Fig 2: uPAR deficiency led to uPA accumulation and increased IL-6 and ACE2 expression in lung tissue following bleomycin-induced lung damage: (A) Representative sections of lung tissue from wild-type (WT) mice 7, 14 or 28 days after instillation with bleomycin, stained for uPAR. uPAR was revealed with DAB chromogen substrate (brown). The scale bar in images is 100 µm. (B) Representative sections of lung tissue from wild-type (WT) and Plaur-deficient (Plaur-/-) mice 7 days after instillation with bleomycin, stained for uPA. uPA was revealed with DAB chromogen substrate (brown), and the slides were counterstained with hematoxylin (blue). The scale bar is 100 µm. (C) IL6 mRNA expression analyzed by RT-qPCR. The mRNA level was normalized to Rpl13a expression as a housekeeping gene; the normalization was carried out assuming the mean level of transcript in WT cells as 1. Data are presented as the mean ± SEM. * p < 0.05, 2-way ANOVA, Holm–Šídák’s test. (D) Representative sections of lung tissue from wild-type (WT) and Plaur-deficient (Plaur-/-) mice 28 days after instillation with bleomycin, stained for ACE2. ACE2 was revealed with DAB chromogen substrate (brown), and the slides were counterstained with hematoxylin (blue). The scale bar is 100 µm. (E) Ace2 mRNA expression as analyzed by RT-qPCR. The mRNA level was normalized to Rpl13a expression as a housekeeping gene; the normalization was carried out assuming the mean level of transcript in WT cells as 1. Data are presented as the mean ± SEM. ** p < 0.01, 2-way ANOVA, Holm–Šídák’s test.

Fig 3: PLAU and PLAUR expression in the mesenchymal cells of control, COVID-19 and PF lung samples: (A) UMAP embedding of jointly analyzed single-cell transcriptomes annotated by cell type (color) and disease status. Lung tissue transcriptomes of patients with COVID-19-associated pneumonia, pulmonary fibrosis (PF) and control lung samples from healthy donors, reported in Habermann et al. and Bharat et al. (GSE158127, GSE135893 [35,36]), were analyzed. Normalized expression levels of PLAU (B) and PLAUR (C) in different cell types of mesenchymal cells; statistics were calculated only for the cell types with the median normalized expression value > 0. *** padjusted < 0.0001 as assessed by FDR. IPF/other PF, idiopathic pulmonary fibrosis and other pulmonary fibrosis; umap, Uniform Manifold Approximation and Projection.

Fig 4: Differential progress of pulmonary fibrosis between wild-type (WT) and Plau-deficient (Plau-/-) or Plaur-deficient (Plaur-/-) mice after instillation with bleomycin. Bleomycin (3 mg/kg) was administered into WT, Plau-/- and Plaur-/- mice by intratracheal instillation at day 0. Instillation of NaCl solution was used as a control. (A) Kaplan–Meier survival curves, % ± SE, for WT, Plau-/- and Plaur-/- mice following intratracheal instillation of bleomycin. *** p < 0.001, log-rank test. (B) Mice were weighed before bleomycin or NaCl administration (day 0) and 28 days after. Body weight at day 28 divided by body weight day 0 (weight ratio) individual values, mean ± SEM are presented. * p < 0.05, 2-way ANOVA, Holm–Šídák’s test. (C) The % of lung tissue with fibrosis at day 0 and day 28 of bleomycin intratracheal instillation in WT, Plau-/- and Plaur-/- mice. * p < 0.05, ** p < 0.01, *** p < 0.001, 2-way ANOVA, Holm–Šídák’s test. (D) Typical MRI images obtained 28 days after bleomycin administration to WT, Plau-/- and Plaur-/- mice, the affected lung areas on the MRI images appear light, and the intact lungs appear dark. Asterisk indicates the area of severe lung damage. (E) Representative micrographs of 5-µm paraffin lung tissue sections stained with hematoxylin and eosin (H&E), Mallory stain (detection of total collagen), picrosirius red stain (selective detection of type I and III collagen) and phosphotungstic acid-hematoxylin stain (PTAH, detection of fibrin). Histological sections (HS) of lung tissues of WT, Plau-/- and Plaur-/- mice 28 days after bleomycin administration are presented. B, bronchus or bronchiole; F, fibrosis; V, vessel. The arrow points to collagen fibers in the pulmonary interstitium in the area of fibrosis. Scale bar—300 µm. (F) The analysis of collagen deposition in WT, Plau-/- and Plaur-/- mice 7, 14, 21 and 28 days after bleomycin administration by picrosirius red stain. The mean % of tissue area ± SEM with collagen deposition is presented. * p < 0.05, *** p < 0.001, **** p < 0.0001, 2-way ANOVA, Holm–Šídák’s test.

Fig 5: The putative scheme of COVID-19-induced pulmonary fibrosis regulation by uPA/uPAR system. In control conditions, only the epithelial basal airway cells express uPA and uPAR, while lung monocytes/macrophages express a considerable amount of uPAR. COVID-19 and pulmonary fibrosis result in uPA and uPAR downregulation in basal cells, uPAR downregulation in lung monocytes/macrophages, and the appearance of pro-fibrogenic KRT17+ KRT5- cells and fibroblasts that express both uPA and uPAR. Additionally, pulmonary fibrosis is associated with the emergence of some alveolar and monocyte-derived macrophages that express uPA. The consequences of uPAR downregulation involve uPA accumulation due to the defects in its internalization and lysosomal degradation. Pro-fibrogenic uPA accumulation in the absence of uPAR leads to IL-6 and ACE2 upregulation and EMT. Excessive lung fibrosis induced by low uPAR levels and the accumulation of inactive uPA can be resolved by plasminogen treatment. AT1 and AT2, alveolar type I and type II cells; AM, alveolar macrophages; EMT, epithelial–mesenchymal transition.