Abstract
Danger signals, or damage-associated molecular patterns (DAMPs), instigate mitochondrial innate immune responses wherein mitochondrial antiviral signaling protein (MAVS) functions as a key platform molecule to mediate them. The role of MAVS in the pathogenesis of idiopathic pulmonary fibrosis (IPF), however, has not yet been identified. Whether MAVS signalling can be modulated by currently existing drugs has also not been explored.
We used an established model of pulmonary fibrosis to demonstrate that MAVS is a critical mediator of multiple DAMP signalling pathways and the consequent lung fibrosis after bleomycin-induced injury in vivo.
After bleomycin injury, MAVS expression was mainly observed in macrophages. Multimeric MAVS aggregation, a key event of MAVS signalling activation, was significantly increased and persisted in bleomycin-injured lungs. A proapoptotic BH3 mimetic, ABT-263, attenuated the expression of MAVS and its signalling and, consequently, the development of experimental pulmonary fibrosis. In contrast, the therapeutic effects of nintedanib and pirfenidone, two drugs approved for IPF treatment, were not related to the modulation of MAVS or its signalling. Multimeric MAVS aggregation was significantly increased in lungs from IPF patients as well.
MAVS may play an important role in the development of pulmonary fibrosis, and targeting MAVS with BH3 mimetics may provide a novel and much needed therapeutic strategy for IPF.
Abstract
MAVS may play a critical role in the development of pulmonary fibrosis, and targeting MAVS or its signalling by proapoptotic BH3 mimetics may be a feasible strategy for the treatment of IPF. https://bit.ly/31rIsmZ
Introduction
Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause and is characterised by the histopathological and/or radiological pattern of usual interstitial pneumonia [1]. IPF is a fatal lung disease; patients with this disorder experience unpredictable lung function decline, have a lower survival rate and often die from respiratory insufficiency within 2–5 years of diagnosis [1–3]. As such, IPF therapy is a major unmet medical need in human health and requires a better mechanistic understanding of IPF pathogenesis to develop novel disease-modifying therapeutics.
Mitochondrial function and behaviour are fundamental to the physiology of cellular and organismal health; consequently, “mitochondrial dysfunction” has been implicated in a wide range of diseases that encompass all aspects of health and disease [4–7]. In accordance with the recent evolution of our understanding of mitochondria's role in human health, attention has increasingly been paid to the functional roles of mitochondrial molecules and the underlying mechanisms by which they contribute to the pathogenesis of IPF [8–10].
During tissue injury or damage responses, mitochondrial functions are influenced by intracellular perturbations. The alteration of mitochondrial functions in turn modulates intracellular signalling to execute appropriate cellular functions [11]. Mitochondrial antiviral signaling protein (MAVS) represents such an example and functions as a platform molecule to mediate mitochondrial innate immune signalling [12–14]. How MAVS contributes to IPF pathogenesis, in which dysregulated tissue damage responses play an important role, has not yet been identified. In addition, whether currently existing drugs can modulate MAVS signalling to offer novel therapeutic strategies has not been explored.
Here, we demonstrate that MAVS plays a critical role in the development of experimental pulmonary fibrosis after bleomycin-induced lung injury in vivo, an established mammalian model of IPF. Bleomycin-induced fibrotic responses and multiple damage-associated molecular pattern (DAMP) signalling pathways were significantly attenuated in a MAVS-dependent manner in murine lungs in vivo. In addition, bleomycin-induced cellular senescence was significantly attenuated in MAVS deficiency. The B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) mimetic ABT-263 induced a significant reduction in MAVS signalling and, consequently, attenuated the development of experimental pulmonary fibrosis in our model. In contrast, the therapeutic effects of nintedanib and pirfenidone, currently approved drugs known to decelerate IPF progression, were not related to MAVS signalling. Finally, human studies revealed significant activation of MAVS in lungs from IPF patients compared to those from controls. Taken together, these data suggest that MAVS and its mitochondrial innate immune signalling, which can be modulated by BH3 mimetics, is activated and may play an important role in the pathogenesis of IPF.
Materials and methods
Animals and experimental design
Wild type (WT) (Mavs+/+; The Jackson Laboratory, Bar Harbor, ME, USA) and Mavs−/− (from Dr Z.J. Chen, University of Texas Southwestern, Dallas, TX, USA) were all kept on C57BL/6J background and bred at Yale University . All animal experiments were approved by the Yale Animal Care and Use Committee. An established mammalian model of IPF was used as described in previous publications [15, 16]. Briefly, to induce pulmonary fibrosis, mice were treated with bleomycin (NDC 61703-0332-18; Pfizer, New York, NY, USA) with PBS by intratracheal (i.t.) administration or oropharyngeal aspiration. At designated times after bleomycin administration, mice were killed by an intraperitoneal (i.p.) injection of urethane, hearts were perfused with PBS and lungs were harvested for further analyses. For bronchoalveolar lavage fluid (BALF) collection, the trachea was cannulated and washed twice with 0.9 mL PBS. Samples were centrifuged at 1500 rpm for 5 min, and the cell-free supernatants were collected for ELISA. The cell pellets were recovered in 200 μL of sterile PBS, and a total cell count of BALF was done using an automated cell count on a Beckman Coulter analyser (Beckman Coulter, Brea, CA, USA).
Therapeutic drugs treatment
To test the therapeutic efficacy of existing drugs in our modelling of IPF, ABT-263 (40 mg·kg−1; MedChemExpress, Monmouth Junction, NJ, USA), nintedanib (40 mg·kg−1; MedChemExpress), pirfenidone (40 mg·kg−1; MedChemExpress) or vehicle control (5% Tween 80) were administered by i.p. injection on days 8, 10, 12, 14, 16, 18 and 20 after bleomycin administration, and the mice were killed on day 21.
