Fig 1: (A, left panel) Serum-starved CD-HIF were treated with 100 µg/mL CDSE for 2 hours. Serum-starved CCD-18Co colonic fibroblasts were treated with 10 ng/mL TGF-ß1 for 2 hours. Conditioned media were loaded to Proteome Profiler Human Protease Arrays (ARY021B; R&D Systems). A Bio-Rad ChemiDoc Imaging system captured the images. The rectangles highlighted the cathepsin S expression. (A, right panel) Quantification of cathepsin S signals (B7-8) and control signals (A1-2 and E1-2) using Bio-Rad Image Lab Software. Results were pooled from 3 independent experiments. Student t test was used to compare no serum exosome and CDSE groups. (B, left panel) Colonic cathepsin S mRNA expression in 40 non-IBD, 52 UC, 28 non-stricturing CD, and 15 stricturing CD patients was determined by real-time reverse transcription polymerase chain reaction. Ordinary one-way ANOVA test did not find any significant differences. (B, middle panel) Colonic cathepsin S mRNA (CTSS) expression in 43 CD patients is not correlated with colonic elafin mRNA expression. (B, right panel) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 media with or without 100 µg/mL CDSE. Two hours later, elafin (1 µg/mL) was added and incubated for 24 hours. Ordinary one-way ANOVA test did not find any significant differences. (C) Cathepsin S activity assay was performed by incubating 2 µL of CS substrate (200 µmol/L final concentration), 94 µL CS reaction buffer, 2 µL cathepsin S inhibitor provided by the assay kit, 1 µL cathepsin S (0.4 µg/mL final concentration), and 1 µL elafin (0.5–10 µg/mL final concentration) at 37oC for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 3 independent experiments. Ordinary one-way ANOVA with Tukey test. (D) Fresh human colonic tissues were pretreated with 100 µg/mL CDSE for 2 hours, followed by elafin 1 µg/mL for 2 hours. Conditioned media were collected. Each piece of tissue was homogenized in 500 µL CS cell lysis buffer. Next, 50 µg of tissue lysate supernatants in 50 µL CS lysis buffer or 50 µL of conditioned media were mixed with 2 µL CS substrates (200 µmol/L final concentration) and 48 µL CS reaction buffer and incubated for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). n = 6 patients. Ordinary one-way ANOVA with Tukey test. (E) CD-HIF in 96-well plates were pretreated with 100 µg/mL CDSE for 2 hours, followed by elafin 1 µg/mL for 2 hours. Conditioned media were collected. Cells were then lysed in 200 µL/well CS cell lysis buffer. Next, 50 µg of cell lysates in 50 µL CS lysis buffer or 50 µL of conditioned media were mixed with 2 µL of CS substrate (200 µmol/L final concentration) and 48 µL CS reaction buffer and incubated at 37oC for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 6 independent experiments. Ordinary one-way ANOVA with Tukey test. (F) Serum-starved CD-HIF were pretreated with either 0.1% TFA r 0.4 µg/mL cathepsin S (1183-CY-010; R&D Systems) for 30 minutes, followed by 100 µg/mL CDSE. Two hours later, elafin (1 µg/mL) was added and incubated for 24 hours. ProCOL1A1 protein was determined by ELISA. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test.
Fig 2: (A) Experimental plan. Control lentivirus, elafin-overexpressing lentiviruses, Ctss-overexpressing lentivirus, Ctss-siRNA lentivirus, Zeb1-shRNA lentivirus, and Zeb1-overexpressing lentivirus were injected into SAMP1/YitFc mice intraperitoneally once at 40 weeks of age. In addition, PAR2 agonist GB110 or PAR2 inhibitor GB88 was given via oral gavage from 40 to 42 weeks of age. Non-fibrotic 10-week-old SAMP1/YitFc mice and parental control 42-week-old AKR strain mice were used for comparison. Ileal tissues were collected for analysis 2 weeks after lentiviral injection. (B) Body weight. Six mice per group. Mean ± standard deviation. (C) H&E staining (upper panels) and Masson Trichrome (MT) staining (lower panels) of ileal tissues from 10 to 42 weeks of age. Blue color in MT staining (arrows) indicated collagen deposition in lamina propria. (D) Ileal histology scores. (E) Ileal fibrosis scores. (F) Ileal overall disease activities. Six mice per group. Ordinary one-way ANOVA with Tukey tests.
Fig 3: (A) H&E staining (upper panels) and MT staining (lower panels) of ileal tissues from SAMP1/YitFc mice at 42 weeks of age. Blue color in MT staining (arrows) indicated collagen deposition. (B) Ileal histology scores. (C) Ileal fibrosis scores. (D) Ileal overall disease activities. Prominent ileal fibrosis was found in elafin-overexpressing groups with lentiviral Ctss and Zeb1 overexpression and oral PAR2 agonist GB110 treatment. Ileal fibrosis was ameliorated with lentiviral Ctss and Zeb1 shRNA inhibition and oral PAR2 inhibitor GB88 treatment. Six AKR or SAMP1/YitFc mice per group. Ordinary one-way ANOVA with Tukey tests.
Fig 4: CTSS-induced Ephrin-B2 liberation accelerates sensory recovery. A Time course of HWT in IANX rats treated with rhCTSS + saline or rhCTSS + EBP. IANX + rhCTSS + saline (n = 7); IANX + rhCTSS + EBP (n = 5), Friedman test post hoc Dunn’s test, *P < 0.05, **P < 0.01 vs. day 2; GEE post hoc Bonferroni’s test. ††P < 0.01. Boxes show the 25th–75th percentiles with the median value as a line within each box, and whiskers indicate the 10th and 90th percentiles of the data. All data points are shown in open circles. B Images showing FG-labeled TG neurons 14 day post-IANX. Column represents the average number of FG-labeled TG neurons out of total TG neurons. n = 5 in each, unpaired t test. ***P < 0.001. C Images show c-Jun and Hoechst-33258 immunofluorescence at the site of IAN injury 14 day post-IANX. Column represents the average number of c-Jun-positive cells at the injured site. n = 5 in each, unpaired t test. ***P < 0.001. Data represent the mean ± SEM in B and C. All data points are shown as open circles
Fig 5: CTSS causes Ephrin-B2 cleavage in the human sensory nerve. A Images showing IBA1, CTSS, Ephrin-B2, αSMA, EphB2, or S100β immunofluorescence and Hoechst-33258 in the human nasopalatine nerve. Arrowheads indicate IBA1/CTSS-double-positive cells (top), Ephrin-B2/αSMA-double-positive cells (middle), and EphB2/S100β-double-positive cells (bottom). B Blots of fEphrin-B2 and cEphrin-B2 in the nasopalatine nerve homogenate after treatment with rhCTSS and Z-FL (20 or 200 μM). C Column represents the average values of fEphrin-B2/β-actin or cEphrin-B2/β-actin. n = 3 patients, one-way ANOVA post hoc Tukey’s test. *P < 0.05, **P < 0.01 All data points are shown in open circles
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