Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorβ1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

Fig. 1. The culture supernatants from three strains of S. haemolyticus induce apoptosis of alveolar epithelial cells.

a A549 alveolar epithelial cells were cultured in the presence of the (1/10 dilution) culture supernatants from strains 1, 7, and 12 of S. haemolyticus for 48 h. A549 cells treated with the culture supernatant from S. nepalensis strain CNDG were the positive controls and cells treated with saline were the negative controls. b The percentage of apoptotic cells was determined by flow cytometry and quantified. N = 4 in each group. Data are expressed as the mean ± S.D. Statistical analysis was performed using ANOVA with a post hoc Newman–Keuls test. ***p < 0.001; ****p < 0.0001. c, d Western blotting of A549 cells cultured in the presence of the culture supernatants of the three S. haemolyticus strains and S. nepalensis. In (d) n = 3 in each group. Representative blots from two independent experiments with similar results are shown. Data are expressed as the mean ± S.D. Statistical analysis was performed using ANOVA with a post hoc Newman–Keuls test. ****p < 0.0001. e–n Transmission electron photo-micrograph of alveolar epithelial cell apoptosis induced by the culture supernatant from Staphylococcus haemolyticus strain 12. A549 cells were cultured in the presence of the bacterial medium (uninoculated) as control and the culture supernatant from S. haemolyticus strain 12 grown for 48 h, and the cells were evaluated by transmission electron microscopy as described under Methods. S., Staphylococcus. Scale bars are 5 µm in e, f, g, j, l, 10 µm in k and 2 µm in h, i, m, n. Representative images from two independent experiments with similar results are shown. The source data underlying (b, d) are provided in the Source Data file.

Fig. 2. Activation of cleaved caspases by corisin.

a Apoptosis occurs by the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by binding of ligands to membrane-bound death receptors that results in activation of intracellular signaling and cleavage of procaspase-8 to caspase-8. The intrinsic pathway is regulated by the mitochondrion. In the presence of the stimulus, perturbation of the mitochondrial membrane increases its permeability and causes the release of suppressors of baculoviral inhibitors of apoptosis repeat-containing (BIRC) proteins and proapoptotic factors, which contribute to apoptosome formation to cleave procaspase-9 to caspase-9. Both caspase-8 and caspase-9 can activate caspase-3, the effector of apoptosis. b, c A549 alveolar epithelial cells were cultured in the presence of 5 µM corisin or scrambled peptide for 48 h. The percentage of cells positive for cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 was determined by flow cytometry and quantified. N = 6 in each group. Data are expressed as the mean ± S.D. Statistical analysis was performed using two-sided unpaired t test. The source data underlying (c) are provided in the Source Data file.

Fig. 3. A bacterial secreted product degrades transglycosylase to induce apoptosis of lung epithelial cells.

a A sodium dodecyl sulfate-polyacrylamide gel stained with silver staining after completing the electrophoresis of a reaction mixture containing digestion buffer, recombinant transglycosylase, and varying doses of the protease fraction (>10 kDa) prepared from Staphylococcus nepalensis culture supernatant and incubated at 37 °C for 2 h. A representative image from two independent experiments with similar results is shown. b A sodium dodecyl sulfate-polyacrylamide gel stained with silver staining after completing the electrophoresis of a reaction mixture containing digestion buffer, recombinant transglycosylase, and a dose of the protease fraction (>10 kDa) prepared from Staphylococcus nepalensis culture supernatant incubated at 37 °C at different time intervals. A representative image from two independent experiments with similar results is shown. c, d Flow cytometry analysis to evaluate apoptosis of A549 alveolar epithelial cells after treatment with the indicated reaction mixture. N = 4 in each group. Data are the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. ****p < 0.0001. The source data underlying (d) are provided in the Source Data file.

Fig. 4. Significant biological activity of native corisin in bacterial culture supernatant and BALF from IPF patients.

a, b A549 alveolar epithelial cells were cultured in the presence of culture diluted supernatants (1:10 dilution) from Staphylococcus (S.) nepalensis (n = 3) in the presence or absence of anticorisin neutralizing mAtb clone 21A (20 µg/ml) for 48 h, and apoptosis was evaluated by flow cytometry. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. ****p < 0.0001. c Concentration of native corisin in undiluted culture supernatant from S. nepalensis and S. haemolyticus. N = 4 in each group examined over two independent experiments. Bars indicate the mean ± S.D. d A549 alveolar epithelial cells were cultured in the presence of diluted bronchoalveolar lavage fluid (BALF) (1:2 dilution) from healthy controls (HC, n = 5) and idiopathic pulmonary fibrosis (IPF, n = 14) patients with acute exacerbation and the anticorisin neutralizing mAtb clone 21A or control IgG for 48 h, and apoptosis was evaluated by flow cytometry. Bars indicate the mean ± S.D. Three replicates performed for the study of each patient. e The mean percentage of apoptotic (Annexin V+) cells induced by diluted BALF from all healthy controls (n = 5) and IPF patients (n = 14) was compared between anticorisin mAtb and control IgG. Statistical analysis by two-sided Wilcoxon signed rank test. f Concentration of native corisin in undiluted BALF from healthy subjects (n = 5) and IPF patients (n = 14). Bars indicate the mean ± S.D. Statistical analysis by two-sided Mann–Whitney U test. g A549 cells were cultured up to subconfluency, synthetic corisin was added to cell culture medium at a final concentration of 10 µg/ml and medium samples were collected after 0, 1, 3, 6, 12, and 24 h to measure corisin levels and calculate the in vitro half-life of corisin in a cell system. N = 4 in each time point examined over two independent experiments. The half-life of synthetic corisin was calculated using an exponential decay equation model available in the GraphPad Prism version 7. Bars indicate the mean ± S.D. The source data underlying (b, c, d, e, f, g) are provided in the Source Data file.