Human sample preparation
To evaluate and compare MAVS aggregation in IPF patients and healthy controls, lung tissue samples were obtained from the laboratory of I. Rosas. Human lung explants were obtained from patients who had signed informed consent forms and undergone lung transplantation at the Brigham and Women's Hospital, Boston, MA, USA, or from donor organs provided by the New England Organ Bank, Waltham, MA, USA, and/or the National Disease Research Interchange, Philadelphia, PA, USA. The study's protocol was approved by the Partners Healthcare Institutional Review Board (IRB Protocol #2011P002419). Briefly, lungs were sliced and washed with cold, sterile PBS several times. Visible airway structures, vessels, blood clots and mucin were removed. Tissues were minced mechanically into small pieces (<5 mm) and then incubated for 45 min in 37°C within a digestion medium, which consisted of 30 U·mL−1 elastase (Elastin Products Company, Owensville, MO, USA), 0.2 mg·mL−1 DNAse I (Sigma-Aldrich, St. Louis, MO, USA), 0.3 mg·mL−1 liberase (Roche, Basel, Switzerland) and 1% penicillin/streptomycin diluted in DMEM/F12 medium (Lonza, Basel, Switzerland). Digested tissues were filtered using a metal strainer (Sigma-Aldrich). Unfiltered tissues were incubated a second time in digestion medium for ≤30 min, followed by repeat filtration and addition of 10% fetal bovine serum (FBS) to stop the enzymatic reaction. Flow-through from both filtrations was combined and centrifuged at 2500 rpm, 4°C for 10 min. The pellet was resuspended in red cell lysis buffer (VWR, Radnor, PA, USA) for <5 min at 37°C and centrifuged again. The pellet was resuspended in DMEM/F12 medium and filtered using a 100 μm strainer (Fisher Scientific, Waltham, MA, USA). Freezing medium (10% FBS and 10% dimethyl sulfoxide in DMEM/F12) was then added to the filtrate. Cell suspensions were aliquoted and stored in liquid nitrogen for future semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) or blue native-PAGE applications.
Statistical analysis
All statistical analyses were executed using GraphPad Prism (version 6; GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed with an unpaired t-test. For the multiple comparisons, two-way ANOVA was used. The significance of the survival rate was analysed with the log-rank test. Values are expressed as mean±sem, sd or minimum to maximum. Statistical significance was defined as p<0.05.
Additional experimental methods are described in the supplementary material and include Sircol assay; flow cytometric analysis; histology, immunohistochemistry and immunofluorescence; isolation of murine alveolar macrophages; ELISA; quantitative reverse-transcriptase PCR (qRT-PCR); Western blot analysis; quantification of cell-free double-stranded DNA (dsDNA); mitochondrial isolation; BN-PAGE and SDD-AGE; in vitro cell culture and isolation; and culture of murine lung fibroblasts (MLFs) and murine embryonic fibroblasts (MEFs).
Results
MAVS plays a crucial role in lung fibrosis after bleomycin administration in vivo
We questioned whether MAVS, a novel adaptor molecule of mitochondrial innate immune signalling, has a functional role in the pathogenesis in a mammalian model of experimental pulmonary fibrosis. When 1.5 U·kg−1 of body weight of bleomycin was administered to male and female mice via i.t. delivery, the bleomycin-induced tissue injury responses revealed striking difference between two groups of C57BL/6J WT or MAVS-null mutant (Mavs−/−) mice. All WT mice died whereas 9 of 14 (64%) Mavs−/− mice survived (figure 1a). With 1.0 U·kg−1 of bleomycin, more than half of the WT mice died while all Mavs−/− mice survived (figure 1b). With 0.4 U·kg−1 of bleomycin, we obtained an ∼3-fold increase in total lung collagen content, which is comparable to known publications (figure 1c) [8, 9]. Therefore, 0.4 U·kg−1 of bleomycin was administered via i.t. delivery for all the following in vivo experiments, unless otherwise specified.
Mitochondrial antiviral signaling protein (MAVS) plays a crucial role in lung fibrosis after bleomycin administration in vivo. The indicated dose of bleomycin was administered through the intratracheal (i.t.) route to C57BL/6J wild-type (Mavs+/+) and MAVS-null mutant (Mavs−/−) mice. a, b) Survival rates during the course of the experiment after a) 1.5 U·kg−1 (n=14 per group) and b) 1.0 U·kg−1 bleomycin administration (n=8 for Mavs+/+ group, n=7 for Mavs−/− group). c–g) Saline (−) or 0.4 U·kg−1 of bleomycin (+) was administered through the i.t. route to Mavs+/+ and Mavs−/− mice. c) Collagen contents of total lung tissues obtained from the mice killed at day 21 after bleomycin administration (n=5 per saline group, n=10 for bleomycin-administered Mavs+/+ and Mavs−/− mice groups). d) Body weight changes during the course of the experiment. e) Representative histological findings after Masson's trichrome staining of collagen on lung sections at day 21 after bleomycin administration. Scale bars: 3000 μm (top), 100 μm (bottom). f) Immunohistochemistry analysis of MAVS on lung sections at indicated time points after saline or bleomycin administration. Representative images of the staining are presented (n=5 per group); scale bars: 100 μm. The red boxed regions are magnified for closer observation; scale bars: 20 μm. Arrows point to alveolar epithelial cells; arrowheads indicate macrophages. g) Representative images from the immunofluorescence analysis of MAVS staining (green) on lung sections at 14 days after bleomycin administration (n=5). CD68 antibody (red) was used for the immunofluorescence staining of macrophages; scale bars: 50 μm. The white boxed region is magnified for closer observation; scale bar: 20 μm. h) The percentage of each cell population among the MAVS-stained lung cells from bleomycin-administered lungs at day 14 was determined by flow cytometry (n=5). Specific surface markers were used to identify lung cell populations: macrophages (CD45+F4/80+), epithelial cells (CD45−CD31−CD49e−EpCAM+) and myofibroblasts (CD45−CD31−EpCAM−CD49e+). Data are presented as mean±sem. Statistical significance was determined using the log-rank (Mantel–Cox) (a, b) or two-way ANOVA with Tukey's multiple comparisons test (c) or multiple t-test (d). *: p<0.05; **: p<0.01; ****: p<0.0001.
