ic  (R&D Systems)

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: a Flow cytometry gating strategy for identifying rTEM neutrophils (ICAM1 high CXCR1 low ) within CD11b⁺Ly6G⁺ lung neutrophils, based on fluorescence-minus-one (FMO) controls. Full gating workflow is shown in Fig. . b Immunofluorescence analysis confirming rTEM neutrophils in lung tissues, with co-localization of Ly6G and ICAM1 signals. Scale bar = 200 μm. c CellChat analysis visualizing cell-cell communication between neutrophil subtypes and fibroblasts. Thicker lines indicate stronger interactions, and darker colors denote higher confidence. d , e Western blots validating the generation of in vitro rTEM neutrophils by showing higher ICAM1 and lower CXCR1 expression in upper chamber cells of the transwell system. f , g Fibroblasts stimulated with upper and lower chamber neutrophil conditioned medium. Western blot analysis shows significant changes in ACTA2 and COL1A2 levels. IL-1β, neutrophils, and mpECs did not show significant effects on fibroblast function in controls. h , i Proliferation and migration of fibroblasts assessed by BrdU incorporation and scratch wound healing assays, respectively. Scale bar = 650 μm. Data are presented as mean ± SEM. n = 5 mice per group for flow cytometry and immunofluorescence, and n = 5 independent experiments for in vitro assays. Biological replicates indicate independent animals or cell cultures. Representative blots/images are shown from n = 5 independent experiments with similar results. Statistical significance was determined using two-sided Student’s t -test with Welch’s correction ( a , compared with NS−56d) or one-way ANOVA with Dunnett’s multiple comparisons test ( e , g , compared with Con-1). Source data are provided as a Source Data file.

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Flow Cytometry, Fluorescence, Immunofluorescence, Western Blot, In Vitro, Expressing, Migration, BrdU Incorporation Assay

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: a Violin plot showing elevated ICAM1 expression in rTEM neutrophils. b Recombinant mouse ICAM1 (rmICAM1, 10 ng/ml) significantly enhances fibroblast viability. c Effects of rmICAM1 on fibroblast activation markers COL1A2 and ACTA2, with quantified data shown in the right panel. d – g BrdU incorporation and scratch wound healing assays demonstrating the effects of rmICAM1 on fibroblast proliferation and migration. d , e Scale bar = 200 μm. f , g Scale bar = 650 μm. e and g quantify BrdU-positive cells and wound area, respectively. h CellChat analysis of communication between ECM-producing fibroblasts and rTEM neutrophils, highlighting ligand-receptor contributions in ICAM1 signaling. i Volcano plot of differential ECM proteins in SiO₂-56d vs. NS−56d, with integrin family proteins (e.g., ITGAM, ITGB2) showing the most significant changes. The gray dots represent all the differentially expressed proteins, while the colored dots represent all the integrin family proteins on the ECM. j Barplot showing KEGG terms enriched for differential ECM proteins in SiO₂-56d compared to NS−56d. k – n HDOCK analysis reveals physical binding sites between ICAM1 and integrin family proteins (ITGAM, ITGB2, ITGAL, ITGAX). Representative blots/images are shown from n = 5 independent experiments with similar results. Data are presented as mean ± SEM. n = 5 independent experiments. b , compared with 0 ng/mL group: Dunn’s post hoc test with Benjamini–Hochberg correction for multiple testing. c , f , g , compared with vehicle group: unpaired two-sided t test with Welch’s correction. Source data are provided as a Source Data file.

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Expressing, Recombinant, Activation Assay, BrdU Incorporation Assay, Migration, Binding Assay

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: 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 .

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Expressing, Fluorescence, Control, Western Blot, Immunofluorescence, Binding Assay, Incubation, Recombinant, Purification

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: a Schematic of the experimental design: wild-type (CTSC +/+ ) and CTSC knockout (CTSC −/− ) mice were administered either normal saline (NS) or SiO₂ via intratracheal instillation and evaluated after 56 days. b Representative CT images of mouse lungs showing reduced high-density fibrotic regions in CTSC −/−__ SiO₂ mice compared to wild-type controls (CTSC +/+__ SiO2). c Representative H&E, Sirius Red, and Masson staining images showing improved alveolar architecture and decreased collagen deposition in CTSC −/−__ SiO₂ lungs. Scale bar 200 μM. d Hydroxyproline quantification indicating decreased collagen content in CTSC −/−__ SiO2 lungs compared with CTSC +/+__ SiO2 group. e – g Western blot and densitometric quantification of fibrosis markers COL1A2 and ACTA2 in lung tissues across all four groups. h , i ELISA quantification of soluble ICAM1 levels in ( h ) lung tissue lysates and ( i ) bronchoalveolar lavage fluid (BALF), showing reduced ICAM1 cleavage in CTSC-deficient mice compared with CTSC +/+__ SiO2 group. Representative images are shown from n = 5 independent experiments with similar results. Data are presented as mean ± SEM. n = 5 mice per group. d , f – i : Statistical significance was determined using two-way ANOVA with Tukey’s post hoc test. Significant comparisons between groups are indicated in the figure panels. Source data are provided as a Source Data file. Created in BioRender. Chao, J. (2025) https://BioRender.com/ua4330j .

