) to 8 (total fibrotic obliteration of tissue in the examined fields) according to Ashcroft [
25]. For immunohistochemical analysis of rat and human lung, the tissues were fixed and embedded in paraffin, cut into sections (4–6 μm), and incubated with JAK2, pJAK2, STAT3, pSTAT3, collagen type I, LC3II, beclin-I, Bcl-2, and p21 antibodies for 24 h at 4 °C. The non-immune IgG isotype control was used as the negative control for all of the samples.
ELISA
IL-6 and IL-13 cytokines were analyzed in the cell culture supernatants of human ATII and fibroblast using commercially available Quantikine® ELISA kits for human IL-6 (Catalog No. D6050; R&D Systems, Madrid, Spain) and IL-13 (Catalog No. D1300B), and in the bronchoalveolar lavage (BAL) fluid of rats using the ELISA rat IL-6 (Catalog No. KRC0061; Invitrogen™, Madrid, Spain) and IL-13 (Catalog No. KRC0132; Invitrogen™) kits according to the manufacturers’ protocols.
Wound repair and cell proliferation assay
Wound repair studies were performed in IPF primary human lung fibroblasts as previously outlined [
26]. The proliferation of IPF primary human lung fibroblasts was measured in a colorimetric immunoassay based on BrdU incorporation during DNA synthesis, which was performed using a cell proliferation ELISA BrdU kit (Roche, Mannheim, Germany) as previously described [
27].
Micro-computed tomography imaging of intratracheal bleomycin animals and BAL
Animal experiments and handling were performed in accordance with the guidelines of the Committee of Animal Ethics and Well-being of the University of Valencia (Valencia, Spain) as previously outlined [
24]. After the rats had been anesthetized with ketamine/medetomidine, a single dose of bleomycin at 3.75 U/kg (dissolved in 200 μL saline) was administered intratracheally via the endotracheal route [
28]. Sham-treated rats received the identical volume of intratracheal saline instead of bleomycin. This procedure defined day 1 of the experiment. The dose of intraperitoneally administered JSI-124 (1 mg/kg/day) was selected based on the results of previous in vivo animal studies [
29]. The inhibitor was administered from day 14 to day 28 as a therapeutic protocol. BAL fluid was processed as previously outlined, and the contents of inflammatory cells, protein, and IL-6/IL-13 were measured [
30]. Micro-computed tomography (micro-CT) analyses were performed as previously reported [
31]. Densitometric analysis of the extension of fibrosis was performed using the micro-CT images, with the density expressed as Hounsfield units.
Statistical analysis
The results were statistically analyzed using non-parametric tests (human tissue studies) and expressed as medians and interquartile ranges. In comparisons of two groups, between-group differences were analyzed using the Mann–Whitney test. Parametric tests were used to analyze the data obtained in animal and cellular in vitro mechanistic experiments; the results are expressed as the mean ± SEM of n experiments. Two-group comparisons were analyzed using a two-tailed Student’s paired t-test for dependent samples, and an unpaired t-test for independent samples. Multiple comparisons were made using a one-way or two-way analysis of variance followed by a Bonferroni post-hoc test. A P value < 0.05 was considered statistically significant.
Discussion
This study examined the role of JAK2 and STAT3 in IPF. Increased STAT3 phosphorylation, which modulates ATII and lung fibroblast plasticity [
11,
12], is characteristically detected in lung biopsy tissue from IPF patients, but there is no evidence implying the involvement of JAK2 in IPF. Our results show overexpression of JAK2 and its phosphorylated form in the fibrotic lungs of IPF patients, thus paralleling previous STAT3 findings. JAK2 and STAT3 activation contributed to cell transformations typical of IPF, including the ATII to mesenchymal and fibroblast to myofibroblast transitions and fibroblast proliferation and migration. Dual JAK2/STAT3 inhibition was more effective for inhibiting both these cellular transitions and lung fibrosis than the individual inhibition of JAK2 or STAT3, which implies synergistic and independent roles of these proteins in IPF.