Bleomycin-induced pulmonary fibrosis and loss of body weight were significantly ameliorated in the lung from Mavs−/− mice compared to those of WT controls (figure 1c, d). These observations were confirmed by histological evaluations (figure 1e and supplementary figure S1a). Male and female mice showed a similar reduction in the level of fibrotic response (data not shown). Immunohistochemistry (IHC) and immunofluorescence evaluations demonstrated significant staining of MAVS molecules after bleomycin administration. The staining was observed most prominently in macrophages but was not restricted to this cell population (figure 1f, g and supplementary figure 1a). Flow cytometric evaluation revealed that CD45+F4/80+ cells occupied ∼80% of the lung cells that were stained with anti-MAVS antibody after bleomycin treatment (figure 1h). The specificity of MAVS staining in our imaging studies as well as our flow cytometric evaluation are presented in supplementary figure S1b, c. The identification of specific cell types and the gating strategy of our flow cytometric evaluation were performed as per previously reported methods (supplementary figure S1d) [17, 18]. To determine whether the bleomycin-induced tissue injury in vivo was altered in the presence of MAVS, a quantification of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and an evaluation of the active form of caspase-3, a marker of apoptotic cell death, were undertaken in the lungs of WT and Mavs−/− mice. The lungs were obtained at day 3 and day 5 after administration of bleomycin in vivo. These results revealed that the bleomycin-induced tissue injury in vivo was not attenuated in MAVS deficiency (supplementary figure S1e–g). Overall, these data reveal that MAVS is expressed mainly in pulmonary macrophages and plays an important pathogenic role in a mammalian IPF model.
MAVS-dependent fibrogenic responses in bleomycin-induced pulmonary fibrosis
Experiments were undertaken to determine the role of MAVS in regulating signalling of transforming growth factor-β (TGF-β), which is often regarded as the master regulator of tissue fibrosis, as well as the consequent fibrogenic responses. Initially, the bleomycin-induced induction of total and active TGF-β showed a trend for attenuation but did not reach statistical significance in the lung from Mavs−/− mice compared to those of WT controls (figure 2a, b). To accurately determine the statistical significance, additional experiments were undertaken with a larger sample size of mice. The results revealed that the bleomycin-induced induction of total and active TGF-β was significantly attenuated in MAVS deficiency in vivo (figure 2c, d). Evaluating gene expression for the molecules involved in TGF-β superfamily signalling showed that the mRNA expression of connective tissue growth factor (Ctgf) and interleukin (IL)-13 (Il13) was significantly attenuated in MAVS deficiency (supplementary figure S2a–f). Both Ctgf and Il13 also regulate the phosphorylation status of Smad2/3, a critical event in TGF-β super family signalling [19–21]. The intra-alveolar inflammatory response was not significantly altered between the two groups (supplementary figure S2g). Additional evaluations of molecular markers that are differentially expressed in macrophages during distinct types of classical, alternative and fibrotic/reparative activations also did not reveal specific differences between the two groups (supplementary figure S2h). Importantly, Western blot evaluation of the activation status of SMAD-2/3, canonical markers of TGF-β distal signalling molecules, revealed marked attenuation in MAVS deficiency (figure 2e). Moreover, well-known marker molecules of tissue fibrotic responses were significantly altered in a MAVS-dependent manner. Specifically, the molecular expression of fibronectin, α-smooth muscle actin (a-SMA) and collagen 1α (Col1a), which were induced after bleomycin injury, were markedly attenuated in MAVS-deficient lungs (figure 2f–i). These observations were further validated by densitometry evaluations of these molecules (supplementary figure S2g–l). IHC evaluation of Col1α confirmed the above findings, revealing that bleomycin injury-induced expression of Col1α was markedly attenuated in lung tissues from Mavs−/− mice (figure 2j). Collectively, these data suggest that distinct MAVS-dependent and MAVS-independent mechanisms exist for the pathogenesis of pulmonary fibrosis in our model.
