CD147 contributes to SARS-CoV-2-induced pulmonary fibrosis

Figures

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  Fig. 1. hCD147 transgenic mice infected with SARS-CoV-2 and delta variant mimicked pulmonary fibrosis pathology. a hCD147 mice or C57BL/6J mice were inoculated via the intranasal with 3 × 105 TCID50 of SARS-CoV-2 or delta variant, and samples were collected at 2, 6, 13, 20 and 27 d.p.i. b RT-qPCR for viral RNA levels in lung tissues at 2 d.p.i. n = 3 for C57BL/6J group, n = 6 for hCD147 groups. c H&E staining of lung tissue sections from 2 to 27 d.p.i. Scale bars, 50 μm. d TEM analysis of lung samples from 2 to 27 d.p.i. The stars indicate collagen fibrils; the arrows indicate elastic fibers. Black scale bars, 20 μm. Yellow scale bars, 2 μm. e Statistics of the percentage of cells positive for α-SMA (fibroblasts), F4/80 (macrophages), CD3 (T cells), CD4, CD8, CD19 (B cells), Ly6G (neutrophils), and NCR1 (NK cells). n = 12 images for each group, one-way ANOVA followed by multiple comparisons

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  Fig. 2. hCD147 mice with SARS-CoV-2 infection showed fibrotic characteristics revealed by RNA-seq analysis. a KEGG enrichment analysis of pathways enriched in differentially expressed genes in three profiles showing increasing trends. b Heatmap of significantly enriched genes involved in cytokine-cytokine receptor interaction and ECM remodeling in the three profiles. c Heatmap of significantly enriched genes involved in TGF-β pathway. d Immunofluorescence staining and quantification of TGF-β in lung tissue sections from SARS-CoV-2-infected hCD147 mice and bleomycin-induced pulmonary fibrosis model at indicated time points. The rabbit IgG isotype control was used along with the staining of TGF-β. Scale bars, 50 μm. n = 12 images for each group, one-way ANOVA followed by multiple comparisons

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  Fig. 3. Activation of TGF-β-CD147 axis in fibroblasts in lungs of SARS-CoV-2-infected hCD147 mouse model. a Multiplex immunohistochemistry of lung tissue sections for F4/80, α-SMA, SPC and TGF-β from SARS-CoV-2-infected hCD147 mice at 13 d.p.i. and bleomycin-induced pulmonary fibrosis model at 13 days post-bleomycin administration. Scale bars, 50 μm. b Statistics of the percentages of cells expressing TGF-β in cells positive for F4/80, α-SMA or SFTPC (SPC). n = 12 images from three mice for each group, one-way ANOVA followed by multiple comparisons. c Representative multiplex immunohistochemistry staining of lung tissue sections from SARS-CoV-2-infected hCD147 mice for F4/80 and α-SMA from 2 to 13 d.p.i. Scale bars, 25 μm. d The number of F4/80+ macrophages within the 100 μm radium of α-SMA+ fibroblasts. n = 6 images (100 ×) from 3 mice for each group, one-way ANOVA followed by multiple comparisons. e Multiplex immunohistochemistry of lung tissue sections for CD147, F4/80, α-SMA and SPC from SARS-CoV-2-infected hCD147 mice at 13 d.p.i. and bleomycin-induced pulmonary fibrosis model at 13 days post-bleomycin administration. For samples from SARS-Cov2-infected hCD147 mice and…

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  Fig. 4. CD147 knockdown inhibits activation of lung fibroblasts upon TGF-β stimulation. a MRC-5 cells were transfected with shRNA for CD147 gene silence and stimulated with TGF-β (10 ng/mL) for 24 h. The gene expression levels of CD147, α-SMA and COL1A1 were detected by real-time PCR. n = 3 samples for each group, one-way ANOVA followed by multiple comparisons. b The statistically significant profile by trend analysis of differentially expressed genes in MRC-5 cells transfected with CD147 shRNA with or without stimulation of TGF-β. c KEGG enrichment analysis of pathways enriched in the profile in b. d Heatmap of significantly enriched genes in the profile. Columns represent samples and rows represent genes. Gene expression levels in the heat maps are z score–normalized values determined by log2[CPM] values

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  Fig. 5. Ablation of CD147 in fibroblasts reverses pulmonary fibrosis in the bleomycin model. a Representative multiplex immunohistochemistry staining of lung tissue sections from bleomycin-induced pulmonary fibrosis model (13 d) for α-SMA and FSP1. Scale bars, 200 μm. b Representative multiplex immunohistochemistry staining of lung tissue sections from bleomycin-induced CD147f/fFSP1-Cre and CD147f/f mice treated with or without tamoxifen (TAM) for α-SMA and CD147. Scale bars, 50 μm. c Statistics of the percentages of cells expressing CD147 in cells positive for α-SMA. n = 9 images from three mice for each group, one-way ANOVA followed by multiple comparisons. d Percentages of α-SMA fibroblasts+ and CD147-expressing α-SMA+ fibroblasts detected by flow cytometry in lung tissues of bleomycin-induced pulmonary fibrosis model. e H&E staining and Masson’s trichrome staining of lung tissues from CD147f/fFSP1-Cre and CD147f/f mice treated with or without tamoxifen at indicated time points after bleomycin administration. Scale bars, 1 cm. f Statistics of the positive area of Masson’s trichrome staining. n = 3 mice for each group, one-way ANOVA followed by multiple comparisons. g Gene expression…

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  Fig. 6. Meplazumab prevents pulmonary fibrosis caused by SARS-CoV-2. a hCD147 mice were inoculated via the intranasal with 3 × 105 TCID50 of SARS-CoV-2 and treated with CD147 antibody meplazumab at 1 d.p.i. and 7 d.p.i. Samples were collected at 2, 6, 13, 20 and 27 d.p.i. b H&E staining and Masson’s trichrome staining of lung tissue sections from the IgG and meplazumab groups at different time points. Black scale bars, 2 mm. Yellow scale bars, 100 μm. c Statistics of the positive area of Masson’s trichrome staining. n = 3 mice for each group, two-tailed unpaired t-test. d TEM analysis of lung tissues from the IgG and meplazumab groups. Black scale bars, 20 μm; yellow scale bars, 2 μm. The stars indicate collagen fibrils. e Immunofluorescence staining and quantification of α-SMA in lung tissue sections from the IgG and meplazumab groups. Scale bars, 100 μm. n = 12 images from three mice for each group, one-way ANOVA followed by multiple comparisons. f Gene expression of TGF-β, PDGF, MMP2, MMP9, TIMP1, TIMP2, COL1A1 and COL1A2 in lung homogenates determined by RT-qPCR, compared with the corresponding IgG controls at indicated time points. GAPDH is used as a reference gene. g Gene…