Upregulation of activin-B and follistatin in pulmonary fibrosis – a translational study using human biopsies and a specific inhibitor in mouse fibrosis models

An approval for collecting tissue for research purposes from diagnostic clinical samples was received from the Helsinki University Hospital Ethics Board (HUS 426/13/03/01/09). All patients that participated to the study gave written informed consent except for the lung transplant donors that were deceased. A permission to use tissue samples from deceased patients was obtained from the national supervisory authority of welfare and health (3317/05.01.00.06/2011).

For RNA isolation and expression array analysis, four IPF and four control specimens were carefully chosen in order to minimize variability due to confounding factors. Each patient’s medical history, HRCT evaluation and clinical course of the disease indicated that they had classical IPF; this was confirmed with lung biopsy. Three IPF biopsies were collected from explanted lungs at the time of transplantation, one from a diagnostic biopsy procedure. The mean age of the IPF patients was 55 (range 49–57), and all patients were nonsmokers. Control specimens in the array analysis consisted of patients who underwent surgical lobectomy due to benign pulmonary tumor (hamartoma), showed normal pulmonary histology, and had normal lung function (all patients were nonsmokers, mean age 64, range 59–70).

For immunohistochemical analyses, samples from diagnostic biopsies or lung transplantation, or both, were obtained from the University of Helsinki, Department of Pathology. Clinical records and HRCT scans were evaluated from all the patients according to the current guidelines to ensure correct diagnosis. Control lung samples were biopsies or segments of healthy donor lung that was used for transplantation. All the IPF patients had typical, advanced UIP pattern in the biopsy of the explanted lung. All the IPF patients were male. Their mean forced vital capacity (FVC % from predicted) before the operation was 47, and the mean volume-adjusted diffusing capacity was 60.5. Mean patient age was 52 (range 40–59).

Mouse monoclonal antibodies (mAbs) against human activin-A, activin-B and follistatin subunits (encoded by INHBA, INHBB and follistatin genes, respectively) were generated by AnshLabs LLC (Webster, TX) by using peptide-conjugates that contain peptide sequences of the C terminal portion of the mature region of each activin. The activin-reacting mAbs were initially selected against commercially available human recombinant activin-A and -B (R&D Systems, Minneapolis, MN) and internally produced chinese hamster ovary (CHO) cell derived human recombinant activins at AnshLabs (not shown). The mAb 18/26A (for activin-A/INHBA), mAb 12/9A (for activin-B/INHBB) and mAb 4/73C for follistatin were selected for immunohistochemistry based on their specificity in Western blots and specific reactivity towards granulosa cells in human ovarian sections (not shown).

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded alcohol. Antigens were retrieved by heating the sections in 0.01 M citrate buffer (pH 6.0). For immunostaining, Novolink Polymer Detection System (Novocastra, Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK) was used according to the manufacturer’s protocol. The sections were exposed to the primary antibodies at room temperature for 1 h. The bound antibodies were visualized by DAB. The sections were counterstained with Mayer’s haematoxylin and mounted on glass slides.

Snap-frozen and subsequently pulverized mouse lung was lysed on ice for 15 min in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2% sodium deoxycholate) that contained protease inhibitors (Pierce, Rockford, IL). Protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE using 4-20% gradient Tris-glycine gels (Lonza, Basel, Switzerland) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) using a semi-dry blotting system (Bio-Rad). Membranes were blocked with 5% non-fat milk in TBS/0.05% Tween-20 to prevent non-specific binding of the antibodies. Next, they were incubated with anti-inhibin ß_B monoclonal antibody (46A/F) [18], and then with biotin-conjugated anti-mouse secondary antibody (DAKO, Glostrup, Denmark) in TBS/0.05% Tween-20 containing 5% bovine serum albumin at room temperature. After several washing steps, the final detection was performed using horseradish peroxidase-conjugated streptavidin and an enhanced chemiluminescence Western blotting detection system (Amersham, Freiburg, Germany). Analyses of protein band intensities were performed using the Scion Image analysis program (Scion Corporation).

