Fig. 4: ECM-accumulated CTSC cleaves ICAM1 into a soluble form.

a Workflow showing the seeding of rTEM neutrophils on fibrotic ECM (Fib-ECM from SiO₂-56d lungs) and normal ECM (Nor-ECM from NS−56d lungs). b, c IF analysis of Ly6G and ICAM1 expression on Fib-ECM and Nor-ECM, with (c) quantifying mean fluorescence intensity. Scale bar = 200 μm. d GO enrichment of upregulated ECM proteins in SiO₂-56d compared to NS−56d. e Volcano plot showing differential ECM proteins, with CTSC exhibiting the most significant changes among hydrolytic enzymes. The gray dots represent all the differentially expressed proteins, while the colored dots represent all the hydrolysis-related proteins on the ECM. The p-values shown in the proteomics results were calculated using a two-sided Welch’s t-test, comparing normalized protein intensities between SiO₂-treated samples and control samples. f, g Western blot and immunofluorescence analyses of CTSC expression in lung ECM from NS-56d and SiO₂−56d mice. Scale bar = 200 μm. h HDOCK suggesting CTSC binding to ICAM1, indicating potential cleavage. i Experimental workflow illustrating CTSC-mediated cleavage of ICAM1. j, k Western blots showing ICAM1 cleavage fragments after incubation with recombinant CTSC (j) or lung ECM (k). As purified proteins (200 ng) were used, traditional loading controls (e.g., GAPDH) were not applicable (k). No loading control was applied due to the acellular nature of the ECM (k). Representative images are shown from n = 5 independent experiments with similar results. Data are presented as mean ± SEM. n = 5 independent experiments. c, compared with Nor-ECM group: two-way ANOVA, followed by Šídák’s multiple comparisons test. Source data are provided as a Source Data file. Created in BioRender. Chao, J. (2025) https://BioRender.com/ua4330j.