Mitochondrial antiviral signaling protein (MAVS)-dependent and independent fibrogenic responses in bleomycin-induced pulmonary fibrosis. a–d) Saline or bleomycin was administered to wild-type (Mavs+/+) and MAVS-null mutant (Mavs−/−) mice. a) Active and b) total transforming growth factor-β1 (TGF-β1) levels at indicated time points after bleomycin administration were measured from the bronchoalveolar lavage fluid (BALF) by ELISA (n=6 per saline group, n=8 per bleomycin-administered group). c) Active and d) total TGF-β1 levels at day 14 after bleomycin administration were measured from BALF by ELISA (n=5 per saline group, n=15 per bleomycin-administered group). e) Western blot analysis of p-SMAD2, SMAD2/3, p-SMAD1/5/9 and SMAD1 expressions in whole lung tissue lysates at day 14 after saline (−) or bleomycin (+) administration. β-Actin was used as a loading control. f–h) Quantitative reverse-transcriptase PCR analysis of the expression of f) fibronectin, g) α-smooth muscle actin (α-SMA) and h) Collagen 1a (Col1a) mRNAs in lung tissues at day 14 after saline or bleomycin administration (n=6 per saline group, n=8 per bleomycin-administered group). i) Western blot analysis of fibronectin and α-SMA expressions in lung tissue lysates day 14 after saline or bleomycin administration. β-Actin was used as a loading control. j) Representative images from immunohistochemistry analysis of Col1a on lung sections at day 14 after bleomycin administration (n=5 per group). Scale bars: 2000 μm. The red boxed regions are magnified on the right for closer observation. Data are presented as the mean±sem or minimum to maximum. Statistical significance was determined using two-way ANOVA with Tukey's multiple comparisons test (a–d, f–h). *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
MAVS amplifies multiple DAMP signalling during the fibrotic phase after bleomycin injury
The above findings led us to investigate what role MAVS may play in the development of experimental pulmonary fibrosis. Because MAVS determines organismal death or survival after severe bleomycin injury, experiments were undertaken to investigate whether DAMP signalling is regulated in a MAVS-dependent manner after bleomycin injury. To this end, multiple molecules related to various DAMP signalling pathways were induced in a time-kinetic manner after bleomycin injury in vivo (figure 3a–g). There was a significant increase in cell-free dsDNA in BALF after bleomycin injury in vivo (figure 3g). Among these multiple molecules, dsDNA, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon response cGAMP interactor (STING) were significantly attenuated in a MAVS-dependent manner after bleomycin injury in vivo (figure 3h, i). Recently, polymerisation of STING was identified as a critical event for its activation [22]. In line with this, SDD-AGE revealed a significant induction of STING aggregation at day 14, a time point often regarded as the peak of fibrogenesis in our IPF murine model (figure 3j). Intriguingly, bleomycin-induced STING aggregation was markedly attenuated in MAVS-deficient lungs (figure 3j). Similar observations were further accentuated at a higher dose (1.0 U·kg−1) of bleomycin injury in vivo. Specifically, gene expression of additional molecules of DAMP signalling pathways, including Sting, high mobility group box 1 (Hmgb1), NLR family, pyrin domain containing 3 (Nlrp3) and purinergic receptor P2Y2 (P2ry2), was significantly attenuated in MAVS-deficient lungs at the higher dose (1.0 U·kg−1) (supplementary figure S3a–d). In accordance with this, bleomycin injury-induced induction of dsDNA from BALF as well as cGAS protein and STING aggregation were also significantly attenuated in a MAVS-dependent manner at this high dose (1.0 U·kg−1) (supplementary figure S3e–g). Taken together, these results suggest that multiple DAMP signalling pathways, which are induced or activated by bleomycin injury in vivo and persist during the fibrotic phase of IPF murine modelling, are critically regulated in a MAVS-dependent manner.
Mitochondrial antiviral signaling protein (MAVS) amplifies multiple damage-associated molecular pattern (DAMP) signalling during fibrotic phase. a–g) Saline or bleomycin was administered to wild-type mice. Expression levels of a) Sting, b) Hmgb1, c) Nlrp3, d) P2ry2, e) P2rx7 and f) Rig1 mRNAs in whole lung tissue taken at the indicated times were evaluated by quantitative reverse-transcriptase PCR. g) The amount of cell-free double-stranded DNA (cfDNA) in bronchoalveolar lavage fluid (BALF) was evaluated by fluorometric assay. h–j) Saline (−) or bleomycin (+) was administered to wild-type (Mavs+/+) and MAVS-null mutant (Mavs−/−) mice. h) Total cfDNA from BALF and i) Western blot evaluations for cyclic GMP-AMP synthase (cGAS) and stimulator of interferon response cGAMP interactor (STING) proteins from whole lung tissue lysates at day 14 after bleomycin administration. β-Actin was used as a loading control. j) The result of STING aggregation. Lung tissue lysates at day 14 after bleomycin administration were separated by semi-denaturating detergent-agarose gel electrophoresis (SDD-AGE) and detected with STING antibody. Data are presented as the mean minimum to maximum. Statistical significance was determined using unpaired t-test (a–g) or two-way ANOVA with Tukey's multiple comparisons test (h). *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
A proapoptotic BH3 mimetic, ABT-263, inhibits MAVS and lung fibrosis in vivo
While we were undertaking experiments to define the role of apoptosis in the regulation of DAMP signalling in our system, we observed a previously unidentified effect of proapoptotic BH3 mimetics. Specifically, BH3 mimetic drugs could reduce MAVS expression. In murine lung epithelial-12 (MLE12) cells, application of several BH3 mimetics, including ABT-263 (a Bcl-2 and Bcl-xl inhibitor), ABT-199 (a Bcl-2 inhibitor) and A1155483 (a Bcl-xl inhibitor), after bleomycin injury in vitro caused reduced expression of MAVS (figure 4a, b). In addition, the ABT-263-induced reduction of MAVS was not related to a decrease in mitochondrial proteins: the components of mitochondrial oxidative phosphorylation were not altered in our experiments (figure 4b). A similar finding was observed when we expanded the evaluation to different types of primary cells, including MEFs, peritoneal macrophages and primary MLFs (figure 4c–e). Inspired by these observations, a pharmacological approach was undertaken in vivo to evaluate potential therapeutic effects of ABT-263 in our murine pulmonary fibrosis model (figure 4f, g). Specifically, from day 8 after bleomycin injury in vivo, ABT-263 (40 mg·kg−1 of body weight) was applied via i.p. administration every other day and the fibrotic responses were evaluated. The bleomycin-induced increase of total lung collagen contents was significantly attenuated with ABT-263 treatment in vivo (figure 4f, g and supplementary figure S4). In line with this, Western blot evaluation revealed that the activation status of SMAD-2/3 and the expression of fibronectin and α-SMA were significantly ameliorated with ABT-263 treatment in vivo (figure 4h). These observations were supported by in vitro experiments: in MEF, MLF and normal human lung fibroblast cells, TGF-β1-induced activation of SMAD-2/3 was significantly reduced after ABT-263 treatment (figure 4i). Overall, these results provide compelling evidence that the BH3 mimetic ABT-263 can attenuate the expression of MAVS and, consequently, the development of experimental pulmonary fibrosis.