Total RNA was extracted from homogenized lung tissue samples with an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and reverse transcribed using iScript cDNA synthesis kit (Bio-Rad). The cDNAs were amplified using TaqMan Assays-on-Demand gene expression products (Applied Biosystems) and CFX96 Real-time PCR detection system (Bio-Rad). Control amplifications directly from RNA were performed in order to rule out DNA contamination. The relative gene expression differences were calculated with the comparative delta delta cycle threshold (ΔΔCT) method, and the results have been reported as mRNA expression levels normalized to the levels of a gene with a constant expression (TATA-binding protein).

Pathway-specific PCR array (#PAHS-035; SABiosciences) was used to analyze mRNA expression levels of genes associated with the TGF-ß/BMP signaling pathway. Following the manufacturer’s instructions, reverse transcription was performed using DNase I treated RNA and RT2 First Strand Kit (SABiosciences) followed by PCR amplification using CFX384 real-time PCR detection system (Bio-Rad). Gene expression levels in control lung tissue (n = 4) were compared to the levels in IPF lung tissue (n = 4) using SABioscience PCR data analysis tools.

The recombinant fusion protein containing the ectodomain of human ActRIIB fused to the Fc domain of human IgG1 (sActRIIB-Fc) was produced in-house as described previously [12, 17]. For the final production of the chimeric protein, CHO cells were transfected with the expression construct via lipofection (Fugene 6; Roche) and selected with puromycin (Sigma-Aldrich). During selection, cells were grown in DMEM supplemented with 2 mM L-glutamine, 100 μl/ml streptomycin, 100 IU/ml penicillin, and 10% fetal calf serum. For large-scale expression, cells were adapted to CD OptiCHO medium (Gibco) supplemented with 2 mM L-glutamine and grown in suspension in an orbital shaker. Cell culture supernatants were clarified by filtration through 0.22 μm membrane (Steritop, Millipore). 1 M NaCl, 5 mM imidazole, and Protease Inhibitor Cocktail (Thermo Scientific) were added into the clarified supernatants, and the solution was pumped through a HisTrap FF Crude column (GE Healthcare Life Sciences, Uppsala, Sweden) at 4°C. Protein was eluted with increasing imidazole concentrations, dialyzed against phosphate-buffered saline (PBS), and finally concentrated with Amicon Ultra concentrator (30 000 MWCO, Millipore).

A permission to use experimental animals was received from the regional state administrative agency of Southern Finland (ESAVI/871/04.10.03/2012) and their ethical board. The reporting on animal experiments was done in accordance to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (http://​www.​nc3rs.​org.​uk/​page.​asp?​id=​1357). Male C57b6J mice were used for the experiments. The mice were exposed to either crocidolite asbestos fibers (0.5 mg/50 μl sterile PBS) or silicon dioxide (SiO₂) (Sigma; 2.5 mg/50 μl sterile PBS) as described previously [19]. Two subsequent oropharyngeal aspiration doses were given on days 0 and 7 of the experiment to ensure even exposure of all animals. The mice were harvested on day 28 after exposure. sActRIIB-Fc was administered via i.p. injections at a dose of 5 mg/kg twice a week during the entire observation period. Knowing that sActRIIB-Fc increases body mass, the mice were weighed at each drug injection to monitor the drug effect [17, 20, 21]. At the time of harvest, mice were euthanized with carbon dioxide, after which BALF samples were collected by injecting 2×300 μl of sterile PBS to the trachea. The BALF cells were counted using Bio-Rad TC10 automated cell counter. Differential cell counts were obtained by microscopy of May-Grünwald-Giemsa-stained cytocentrifuge preparates: 150 μl BALF was loaded to cytospin chambers containing Superfrost Ultra Plus glass slides (Menzel GmbH & Co KG, Braunschweig, Germany) and centrifuged for 8 minutes, 500 rpm. The right lung was snap-frozen and subsequently pulverized for hydroxyproline and RNA and/or protein analysis. The left lung was fixed with 3% paraformaldehyde for histological analysis. For sActRIIB-Fc administration and analysis of possible alterations in body composition due to asbestos or silica exposure or both, hindlimb skeletal muscles and epididymal/retroperitoneal fat was prepared and weighed using an analytical scale. Histological evaluation was performed as described and validated previously [22].