Fig. 5. Monoclonal anticorisin antibody inhibits acute exacerbation of pulmonary fibrosis in TGFβ1 TG mice.

TGFβ1 transgenic (TG) mice were randomly allocated into two groups with a matched grade of lung fibrosis and one group without lung fibrosis. A group of TGFβ1 TG mice (n = 6) with lung fibrosis received an intraperitoneal injection of anticorisin monoclonal antibody (mAtb) and another group (n = 6) with lung fibrosis received control IgG five times every 2 days before intratracheal instillation of corisin. The group of TGFβ1 TG mice without fibrosis (n = 5) received only intratracheal corisin. After euthanasia by an overdose of anesthesia,bronchoalveolar lavage fluid (BALF) was drawn from mice of each group, the BALF fluid was centrifuged and the pellet was used to evaluate the total cell count and differential cells. a Giemsa staining for differential cell count. Scale bars indicate 50 µm. b Count of total number of cells and neutrophils in BALF (total lymphocytes, macrophages and all cells). Data are the mean ± S.D. Statistical analysis was performed by ANOVA with a post hoc Neuman–Keuls test. *p < 0.05; **p < 0.01; ***p < 0.001. c The levels of surfactant protein-D (SP-D), MUC5B protein, matrix metalloproteinase-1 (MMP-1), and MUC-1 were measured by commercially available enzyme immunoassay kits. Data are the mean ± S.D. Statistical analysis was performed by ANOVA with a post hoc Neuman–Keuls test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. TGFβ1 transforming growth factor β1. The source data underlying (b, c) are provided in the Source Data file.

Fig. 6. Amelioration of acute tissue injury, and apoptosis in the lungs of mice with bleomicin-induced pulmonary fibrosis treated with anticorisin monoclonal antibody.

Wild-type (WT) mice received bleomycin (BLM) by osmotic mini-pumps and treated with anticorisin monoclonal antibody (mAtb) (WT/BLM/anticorisin) or control IgG (WT/BLM/control IgG) by intraperitoneal route three times a week for 3 weeks. WT mice receiving saline (SAL) by osmotic mini-pumps and treated with anticorisin mAtb (WT/SAL/anticorisin) or control IgG (WT/SAL/control IgG) by intraperitoneal route three times a week for 3 weeks were the control mice. a The levels of osteopontin, MUC-1 and MUC5B were measured by enzyme immunoassays using commercial kits. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns not significant. b, c DNA fragmentation was evaluated by staining through terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL). Scale bars indicate 20 µm. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. ***p < 0.001. d, e Cleavage of caspase-3 confirmed by western blotting and quantified by an image software. Representative blots from two independent experiments with similar results are shown. N = 3 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 4 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. *p < 0.05; ****p < 0.0001. The source data underlying (a, c, e) are provided in the Source Data file.

Fig. 7. Amelioration of tissue fibrosis in mice with bleomycin-induced pulmonary fibrosis treated with anticorisin monoclonal antibody.

Wild-type (WT) mice received bleomycin (BLM) by osmotic mini-pumps and treated with anticorisin monoclonal antibody (mAtb) (WT/BLM/anticorisin) or control IgG (WT/BLM/control IgG) by intraperitoneal route three times a week for 3 weeks. WT mice receiving saline (SAL) by osmotic mini-pumps and treated with anticorisin mAtb (WT/SAL/anticorisin) or control IgG (WT/SAL/control IgG) by intraperitoneal route three times a week for 3 weeks were the control mice. a, b Computed tomography (CT) was performed 1 day before mouse euthanasia. The radiological findings of lung fibrosis were evaluated using a CT fibrosis score as described under Methods. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Data are the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. ****p < 0.0001. c, d The grade of lung fibrosis in hematoxylin & eosin (H&E) stained lung tissue were evaluated using the Ashcroft score. Scale bars indicate 200 µm. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. **p < 0.01; ****p < 0.0001. e, f Lung collagen deposition was evaluated by Masson’s trichrome staining, and the percentage of trichrome stain (+) area was measured using the WinRoof Image Processing Software. Scale bars indicate 200 µm. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. **p < 0.01; ****p < 0.0001. g The lung tissue content of hydroxyproline was measured by a colorimetric assay using a commercially available kit following the manufacturer instructions. N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups and n = 9 in WT/BLM/control IgG and WT/BLM/anticorisin groups. Bars indicate the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls test. ***p < 0.001; ****p < 0.0001. The source data underlying (b, d, f, g) are provided in the Source Data file.