The BH3 mimetic ABT-263 attenuates the expression of mitochondrial antiviral signaling protein (MAVS) and lung fibrosis. a–e) Treatment was given for 3 h using 100 μM of the indicated BH3 mimetics. a) Murine lung epithelial-12 (MLE12) cells were treated with the indicated BH3 mimetics after 10 mU·mL−1 bleomycin treatment for 3 days. MAVS expression was evaluated by Western blot. b) The mitochondrial fractions of MLE12 cells treated with the indicated BH3 mimetics after 10 mU·mL−1 bleomycin treatment for 3 days. MAVS and oxidative phosphorylation (OXPHOS) complex expression was evaluated by Western blot. Voltage-dependent anionic channel (VDAC) protein was evaluated as the loading control of mitochondria. c) Murine embryonic fibroblast (MEF) cells were treated with the indicated BH3 mimetics. MAVS expression was evaluated by Western blot. d) Primary peritoneal macrophages (PM) were isolated from the peritoneal cavity of wild-type mice. The cells were treated with 10 μM and 100 μM ABT-263. MAVS expression was evaluated by Western blot. e) Primary murine lung fibroblast (MLF) cells were isolated from wild-type murine lungs. The cells were treated with ABT-263. MAVS expression was evaluated by Western blot. f) Scheme of the experimental approach. The mice were administered bleomycin and treated after day 8 with ABT-263 (40 mg·kg−1, every 2 days, intraperitoneal), and killed at day 21. g) Evaluation results of total lung collagen contents from wild-type murine lungs. Each circle indicates the individual mouse used for the experiment (n=5 for each control group, n=23 for bleomycin-only treatment group, n=20 for bleomycin+ABT-263 treatment group). h) Western blot evaluations for p-SAMD2, SMAD2/3, fibronectin and α-SMA proteins from whole lung tissue lysates at day 14 after bleomycin administration (n=5 per group). i) After stimulation with 20 ng·mL−1 transforming growth factor-β1 (TGF-β1) for 24 h, MEF, MLF and normal human lung fibroblast (NHLF) cells were treated with 100 μM ABT-263 for 3 h. p-SMAD2 and SMAD2/3 expression was evaluated by Western blot. For panels (a), (c–e) and (h, i), β-Actin was used as a loading control. Data are presented as mean±sem. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparisons test (g). ****: p<0.0001.
Therapeutic effects of nintedanib and pirfenidone are not related to MAVS
To date, nintedanib and pirfenidone are the only approved drugs known to decelerate IPF disease progression [23, 24]. However, it is not known whether these drugs can modulate MAVS signalling in vitro or in vivo. Our in vitro experiments revealed that these two drugs failed to reduce the expression of MAVS, whereas ABT-263 could significantly reduce it (figure 5a, b). Overall, treatment with nintedanib or pirfenidone did not alter the expression of mitochondrial proteins such as the oxidative phosphorylation components (figure 5b). In our IPF modelling system, treatment with ABT-263, nintedanib and pirfenidone significantly ameliorated the bleomycin-induced increase of total lung collagen contents and semi-quantitative severity scores of the microscopic changes (figure 5c, d and supplementary figure S5); there was no statistical difference among the three treated groups (figure 5c, d). In line with this, Western blot evaluation revealed that the activation status of SMAD-2/3 and expression of fibronectin and α-SMA were significantly ameliorated after treatment with these three drugs (figure 5e). Overall, these data suggest that the pharmacological effects of nintedanib and pirfenidone for the treatment of IPF are not directly related to MAVS.
Therapeutic effects of nintedanib and pirfenidone are not related to mitochondrial antiviral signaling protein (MAVS). a, b) Murine lung epithelial-12 cells were treated with ABT-263 (100 μM), nintedanib (100 μM) and pirfenidone (100 μM) for 3 h after 10 mU·mL−1 bleomycin treatment for 3 days. MAVS expression in a) whole cell lysates and b) mitochondrial fractions was evaluated by Western blot. b) Expression of oxidative phosphorylation (OXPHOS) complexes and voltage-dependent anionic channel (VDAC) protein as loading controls of mitochondria. c, d) C57BL/6J wild-type mice were administered with bleomycin (+) and treated after day 8 with ABT-263 (40 mg·kg−1, every 2 days, intraperitoneal (i.p.), nintedanib (40 mg·kg−1, every 2 days, i.p.) and pirfenidone (40 mg·kg−1, every 2 days, i.p.), and killed at day 21. c) Total lung collagen contents. Each data point indicates the individual mouse used for the experiment (n=10 per bleomycin treatment group (three mice dead), n=10 per bleomycin+ABT-263 treatment group, n=10 per bleomycin+nintedanib treatment group (two mice dead) and n=10 per bleomycin+pirfenidone treatment group). d) Representative images of Masson's trichrome staining. Scale bars: 500 μm. e) Western blot evaluation for p-SAMD2, SMAD2/3, fibronectin and α-SMA proteins from whole lung tissue lysates at day 14 after bleomycin administration. For panels (a) and (e), β-Actin was used as a loading control. Data are presented as mean±sem. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparisons test (c). *: p<0.05; **: p<0.01; ***: p<0.001.