All comparisons were made using nonparametric tests with SPSS software (IBM). Multiple group comparisons were made using Kruskal-Wallis test, and two-group comparisons were made using Mann-Whitney test. If multiple comparisons were made between two groups, the Bonferroni correction was applied to P values. P values below 0.05 were considered statistically significant.

Total RNA was isolated from frozen human lung tissue samples from control subjects and IPF patients. A commercial PCR array was used to analyze the mRNA expression levels of the TGF-ß/BMP pathway genes (see Methods). As expected, the major fibrotic collagen genes COL1A1 (p = 0.011), COL1A2 (p = 0.005) and COL3A1 (p = 0.018) were significantly upregulated in the IPF patient lungs (Figure 1A). In addition, LTBP-1 (p = 0.002), IGF-1 (p = 0.011) and IGFBP3 (p = 0.002) were found upregulated as previously described [23‐25]. A trend for increased levels of INHBB mRNA (p = 0.088), which encodes activin-B subunit was observed. The mRNA expression of the activin inhibitor protein follistatin was also notably increased in the IPF lungs (p = 0.005). Upregulation of INHBB and FST mRNA levels were confirmed by quantitative RT-PCR (Figure 1B). The TGF-ß/BMP pathway genes BMP2 (p = 0.072), BMP3 (p = 0.005), NOG (p = 0.04), FOS (p = 0.087) and JUN (p = 0.083) were downregulated in the IPF lungs.

Immunoreactivity for activin subunits was markedly increased in human IPF/UIP tissue when compared to controls, especially in the IPF/UIP lungs’ hyperplastic alveolar epithelial cells (Figure 2 and Table 1). Follistatin immunoreactivity was seen in the control and IPF lungs at high intensity. In the IPF/UIP tissue, high-intensity follistatin staining was seen in the activated alveolar epithelium and parenchymal macrophages. In control lungs, immunoreactivity was seen in macrophages or type-II-appearing cells or both. In control samples, immunoreactivity was observed in some alveolar macrophages and the bronchial epithelium for inhibin ß_A and follistatin, and for the inhibin ß_B subunit mainly in the bronchial epithelium. In IPF/UIP tissue, immunoreactivity for inhibin ß_A subunit was observed in the activated alveolar epithelial cells, macrophage-appearing cells in the lung parenchyma, and alveolar space, whereas fibroblastic foci in the lung parenchymal tissue were negative (Figure 2). Inhibin ß_B antibody showed immunoreactivity specifically in hyperplastic alveolar epithelial cells and bronchial epithelial cells. Macrophages remained mostly negative for inhibin ß_B in the IPF/UIP lung.

Next, we analyzed the expression of activin subunits in two different mouse models of IPF, asbestos-induced fibrosis and silicon dioxide (silica)-induced fibrosis. In both models progressive fibrosis is observed, and the histopathological alterations resemble human IPF/UIP: patchy areas of fibrosis and inflammatory cell aggregates and areas of preserved normal lung architecture. In control mice, Inhbb mRNA was expressed at 7–10 fold higher levels than Inhba mRNA (Figure 3A). In both models, Inhba mRNA was markedly increased in the fibrotic lung tissue, whereas Follistatin levels remained essentially unaltered (Figure 3A). Interestingly, in the silica-induced fibrosis model also Inhbb mRNA levels were significantly elevated in the lung tissue (Figure 3A). The upregulation of activin-B in the silica-induced fibrosis model was confirmed at the protein level (Figure 3B and C).

