Fig. 6: Mesothelial Cathepsin B drives pleural matrix transfer and pulmonary fibrosis.

a scRNA-Seq data from bleomycin-installed mice and ILD patients. b Representative immunolabeling of Cathepsin histology images of ex vivo mouse lungs treated with IL-18 or transwell coculture with IL-18^high Macrophages. n = 10 biological replicates (C57BL/6J WT mice) and 5 independent experiments. Scale bars: 20 µm. c, d Representative immunolabeling histology images of Cathepsin B in mouse lungs 7, 14 and 45 days p.b.i. or 15 days post-herpes virus. n = 10 biological replicates (C57BL/6J WT mice or IFN-γ-R⁻/⁻ mice) and 5 independent experiments. p.b.i.: Day 7: p = 5.71e-10, Day14: p = 8.59e-11; post-herpes virus: Day15: p = 7.32e-12, Day 45: p = 1.59e-16. Scale bars: 50 µm. e Representative histology images of mouse lungs 14 days p.b.i. Mice were intrapleurally injected with NHS-FITC labelling mix. The next day bleomycin was installed and cathepsin B inhibitor were applied every other day in 10 µm concentration, DMSO acted as control. Log-rank test was used for statistical comparison. n = 6 biological replicates (C57BL/6J WT mice) and 3 independent experiments. Scale bars: 1000 µm (Fluorescence); 100 µm (Histology). f Survival of mice in (e). Log-rank test was used for statistical comparison. g Representative immunolabeling histology images of Cathepsin B and its inhibitor Cystatin A in mouse lungs 3 days post-intrapleural AAV injection. n = 6 biological (C57BL/6J WT mice) and 6 independent experiments. Scale bars: 500 µm; high magnification: 20 µm. h Representative histology images of mouse lungs 14 days p.b.i. Mice were intrapleurally injected with NHS-FITC labelling mix. AAV particles encoding for Cathepsin B inhibitor Cystatin A were also applied intrapleurally; then, five days later bleomycin was installed. For Cathepsin B coding AAVs no bleomycin was applied. n= six biological replicates (C57BL/6J WT mice) and 3 independent experiments. Masson trichrome was used to visualize structural changes, pSMAD2/3 staining visualized fibroblast activation. FITC: p = 1.69e-07, pSAMD: p = 6.84e-13. Scale bars: Fluorescence 1000 µm (Overview); 100 µm (pSMAD2/3 staining); Masson Trichrome 100 µm. i Bodyweight and survival of mice in (h). Log-rank test was used for statistical comparison. j Schematic representation of AAV-Cystatin mediated intervention in bleomycin lung fibrosis model experiment. Mice were treated with bleomycin and seven days later, mice were intrapleurally injected with AAV-Cystatin A. k Representative histology- and pSMAD2/3 immuno- staining images 24 days and bodyweights of animals after bleomycin treatment. n = 6 biological replicates (C57BL/6J WT mice) and 3 independent experiments. Scale bars: immunostainings: 100 µm; Masson trichrome 100 µm. Log-rank test was used for statistical comparison of bodyweights. pSAMD: p = 9.46e-11. l Schematic representation of AAV-Cystatin mediated intervention in herpes virus lung fibrosis model. Mice were treated with herpes virus and 45 days later, mice were intrapleurally injected with AAV-Cystatin A. m Representative histology- and pSMAD2/3 immuno- staining images 90 days after Herpes virus treatment. n = 6 biological replicates (IFN-γ-R⁻/⁻ mice) and 3 independent experiments. Scale bars: immunostainings: 100 µm; Masson trichrome 50 µm. pSAMD: p = 2.54e-09. All data represented in Fig. 6 are mean ± SD. One-way ANOVA was used for the multiple comparison. Single comparison was performed by two-sided independent T-test. (***P < 0.001; NS= not significant).