Bleomycin-induced cellular senescence is inhibited in MAVS deficiency
Cellular senescence has recently emerged as a key driving force of IPF pathogenesis [25, 26]. ABT-263 mediates anti-fibrotic effects via its selective removal of senescent cells by inducing their apoptotic cell death, a process called senolysis [25]. Thus, experiments were undertaken to further determine how ABT-263-induced senolysis might be related to the molecular function of MAVS in cellular senescence. In MEF cells, bleomycin-induced cellular senescence, which was evaluated by measuring senescence-associated β-galactosidase (SA-β-Gal) activity, was significantly attenuated after treatment with ABT-263 (figure 6a, b). As expected, the reduction of cellular senescence was related to the induction of apoptosis and the consequent decrease of cell viability in a dose-dependent manner (figure 6c, d and data not shown). Notably, there was a significant reduction of MAVS protein expression in association with ABT-263-induced senolysis (figure 6d). Similar results were obtained from MLE12 cells, further confirming the reduction of MAVS in association with ABT-263-mediated senolysis (supplementary figure S6a–d).
Bleomycin-induced cellular senescence is inhibited in mitochondrial antiviral signaling protein (MAVS) deficiency. a–d) Murine embryonic fibroblast (MEF) cells were treated with indicated concentration of ABT-263 for 30 h after 50 mU·mL−1 bleomycin treatment. a) Senescence-associated β-galactosidase (SA-β-Gal) staining. Scale bars: 100 μm. b) Quantification of SA-β-gal+ cells. c) Cell viability was measured by XTT assay. Ble-SC: bleomycin-induced senescent cells. d) Western blot results of MAVS and cleaved caspase-3 (cas-3) proteins. e) Murine lung epithelial-12 (MLE12), f) MEF and g) normal human lung fibroblast (NHLF) cells were treated with the indicated concentration of ABT-263 for the indicated times. MAVS expression was evaluated by Western blot. h, i) Wild-type (Mavs+/+) and MAVS-null mutant (Mavs−/−) MEF cells were treated with 1 μM ABT-263 for 30 h after 50 mU·mL−1 bleomycin treatment. h) SA-β-Gal staining. Scale bars: 100 μm. i) Quantification of SA-β-gal+. j) Western blot for MAVS and cas-3 proteins in Mavs+/+ and Mavs−/− MEF cells treated with 50 mU·mL−1 bleomycin for 30 h. k–m) Saline (−) or bleomycin (+) was administered to Mavs+/+ and Mavs−/− mice. Expression levels of k) P16INK4a and l) P19ARF in whole lung tissues at day 14 after bleomycin administration were evaluated by quantitative reverse-transcriptase PCR. m) Western blot of P16INK4a. For panels (d–g), (j) and (m), β-Actin was used as a loading control. Data are presented as mean±sd. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparisons test. *: p<0.05; **: p<0.01; ***: p<0.001.
To clarify whether the inhibitory effect of ABT-263 on MAVS protein expression is mainly related to its selective removal of senescent cells (senolysis) in the setting of cellular senescence or via a senolysis-independent mechanism, further experiments with no stimulation of bleomycin were undertaken, eliminating the bleomycin-induced cellular senescence. After treatment with ABT-263, a significant reduction of MAVS protein was observed in MLE12, MEF and normal human lung fibroblast (NHLF) cells (figure 6e–g).
To define the functional role of MAVS in bleomycin-induced cellular senescence in vitro, SA-β-Gal activity was evaluated and compared in the presence and absence of MAVS. Bleomycin-induced cellular senescence was significantly attenuated in MAVS deficiency (figure 6h, i). The level of apoptotic cell death or cell viability was not significantly different in the presence or absence of MAVS (figure 6j and data not shown), revealing that the reduction of bleomycin-induced cellular senescence in MAVS deficiency cannot be attributed to senolysis. Rather, these results demonstrate that bleomycin-induced cellular senescence is inhibited in MAVS deficiency. The functional significance of MAVS in the regulation of bleomycin-induced senescence was also evaluated in our in vivo model. When gene expression of known senescence markers was evaluated, P16INK4a, P19ARF and P21Cip1 mRNAs were significantly induced in our murine pulmonary fibrosis model (supplementary figure S6e–g). Indeed, bleomycin injury-induced mRNA expression of P16INK4a and P19ARF was significantly attenuated in a MAVS-dependent manner (figure 6k, l). In accordance with the time-kinetic patterns of its mRNA expression, P16INK4a protein expression was significantly induced, reaching a peak at day 14 after bleomycin injury in vivo (supplementary figure S6h). Induction of P16INK4a protein was significantly reduced in MAVS deficiency at day 14 and day 21 after bleomycin administration in vivo (figure 6m and supplementary figure 6i). Collectively, these results suggest that, in addition to its known senolytic effect, ABT-263 may exert its anti-fibrotic effects by the reduction of MAVS. In addition, MAVS may play a functional role in the induction of bleomycin-induced cellular senescence.
Multimeric aggregation of MAVS, a critical event of MAVS signalling, is significantly activated in humans as well as in mice
Because prion-like multimeric aggregation of MAVS molecules on the mitochondrial outer membrane was identified as a critical event for its proper signalling [27], experiments were undertaken to evaluate this in our system. SDD-AGE and BN-PAGE evaluation revealed that bleomycin injury induces significant MAVS aggregation in vivo (figure 7a). This was observed in the mitochondrial fraction and in whole lung lysates (figure 7a). Surprisingly, this phenomenon persisted even at day 14 after bleomycin administration. An additional finding was that although the expression of MAVS was markedly enhanced after bleomycin administration in IHC evaluation (figure 1e), the absolute amount of MAVS protein in the lung tissue seemed to decrease during bleomycin-induced fibrogenesis in vivo (figure 7a). We interpret the data as additional evidence for the multimeric aggregation of MAVS during fibrogenesis. Specifically, the strong positive staining of MAVS obtained from IHC evaluation (figure 1e) may reflect the multimeric aggregation of MAVS, which is not related to its molecular induction after bleomycin injury in vivo.