As marked upregulation of activin subunits was observed both in human and mouse IPF tissue, we explored the effects of inhibition of activin function on inflammation, fibrosis, and body composition using a soluble inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of human IgG. Treatment with sActRIIB-Fc started immediately after exposure of mice either to asbestos or silica (day 1). After exposure, there was a transient reduction or slowing down in the weight gain of the animals (Figure 4A). After two weeks, the mice treated with sActRIIB-Fc showed a clear increase in body weight when compared to both non-treated and non-treated, non-exposed controls. By the end of the follow-up time, the sActRIIB-Fc treated mice were approximately 4 g heavier than the non-treated, exposed control animals in both models. Since sActRIIB-Fc is known to block myostatin and activin-A function as an inhibitor of muscle growth [17, 20, 21] weight gain was expected. Measurements of quadriceps femoris muscle and gastrocnemius muscles (Figure 4B) as well as extensor digitorum longus, soleus and tibialis anterior muscle mass (not shown) showed significant increase in sActRIIB-Fc-treated animals, while no alterations were observed in non-treated, exposed animals (Figure 4B). On average, muscle masses were increased by 35.9% (asbestos-exposed) and 30.7% (silica-exposed) in the sActRIIB-Fc-treated animals. Heart mass was comparable in all the animal groups (not shown). These results confirm the expected function of the soluble inhibitor in both models and provide evidence that exposure of mice to significant amounts of silica or asbestos do not lead to permanent muscle atrophy.Epididymal fat mass was measured in all the animal groups and retroperitoneal fat mass in the silica model. In mice exposed to asbestos, epididymal fat mass decreased (Figure 4C) in both the treated and non-treated groups. In silica-exposed mice, epididymal fat mass decreased marginally with a further decrease in the sActRIIB-Fc-treated mice (Figure 4C). Furthermore, retroperitoneal fat mass decreased in both silica-exposed animal groups (not shown). These results suggest that fibrotic mice have a reduced fat mass, which may reflect increased energy consumption due to either inflammation or increased respiratory work.

BALF total cell counts increased in mice exposed to asbestos or silica as expected (Figure 5A). In both models, there was a clear decrease of alveolar total cell numbers in the animals treated with the inhibitor, suggesting that neutralization of activins has an attenuating effect on the cellular response to alveolar injury. The relative numbers (percentages) of neutrophils and lymphocytes increased significantly in asbestos-exposed mice, and this was not altered by treatment with sActRIIB-Fc (not shown). There were no significant changes in tissue inflammatory cell numbers (Figure 5B).

As the upregulation of activins was observed in human lung tissue in relationship to lung fibrosis, we wanted to test whether efficient blockade of activins could modulate the fibrotic response in these two different mouse models of progressive lung fibrosis. Lung tissue mRNA expression levels of Inhba were not altered by treatment with sActRIIB-Fc (not shown). There was a slight, but statistically nonsignificant, decrease in Inhbb expression in sActRIIB-Fc-treated asbestos-exposed mice (Figure 6A). In agreement with the known regulatory effect of activin on follistatin expression [26], treatment with the sActRIIB-Fc decreased Follistatin mRNA expression in the lung tissue of these mice (Figure 6A). Interestingly, there was also a trend towards decreased mRNA expression of major fibrotic genes (Col1A1, Col3A1, and tenascin-C) in sActRIIB-Fc treated asbestos-exposed animals (Figure 6B). However, these alterations were not reflected in tissue hydroxyproline levels (Figure 6C), but there was a 20% decrease in the histological fibrosis score evaluated from haematoxylin-eosin-stained lung tissue sections in the asbestos-exposed sActRIIB-Fc treated animals in comparison to controls (Figure 7). In contrast to what was observed in the asbestos-exposed animals, there was no reduction of lung tissue Follistatin mRNA expression in silica-exposed, sActRIIB-Fc-treated mice (Figure 6A). Expression of fibrotic genes and histological fibrosis score tended to further increase slightly in the treated animals (Figures 6B,D). This suggests that the mechanism of fibrosis formation in these two mouse models is different. Hydroxyproline levels were unaltered by sActRIIB-Fc (Figure 6C).