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Knock-Out, Saline, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: a , b Dot plots from GSE183682 and GSE174725 showing highest CTSC expression in macrophages. c Western blot of CTSC levels in RAW264.7 cells treated with SiO₂. d , e Spatial transcriptomics and IF show elevated CTSC expression in SiO₂-56d mouse lungs. Scale bar = 200 μm. f Clodronate liposome workflow for macrophage depletion to investigate effects on ECM-associated CTSC and rTEM neutrophils. g , h IF showing Ly6G and ICAM1 levels in ECM after macrophage depletion, with Nor-ECM as the control group. Scale bar = 200 μm. i Experimental workflow exploring CTSC’s role in ICAM1 cleavage. PI represents a protease inhibitor that inhibits CTSC function, while Ab refers to an anti-CTSC antibody that blocks CTSC function. j ELISA detecting ICAM1 levels in CM-2, confirming CTSC cleaves neutrophil-derived ICAM1. k , l Fibroblasts co-cultured with CM-2 show elevated COL1A2 and ACTA2 levels. m – o BrdU assay and wound healing assessing fibroblast proliferation ( m , n ) and migration ( o , p ) after CM-2 treatment. Representative images are shown from n = 5 independent experiments with similar results. Scale bar = 650 μm. Data are presented as mean ± SEM. n = 5 independent experiments. h : two-way ANOVA, followed by Šídák’s multiple comparisons test. j , compared with Con-CM2: one-way ANOVA, followed by Tukey’s test. l , n , p : Unpaired t test with Welch’s correction. Source data are provided as a Source Data file. Created in BioRender. Chao, J. (2025) https://BioRender.com/ua4330j .

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Expressing, Western Blot, Control, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, BrdU Staining, Migration

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: a Workflow of anti-Ly6G antibody treatment for neutrophil depletion; anti-IgG2a served as the isotype control. b Flow cytometry detecting rTEM neutrophil levels in lungs 10 days after anti-Ly6G treatment. c CT imaging and 3D reconstruction showing reduced high-density lung areas in neutrophil-depleted mice. d Histological analysis (H&E, Masson, Sirius Red) of lung tissues showing improved morphology and reduced collagen deposition. Scale bar = 200 μm. e Hydroxyproline assay quantifying collagen content in lung tissues. f – h Western blot analysis of COL1A2 and ACTA2 levels in lung tissues. i Immunofluorescence showing Ly6G and ICAM1 co-localization in lung tissues. Yellow arrows indicate cells with strong co-localization. Scale bar = 200 μm. j , k ELISA showing reduced sICAM1 levels in lung tissues and BALF after neutrophil depletion. Representative images are shown from n = 5 independent experiments with similar results. Data are presented as mean ± SEM. n = 5 mice. The unit of study was the individual mouse, and samples (lung tissue or protein extracts) were collected from each animal independently. All replicates are biological replicates derived from distinct experimental subjects. b , e , g , h , j , k : two-way ANOVA, followed by Tukey’s test. Groups compared include NS (normal saline control) and SiO₂ (silica-induced fibrosis) treatments, each assessed with two different antibody labels (αIgG2a and αLy6G). Source data are provided as a Source Data file. Created in BioRender. Chao, J. (2025) https://BioRender.com/ua4330j .

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Control, Flow Cytometry, Imaging, Hydroxyproline Assay, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Derivative Assay, Saline

Journal: Nature Communications

Article Title: Extracellular matrix anchored neutrophils drive pulmonary fibrosis in mice

doi: 10.1038/s41467-025-66633-8

Figure Lengend Snippet: Fibrotic insults induce reverse transendothelial migration (rTEM) of neutrophils①. ICAM1high CXCR1low rTEM neutrophils adhere to and are retained by lung ECM via integrins②. Macrophage‑derived CTSC accumulates in the ECM③ and cleaves neutrophil ICAM1 to generate sICAM1④, which activates fibroblasts and drives collagen deposition⑤. This CTSC–ICAM1 axis links ECM retention of rTEM neutrophils to fibroblast activation in pulmonary fibrosis. Schematic illustration created with Figdraw.com (license ID: SIUUY789f7).

Article Snippet: For recombinant CTSC cleavage of ICAM1, recombinant mouse ICAM1 protein (R&D Systems) was incubated with recombinant CTSC (R&D Systems) in cleavage buffer containing 25 mM MES, 50 mM NaCl, and 5 mM DTT at pH 5.5.

Techniques: Migration, Activation Assay