Multimeric aggregation of mitochondrial antiviral signaling protein (MAVS), a critical event of MAVS signalling, is significantly activated in humans as well as in mice. a) Saline or bleomycin was administered to wild-type (Mavs+/+) mice. The multimeric MAVS aggregation from whole lung tissue lysates (WL) and mitochondrial fractions at indicated time points was evaluated by blue native (BN)-PAGE and semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE). b, c) Wild-type mice were administered with bleomycin (+) and treated after day 8 with ABT-263 (40 mg·kg−1, every 2 days, intraperitoneal (i.p.)), nintedanib (40 mg·kg−1, every 2 days, i.p.) and pirfenidone (40 mg·kg−1, every 2 days, i.p.). The multimeric MAVS aggregation in WL at day 14 was evaluated by SDD-AGE. d) The mitochondrial fractions of murine lung epithelial-12 cells treated with 100 μM ABT-263, ABT-199, A-1155463, nintedanib and pirfenidone for 3 h after 10 mU·mL−1 bleomycin treatment for 3 days. The multimeric MAVS aggregation and expression of MAVS and oxidative phosphorylation (OXPHOS) complexes were evaluated by BN-PAGE and SDS-PAGE, respectively. Voltage-dependent anionic channel (VDAC) protein was evaluated as loading controls of mitochondria for panels (a) and (e). e) Multimeric MAVS aggregation was evaluated and compared by the application of BN-PAGE on lung specimens obtained from idiopathic pulmonary fibrosis (IPF) patients or healthy controls (n=9 per control group, n=13 per IPF group; each number indicates an individual subject). The cell lysate from human embryonic kidney 293 cells treated with nigericin was used as a positive control to evaluate MAVS aggregation. β-Actin was used as a loading control for panels (a–c) and (e). f) Fisher's exact test was applied to determine statistical significance of the result presented in panel (e).
In experiments to determine whether the bleomycin-induced multimeric aggregation of MAVS in vivo can be modulated by ABT-263, nintedanib or pirfenidone, the multimeric aggregation of MAVS was markedly attenuated only after ABT-263 treatment (figure 7b, c). Among multiple drugs tested, all BH3 mimetics could ameliorate the multimeric aggregation of MAVS (figure 7d). In contrast, nintedanib and pirfenidone failed to ameliorate it (figure 7d). Finally, to determine the clinical relevance of the above finding observed in murine disease modelling, an experiment was undertaken to test whether MAVS aggregation was observed in the lung tissue from human IPF patients. A significant increase in MAVS aggregation was observed in 11 of 13 specimens from IPF patients, while only two of nine specimens showed MAVS aggregation among controls, demonstrating a compelling statistical significance (figure 7e, f). Overall, these data demonstrate that persistent MAVS aggregation, a key event of MAVS signalling activation, is observed in lungs from IPF patients as well as in our mammalian model of IPF. Importantly, MAVS aggregation can be therapeutically targeted by BH3 mimetics.
Discussion
The current studies are novel because they demonstrate that 1) MAVS, a key adaptor of mitochondrial innate immune signalling, is significantly activated after fibrogenic injury; 2) the activation of MAVS is associated with various DAMP signalling pathways, especially cGAS-STING innate immune signalling; 3) proapoptotic BH3 mimetics can reduce MAVS expression as well as its multimeric aggregation; and, most importantly, 4) the multimeric aggregation of MAVS, a key event during MAVS signalling activation, is markedly enhanced in lungs of IPF patients. Overall, these studies suggest that MAVS plays a critical pathogenic role in experimental pulmonary fibrosis.
MAVS is the first mitochondria-localised protein to be linked to innate immunity [12] and is involved in multiple biological phenomena, including mitochondrial innate immune signalling, apoptosis, autophagy and metabolic functions [28–30]. Given that recent studies continue to highlight the functional roles of mitochondria and the underlying mechanisms by which mitochondrial dysfunction contributes to the pathogenesis of IPF [6, 31], the current studies emphasise mitochondrial significance by illuminating the role of MAVS-mediated DAMP signalling in fibrotic tissue injury/damage responses.
Danger theory has been implicated in the pathogenesis of many chronic disorders for which effective therapeutics are not available [32–34]; IPF is such an example. Intriguingly, MAVS, a molecule mostly studied in the context of viral pathogen-associated molecular pattern (PAMP) signalling, is significantly activated by bleomycin-induced injury, which is obviously not related to viral infection. In addition, persistent MAVS aggregation is observed in the lungs of IPF patients. Fibrogenic tissue injury may drive fibrosis by inducing various endogenous DAMP signalling molecules, which could be converged to MAVS. The potential significance of MAVS signalling in the pathogenesis of non-viral injury-induced chronic diseases has previously been reported [35].
The phosphorylation state of receptor-regulated Smads (R-Smads) such as Smad2/3 determines the most critical event in TGF-β super family signalling [36]. Multiple TGF-β-dependent and -independent complex regulatory mechanisms have been identified that dynamically and tightly control the phosphorylation status of R-Smads [36, 37]. Our current study adds further knowledge to this important research area by revealing the regulation of R-Smads phosphorylation in a MAVS-dependent manner. In addition, there have been few reports on whether MAVS plays a role in cellular senescence. By demonstrating that bleomycin injury-induced cellular senescence is significantly attenuated in a MAVS-dependent manner, our study provides additional mechanistic insight into how MAVS contributes to the development of pulmonary fibrosis. The significance of these novel observations should be explored in future studies, considering that cellular senescence has emerged as a crucial driving force of IPF pathogenesis [25, 26, 38].
