Fig 2: GDF15 promotes profibrotic responses in lung fibroblasts.(A) TMLC reporter cells were stimulated with rGDF15 or TGF-ß for 24 hours, and luminescence corresponding to PAI1 receptor activity was measured; n = 3 biological replicates. (B) NHLF were incubated for 48 hours with rGDF15 alone, and/or with 0.5 ng/mL of TGF-ß, or with SB525334 inhibitor. Next, aSMA and nuclei were immunofluorescence labeled, and the percentage of aSMA+ cells was quantified using MetaXpress High Content Image Analysis Software. Data are expressed as mean ± SD of n = 3 biological replicates. (C and D) The expression of COL1A1 (C) and COL3A1 (D) in rGDF15-stimulated NHLF was calculated after the normalization to GAPDH housekeeping gene expression and compared with untreated cells, shown as 2–??CT (fold change) and presented using box-and-whisker plots, with the line in the middle plotted at the median. *P < 0.05 and ****P < 0.000 by 1-way ANOVA; n = 4 biological replicates.

Fig 3: GDF15 expression increases in IPF lung and in histopathological lesions.(A) Representative IHC GDF15 staining of human lung tissue derived from healthy individual (n = 10). (B) Representative IHC GDF15 staining of human lung tissue from patients with IPF (n = 9) and corresponding MTC-stained sections. Arrows indicate GDF1+ cells in fibroblast foci (B, middle panel) and hyperplastic epithelium of small airways (B, bottom panel) in lung peripheral tissue. Scale bars: 100 µm (A) and 250 µm (B). (C) GDF15 expression in whole sections from healthy (n = 10) and IPF (n = 9) lung biopsies was quantified using Visiopharm software. Data are shown as mean ± SD. ***P = 0.0004, using 2-tailed unpaired Mann-Whitney U test.

Fig 4: Neutralization of GDF15 attenuates bleomycin-induced collagen deposition.(A) Schematic representation of experimental procedure for GDF15 mAb administration in bleomycin-induced fibrosis model. (B) Lung tissue hydroxyproline content was measured in differently treated animals. (C) Representative MTC-stained sections (top panel; collagen in blue, nuclei in red) and the respective digital reconstructed images (bottom panel) in Visiopharm software correspond to the lung sections from differently treated groups (n = 6–8 animals per group). Healthy parenchyma (cyan) and dense damaged area (purple) are marked. (D) Imaged-based quantification of dense damaged area in differently treated groups. (E) High magnification of dense MTC-stained damaged area (top panel) in bleomycin-treated (bleo) lungs with and without anti-GDF15 antibody, and corresponding classifiers obtained by Visiopharm software (bottom panel; collagen marked in red). (F and G) Quantification of collagen (after MTC staining) in dense damaged area and in healthy parenchymal area in murine lungs from differently treated groups. Data are presented as box-and-whisker plots. n = 6–8; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (analyzed using multiple Kruskal-Wallis test). Scale bars: 1 mm (B) and 50 µm (E).

Fig 5: Diseased lung ECM promotes fibroblast-to-myofibroblast transition.(A and B) Decellularized and weight-normalized ECM from healthy or IPF lung (n = 8 donors) was cocultured with NHLF using 3D spheroid hanging drop system, and aSMA and nuclei were immunofluorescence labeled. Image analysis–based aSMA quantification was performed using ImageXpress Micro system, and the percentage of aSMA+ cells was calculated in each spheroid (>14 spheroids per patient) using MetaXpress High Content Image Analysis Software at day 4 (A) and day 8 (B) of culture. The data are presented as mean ± SD, with dots representing single patients, and they were statistically analyzed with 2-tailed Mann Whitney U test in comparison with healthy subjects; *P < 0.05 and ****P < 0.0001. (C) Representative immunofluorescence staining (5 experiments conducted) of aSMA (red) and nuclei (blue) in 3D spheroids, day 8. Magnified area is marked with dotted line. Scale bar: 100 µm. (D) PCA plot representing the data from proteomic analysis of decellularized ECM derived from healthy lung (blue; n = 7 donors) and IPF lung (pink; n = 7 donors). (E) IPA analysis of the top 10 significantly (–log[P value] > 1.30) enriched canonical pathways based on differentially expressed proteins in IPF ECM compared with healthy ECM. The data are presented as IPA –log(P value), and the ratio of proteins is represented in each pathway. (F) Heatmap comparison of significantly altered proteins (FC > 2.8 and FC < 0.36, q < 0.05, statistically analyzed with unpaired Student’s t test), identified in the proteome of IPF (pink) and healthy (blue) ECM from patient lung-derived samples. (G) The top-ranked proteins identified in IPF ECM plotted based on abundance and compared with healthy ECM. The data are expressed as log2FC. (H and I) Box-and-whisker plots representing FC over internal control (ref) for TGF-ß1 (H) and GDF15 (I) identified in the proteome of healthy and IPF lung ECM (n = 7, dots represent single patients). Statistically analyzed with Mann Whitney U test; ***P < 0.001.