Fig 2: PAI-1 regulates neutrophil infiltration into the colon lamina propria and epithelium through the chemokines CXCL1 and CXCL5 and their receptor CXCR2. (A) Top 20 significantly altered genes in the colon during DSS-induced colitis in WT mice revealed by PCR array. (B) Top 12 co-occurrence networks deduced from 108 hub genes (fold change=2) enriched in the PAI-1 and control groups after DSS by cytoHubba. (C to K) Comparison of the levels of 9 proteins in the colon during DSS-induced colitis measured by ELISA (mean±SEM, Student's t test, *P=0.05, **P=0.01, ***P=0.001, n=4-5 per group). (L and M) Enriched GO pathways among the upregulated and downregulated genes.

Fig 3: PAI-1 deficiency ameliorated DSS-induced colitis. Both WT and PAI-1 KO mice were treated with 2% DSS for 7 days. (A) Representative immunoblot results of PAI-1 and ß-actin staining in colons showed WT mice treated with DSS had significantly increased PAI-1 expression in the colon. (B) Quantification of the PAI-1 expression results shown in A. (C) PAI-1 expression in the colon measured by ELISA. (D) Representative immunofluorescence images showing increased PAI-1 expression in the epithelial cells and crypts of the colon during DSS-induced colitis. Images were taken at 10× magnification (top panels) or 40× magnification (middle panels). (E) Weight loss compared to body weight on day 0. (F) Disease activity scores in the WT and PAI-1 KO DSS groups. (G) Colon length at 7 days after DSS administration. (H) Quantification of the normalized colon length. The length of the colon was adjusted to the initial body weight of individual mice. (I) Representative histological images of colon tissues from PAI-1 KO and WT mice were collected on day 7 after water or DSS treatment. (J) Histology scores in the two groups. (K) Representative flow plots of the CD11b+Ly6G+ neutrophil population within live CD45+ cells. Cells were prepared from cLPCs or cIECs. (L and M): Summary of the percentages of neutrophils in the cLPCs and cIECs of WT and PAI-1 KO DSS mice. (mean±SEM; Student's t test; *P=0.05, **P=0.01, ***P=0.001, ****P=0.0001, n=4-5 per group). cLPCs: colonic lamina propria cells (LPCs), cIECs: colonic intestinal epithelial cells.

Fig 4: PAI-1 was validated as a potential biomarker for the diagnosis of IBD. (A) Representative immunoblot results of PAI-1 and GAPDH expression in colon samples from IBD patients and non-IBD controls. (B) Quantification of PAI-1 expression (mean±SEM, Student's t test, n=3, *P=0.05). (C) Immunohistochemistry images of PAI-1 expression in IBD and control cases. (D) PAI-1 plasma concentrations in IBD patients and the control cohort measured by ELISA (mean±SEM, Mann?Whitney test, ****P=0.0001). (E) AUC value of 82.78% (95% CI: 71.89 to 93.67) using PAI-1 levels to distinguish between IBD patients and non-IBD controls. (F) An illustration of the role of PAI-1 in the pathogenesis of IBD. PAI-1 is expressed in the inflamed epithelium in IBD. It stimulates the expression of the chemokines CXCL1 and CXCL5 and the adhesion molecule ICAM-1 on IECs through activation of the NF-?B pathway. It also affects the balance of the gut microbiome. CXCL1 and CXCL5 signal through CXCR2 to recruit neutrophils to inflamed tissues and cause ongoing colonic damage and inflammation, which is shown by increased expression of IL-6, IL-1ß and TNF-a. Deficiency in PAI-1 decreases the activation of the NF-?B pathway, restores the diversity of the microbiome, reduces the expression of neutrophil-specific chemokines and ultimately reduces disease severity.

Fig 5: DSS-induced neutrophil infiltration is blunted in PAI-1-deficient mice. (A) CD45-CD326+ epithelial cells sorted from the IECs of WT or PAI-1 KO mice treated with DSS. The RNA expression of Cxcl1, Cxcl5, Cxcr2, Tnf, and Icam1 is shown in (B) (n=3 per group). (C) PCR array for the NF-?B pathway was used for sorted epithelial cells, and the DEGs were subjected to KEGG pathway analysis (fold change=2). (D&E) PAI-1 deficiency reduced the nuclear translocation of NF-?B p65 in colon tissue. (F) NF-?B activity analysis via the luciferase reporter assay. Luciferase activity was calculated as Firefly Luc value/Renilla Luc value. (n=3 per group) (G&H) show the changes in ICAM-1 levels in different groups measured by Western blotting. (n=5 per group). (mean±SEM, Student's t test, *P=0.05, **P=0.01, ***P=0.001) DEGs: differentially expressed genes; EPC: epithelial cells; IEC: intestinal epithelial cells.