Fig. 8. Monoclonal anticorisin mAtb attenuates severe lipopolysaccharide-induced acute lung injury.

a Wild-type mice were treated three times with control IgG or anticorisin monoclonal antibody at a dose of 20 mg/kg by intraperitoneal route once a day every other day. Mice received intratracheal instillation of a high-dose (150 µg) of lipopolysaccharide (LPS) or saline (SAL) 2 days after the last treatment with antibody and sacrificed 2 days after LPS instillation. Mice receiving intratracheal saline (SAL) and treated with control IgG. (SAL/control IgG) or anticorisin antibody (SAL/anticorisin) were the control mice. b, c Computed tomography (CT) was performed 1 day after the intratracheal instillation of lipopolysaccharide (LPS). N = 4 in WT/SAL/control IgG and WT/SAL/anticorisin groups, n = 5 in WT/LPS/control IgG group, and n = 8 in WT/LPS/anticorisin group. The radiological findings of LPS-acute lung injury were evaluated by measuring lung opacity on axial CT images using the WinRoof Image Processing Software as described under Methods. Data are the mean ± S.D. Statistical analysis by ANOVA with a post hoc Newman–Keuls’ test. *p < 0.05; ***p < 0.001. d, e Mice were sacrificed on day 2 after intratracheal LPS instillation and bronchoalveolar lavage fluid (BALF) was collected. BALF cells were counted using a nucleocounter and stained with Giemsa for differential cell counting as described under Methods. Scale bars indicate 20 µm. N = 4 in SAL/control IgG and SAL/anticorisin groups, n = 5 in LPS/control IgG group, and n = 8 in LPS/anticorisin group. Data are the mean ± S.D. Statistical analysis by ANOVA with a post hoc Neuman–Keuls test. ****p < 0.0001. ns not significant. f The levels of lactate dehydrogenase A (LDHA), surfactant protein-D (SP-D), matrix metalloproteinase-1 (MMP-1), MUC-1, tumor necrosis factorα (TNFα) and chemokine (C-C motif) ligand 2 (CCL2)) were measured using commercially available immunoassay kit following the manufacturer instruction. N = 4 in SAL/control IgG and SAL/anticorisin groups, n = 5 in LPS/control IgG group, and n = 8 in LPS/anticorisin group. Data are the mean ± S.D. Statistical analysis by ANOVA with a post hoc Neuman–Keuls test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns not significant. The source data underlying (c, e, f) are provided in the Source Data file.

Fig. 9. Significant increase in serum corisin level in idiopathic pulmonary fibrosis patients with acute exacerbation.

a, b Corisin was measured using an enzyme immune assay using rabbit polyclonal antitransglycosylase antibody as coating antibody and rat monoclonal anticorisin antibody as secondary antibody. Healthy controls, n = 6; all stable idiopathic pulmonary fibrosis (IPF) patients, n = 22; IPF patients with acute exacerbation, n = 22. Bars indicate the means ± S.D. Statistical difference between healthy controls and stable IPF patients was evaluated by two-sided unpaired t test, and statistical difference between stable IPF and acute exacerbation-IPF patients by two-tailed paired t test. The source data underlying (a, b) are provided in the Source Data file.

Fig. 10. A model of the process of corisin-induced acute alveolar damage during acute exacerbation of pulmonary fibrosis and the protective role of the anticorisin monoclonal antibody.

a Environmental factors that trigger enhanced fibrosis and injury (e.g., drugs, viral and bacterial infections, radiotherapy, or unknown factors), remodel the lung environment to enrich for a salt-tolerant microbial community. Within this community are Staphylococcus spp (e.g., S. nepalensis and S. haemolyticus) harboring transglycosylases with corisin and corisin-like peptides embedded in their C-terminal region. The corisin-containing bacteria secrete their transglycosylases together with a protease that cleaves the proapoptotic peptides from the transglycosylases. b Increased intra-alveolar concentration of the deadly peptides (corisin and corisin-like peptides) in the predisposed host stimulates secretion of inflammatory cytokines (TNFα, osteopontin) chemokines (MCP-1), profibrotic cytokines (TGFβ1), and periostin from alveolar epithelial cells that enhance the inflammatory response, the recruitment of fibroblasts, and the deposition of extracellular matrix in the lungs. Lung areas with enhanced apoptosis of alveolar wall lining epithelial cells caused by corisin or corisin-like peptides are replaced by lung fibrotic tissue. The process accelerates the clinical progression (acute exacerbation) of the disease that ultimately has a fatal outcome. The anticorisin monoclonal antibody binds to the peptides to block the pro-inflammatory and proapoptotic activity of the deadly peptides, ameliorating acute exacerbation of the disease. mAtb monoclonal antibody, TNFα tumor necrosis factor-α, MCP-1 monocyte chemoattractant protein-1, TGFβ1 transforming growth factor-β1.