In this study, non-phosphorylated forms of STAT3 were overexpressed in lung tissue from IPF patients and localized in the cytoplasm of fibroblasts from fibrotic areas and in hyperplastic ATII cells, as previously reported [
12]. The nuclear localization of p-STAT3 in fibrotic areas of lungs from IPF patients is consistent with its role as a transcription factor modulating the expression of the genes that cause fibrosis [
11]. Similar to STAT3, non-phosphorylated JAK2 was overexpressed in the fibrotic lungs of IPF patients and localized both in fibroblasts from fibrotic areas and in ATII hyperplastic cells, which implies a dominant role for these cells in IPF. Surprisingly, the active phosphorylated form of JAK2 was not detected in control healthy lungs, but was also overexpressed and localized in the nuclei of cells from the fibrotic areas of the lungs of IPF patients and bleomycin-treated animals. These are novel observations, as JAK2 and its phosphorylated form are typically located in the cytoplasm. JAK2 is phosphorylated in response to cell stimulation by different cytokines or growth factors, including TGF-β1, which leads to the translocation of STAT3 to the nucleus, where it activates genes associated with fibrosis [
8]. The similar nuclear localization of p-JAK2 in fibrotic areas implies that it is a non-canonical transcription factor, with a pathway independent of the canonical STAT3 pathway. Previous reports of the nuclear localization of p-JAK2 support our findings [
32,
33], while emerging evidence indicates that nuclear pJAK2 plays important roles in physiological and pathological conditions characterized by heightened cellular growth. Therefore, p-JAK2 activates not only STAT3 but also different intracellular receptors and forms multiprotein complexes [
33]. However the exact role of nuclear p-JAK2 in IPF is beyond the scope of the present work. Consistent with an independent role of p-JAK2 in lung fibrosis, we observed that p-JAK2 inhibition in ATII and lung fibroblasts from IPF patients partially reduced the mesenchymal-myofibroblast transformation induced by TGF-β1 and IL-6/IL-13. The degree of inhibition was similar to that of p-STAT3 inhibition. Moreover, the inhibition of p-JAK2/STAT3, whether by JSI-124 or by gene silencing, was synergistic in its inhibitory effects on cell transformation.
Overexpression of p-JAK2 has also been reported in the cytoplasm of skin fibroblasts from systemic sclerosis (SSc) patients [
8]. In SSc, TGF-β1 independently activates JAK2 and STAT3 via SMAD3, and pharmacologic or genetic inactivation of JAK2 in skin reduces the profibrotic effects of TGF-β1 [
8]. However, recent evidence indicates that in fibroblasts from IPF patients, TGF-β1 activates STAT3 via a SMAD2/3-dependent mechanism, independent of JAK2 [
12]. A physical interaction between STAT3 and the TGF-β receptor I and between STAT3 and SMAD3 in different cell lines has been suggested [
34]. Alternatively, TGF-β1 may activate STAT3 indirectly by inducing IL-6, IL-13, or other activators [
35]. In this study, TGF-β1 was shown to increase IL-6 and IL-13 secretion in IPF ATII and fibroblasts after 24 h, to phosphorylate JAK2 and STAT3, which implies either slow or indirect activation of these proteins. In addition to TGF-β1, other important fibrogenic mediators with increased expression in IPF lung tissue were CTGF, PDGF, FGF-2, ET-1, ANGII, and cytokines such as IL-13, IL-6, and chemokine ligand 2, all of which have been implicated in the pathogenesis of this disease [
2,
3]. Of note, most mediators activate JAK2 or STAT3 [
9,
10]. In this study, bleomycin-induced pulmonary fibrosis was characterized by increases in CTGF, TGFβ1, ET-1, IL-6, and IL-13, as profibrotic mediators. Thus, inhibition of downstream JAK2 and STAT3 signaling by JSI-124 reduced both pulmonary fibrosis and expression of these mediators in the lung. Accordingly, inhibition of JAK2 and STAT3, and thus of several of the cellular pathways implicated in IPF, may be a strategy for treating this complex disease.
Senescence and impaired autophagy are hallmarks of fibroblasts isolated from IPF patients. Autophagy, which helps to maintain the balanced synthesis, degradation, and recycling of organelles and proteins to meet metabolic demands, plays an important regulatory role in cellular senescence and differentiation. Impaired autophagy and increased senescence promote myofibroblast formation in IPF and thus are attractive targets in its treatment. In this work, TGFβ1 increased p21 senescence and Bcl-1 anti-apoptotic markers while decreasing the autophagy marker LC3-I/II in human fibroblasts. Similar results were observed in fibrotic lung tissue from bleomycin-treated rats. As in their cellular transformations, JAK2 and STAT3 exhibited independent effects on autophagy and senescence, with dual JAK2 and STAT3 inhibition leading to greater reductions in cell senescence and higher levels of autophagy than achieved by inhibiting either protein alone. Previous reports have demonstrated a role for STAT3 in fibroblast senescence [
36], consistent with the relevance of the JAK2/STAT3 pathway in IPF. However, the individual mechanisms by which JAK2 and STAT3 cause pulmonary fibrosis are currently unknown, and together with the mechanism underlying the synergistic effects of dual inhibition, remain to be determined in future research.
Acknowledgments
We thank the personnel of the Dept. of Pathology at the General University Hospital of Valencia and the animal housing facilities of the University of Valencia, Spain.