In line with our studies, a recent study demonstrated the potential efficacy of targeting myofibroblast anti-apoptotic proteins with BH3 mimetic drugs in skin fibrosis [39]. In addition, the pharmacological effects of BH3 mimetics that can selectively remove senescent cells, called senolysis, have recently been reported as having therapeutic potential for the treatment of age-associated diseases [40, 41]. Regarding this, our finding that BH3 mimetics can attenuate MAVS expression in vitro and in vivo opens up new research avenues for the underlying mechanisms by which BH3 mimetics function as novel therapeutics. Specifically, when combined with the inhibitory role of MAVS in regulating multiple DAMP signalling as well as cellular senescence, the current study enables us to speculate that BH3 mimetics might exert their anti-fibrotic effects by inhibiting MAVS and the consequent DAMP signalling pathways, in addition to their known senolytic effects [40]. Or, the senolytic effects of BH3 mimetics might be linked to their inhibitory function on MAVS through an intricate regulatory network. These questions about the senescent or non-senescent impact of BH3 mimetics on the regulation of fibrosis, especially in relation to their inhibitory function on MAVS, have rarely been explored. Our novel observations pose intriguing mechanistic questions for exploring the therapeutic potential that BH3 mimetics may have in the treatment of fibrotic disorders, including IPF in humans.
To date, nintedanib and pirfenidone are the only approved drugs known to decelerate IPF disease progression [23, 24]. Nintedanib is a multi-tyrosine kinase inhibitor that inhibits the receptor kinases of platelet-derived growth factor, fibroblast growth factor and vascular endothelial growth factor, which are all thought to play an important role in the pathogenesis of IPF [24]. Pirfenidone is known to have anti-oxidant, anti-fibrotic and anti-inflammatory properties [42]. However, their modulatory effects on the expression of MAVS or its mediated DAMP signalling have not yet been explored. Although this question has not been investigated in a comprehensive manner, our studies suggest that therapeutic effects of nintedanib and pirfenidone for the treatment of IPF are not related to the modulation of MAVS and its signalling.
Several issues remain unanswered in the current studies. Detailed mechanistic explanations are not provided about how endogenous DAMP-mediated innate immune signalling pathways may contribute to the pathogenesis of IPF. The molecular mechanisms by which pro-apoptotic BH3 mimetics function to reduce MAVS expression and aggregation have not been investigated. In addition, an interesting question is raised about whether synergistic therapeutic effects will be observed if a BH3 mimetic is combined with nintedanib or pirfenidone for IPF treatment. Future studies will be required to explore these important questions.
Although bleomycin-induced experimental fibrosis modelling may not fully recapitulate the pathogenesis of human IPF, multimeric aggregation of MAVS was markedly observed in the lungs of IPF patients as well as the late fibrotic phase in our experimental modelling. We believe that a similar observation of multimeric MAVS aggregation in humans and our murine model highlights the potential significance of MAVS in IPF pathogenesis. An enhanced understanding of MAVS and its signalling in fibrotic tissue injury or damage responses may have a significant impact on overall fibrosis biology, including IPF.
In conclusion, these studies demonstrate that MAVS plays an important role in the development of experimental pulmonary fibrosis in a mammalian model of IPF, and provide a rationale that targeting MAVS or its signalling may be a feasible strategy for the regulation of pulmonary fibrosis.
Supplementary material
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material ERJ-00652-2020.SUPPLEMENT
Shareable PDF
Supplementary Material
This one-page PDF can be shared freely online.
Shareable PDF ERJ-00652-2020.Shareable
Acknowledgments
The authors thank Susan Ardito for excellent administrative assistance.
Footnotes
This article has an editorial commentary: https://doi.org/10.1183/13993003.04500-2020
This article has supplementary material available from erj.ersjournals.com
Author contributions: Conceived the idea and designed the experiments (S-H. Kim, J.Y. Lee, M-J. Kang); performed experiments (S-H. Kim, J.Y. Lee, H.J. Shin, C.M. Yoon, S.W. Lee); provided important reagents/tools (S.W. Lee, C.M. Yoon, I. Rosas, N. Kaminski); provided scientific insight (S-H. Kim, E. Herzog, C. Dela Cruz, N. Kaminski, M-J. Kang); analysed data (S-H. Kim, E. Herzog, M-J. Kang); drafted the manuscript (S-H. Kim, J.Y. Lee, M-J. Kang); all of the authors reviewed the manuscript.
Conflict of interest: S-H. Kim has nothing to disclose.
Conflict of interest: J.Y. Lee has nothing to disclose.
Conflict of interest: C.M. Yoon has nothing to disclose.
Conflict of interest: H.J. Shin has nothing to disclose.
Conflict of interest: S.W. Lee has nothing to disclose.
Conflict of interest: I. Rosas has nothing to disclose.
Conflict of interest: E. Herzog reports personal fees from Boehringer Ingelheim, Merck and Genentech, and grants from Boehringer Ingelheim, Sanofi and Bristol Myers, outside the submitted work.
Conflict of interest: C. Dela Cruz has nothing to disclose.
Conflict of interest: N. Kaminski reports personal fees from Biogen Idec (consultant), Boehringer Ingelheim (consultant), Third Rock (consultant), Pliant (Advisory Board), Samumed (consultant), NuMedii (consultant), Indaloo (consultant), Theravance (consultant), LifeMax (consultant) and Three Lake Partners (Scientific Advisory Committee), and non-financial support from Miragen (for pulmonary fibrosis work), outside the submitted work; and has a patent New Threapies in Pulmonary Fibrosis with royalties paid to Biotech, a patent Peripheral Blood Gene Expression issued, and serves as Deputy Editor of Thorax, BMJ.
Conflict of interest: M-J. Kang has nothing to disclose.
Support statement: These studies were supported by National Institute on Aging (grant: R01AG053495 to M-J. Kang) and the National Heart, Lung, and Blood Institute (grant: R01HL130283 to M-J. Kang). Funding information for this article has been deposited with the Crossref Funder Registry.
- Received March 16, 2020.
- Accepted October 12, 2020.
- Copyright ©ERS 2021. For reproduction rights and permissions contact permissions{at}ersnet.org
References
