Research ArticleColitis

PAI-1 augments mucosal damage in colitis

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Science Translational Medicine  06 Mar 2019:
Vol. 11, Issue 482, eaat0852
DOI: 10.1126/scitranslmed.aat0852
  • Fig. 1 The coagulation pathway and SERPINE1 is enriched in IBD and links the epithelium to inflammation.

    (A) Commonly dysregulated genes in colon biopsies between IBD and non-IBD controls (P < 0.05, twofold change) from MIRC (three independent cohorts of patients with UC and CD and non-IBD controls; table S1). (B) Pathway overrepresentation analysis of the commonly dysregulated genes in (A) across databases and identification of the coagulation/hemostasis pathway with false discovery rate (FDR)–adjusted P value scores. The number on the bar indicates ranking position in the top 10 pathways for each specific database. KEGG, Kyoto Encyclopedia of Genes and Genomes. (C) Gene set enrichment analysis (GSEA) quantitatively identifying enrichment among the 1773 commonly dysregulated IBD probes from (A). (D) Bayesian network analysis of a three-step SERPINE1-induced subnetwork among intestinal epithelial and myeloid inflammation–associated gene clusters; SERPINE1 and CEBPB (red dashed circles) are highlighted as genes linking these two clusters. (E and F) Box and whiskers plot of log 2 fold expression of SERPINE1 in colon and ileal biopsies from UC (GSE38713) and CD (GSE16879), using median and interquartile range. Whiskers represent 5 and 95% range. P values are based on one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. (G) Immunofluorescent protein staining and PAI-1–positive (PAI-1+) cell quantification in colon resection cases (non-IBD colon, n = 11; UC uninvolved colon, n = 9; UC involved colon, n = 14). ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test). (H) Immunofluorescent images of PAI-1 (red) and VIMENTIN (green) expression in resection cases from (G); images taken at 10× magnification (top panels; scale bars, 100 μm) and 20× magnification (bottom panels; scale bars, 50 μm). DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 2 PAI-1 exacerbates DSS-induced colitis.

    (A to E) Serpine1+/+, Serpine1+/−, or Serpine1−/− mice were administered DSS for 6 days followed by 6 days of recovery. (A) Weight loss compared to day 0, (B and C) colon damage and healing (% length of healed colon), (D) Ly6G+ neutrophils [immunofluorescence with a minimum of 10 high powered (20×) fields per mouse], and (E) concentration of IL-6 by enzyme-linked immunosorbent assay (ELISA) were quantified in colon tissue on day 12. n = 8 to 10 mice per group from two independent experiments; **P < 0.01 and ***P < 0.001 compared to either Serpine1+/+ or Serpine1+/− using two-way ANOVA and *P < 0.05 and #P < 0.01 using one-way ANOVA. Data are presented as means ± SEM.

  • Fig. 3 SERPINE1/PAI-1 correlation cluster is linked to cytokine signaling, and IL-17A drives expression of the binding target Plat.

    (A) IPA of canonical pathways regulating the SERPINE1 correlation cluster (380 genes). Cytokine-associated pathways are circled in red. (B) Two doses (a, 20 ng/ml; b, 100 ng/ml) of indicated cytokines were screened on primary mouse colon epithelial spheroids, and expression of Plat was measured by quantitative polymerase chain reaction (PCR); >2-fold increase indicated by a red dotted line, n = 3 independent experiments. (C) Commonly altered genes (FDR < 0.05, twofold change) across three mouse colon epithelial states [stem cells, Diff (mid-crypt/differentiating cells), and terminally differentiated colonocytes] after treatment with recombinant IL-17A in vitro. Listed are the five genes commonly altered by IL-17A across all three states as determined by microarray analysis. (D) Plat expression measured in mouse colon epithelial cells in vitro by quantitative PCR. *P < 0.05 and **P < 0.01 using one-way ANOVA; n = 3 to 5 independent experiments. (E) Plat expression measured by quantitative PCR in Diff cells after coculture with TH17 cells ± anti–IL-17RA, TH1 cells, or unstimulated T cells. a and c = P < 0.01 compared to Diff epithelial cells alone, b and d = P < 0.05, and e = P < 0.01 compared to epithelial cells + TH17 cells and epithelial cells + IL-17A, using one-way ANOVA with Tukey’s multiple comparison test; n = 4 independent experiments. Data represent means ± SEM.

  • Fig. 4 Plat/tPA suppresses colitis and mucosal biopsy colon damage.

    (A to E) Plat+/− or Plat−/− mice were administered DSS for 6 days followed by 6 days of recovery. (A) Weight loss compared to day 0, (B and C) colon damage and healing (% healed colon) (scale bars, 0.5 mm), (D) Ly6G+ neutrophils, and (E) concentration of IL-6 by ELISA were quantified in colon tissue on day 12. n = 10 to 14 mice per group from two independent experiments; ***P < 0.001 compared to Plat+/− mice using two-way ANOVA, *P < 0.05, and **P < 0.01 using a two-tailed unpaired t test with 95% confidence intervals. (F) tPA protein expression in a colonic wound from a mucosal colonic pinch biopsy model showing expression in wound-associated epithelial (WAE) cells on top of wound (top) and in normal uninjured crypts (bottom). (G to I) Mucosal colonic pinch biopsy was used to create wounds in Plat+/+ or Plat−/− mice, and colonic damage and repair was measured 4 days later by (G to H) percentage total unhealed wound area (dotted line indicates total wound area and solid line indicates unhealed area) and (I) percentage presence (red) or absence (blue) of a neutrophil/fibrin cap on top of the wounds. n = 8 to 9 mice per group from two independent experiments; ***P < 0.001 using a two-tailed unpaired t test with 95% confidence intervals. Scale bars, 100 μm. Data represent means ± SEM.

  • Fig. 5 A small-molecule PAI-1 inhibitor partially suppresses DSS- and Citrobacter-induced colitis in a tPA-dependent manner.

    (A) Ratio of active tPA to total tPA measured in colon homogenates and plasma. n = 7 to 11 mice per group from two independent experiments; *P < 0.05 using a two-tailed unpaired t test with 95% confidence intervals. (B to G) Mice were administered DSS for 6 days followed by 6 days of recovery; at day 6, mice received daily intraperitoneal injections of vehicle or a small-molecule PAI-1 inhibitor, MDI-2268. (B) Weight loss compared to day 0. n = 20 mice to group from two independent experiments; **P < 0.01 using a two-way ANOVA. (C) Colon damage and healing (% healed colon), (D) Ly6G+ neutrophils, and (E) IL-6 in colon tissue were quantified. n = 9 to 10 mice per group from two independent experiments; **P < 0.01 using two-tailed unpaired t test with 95% confidence intervals. (F) Plat−/− mice weight loss compared to day 0 and (G) colon damage and healing (% healed colon) were quantified. n = 8 mice per group from two independent experiments. (H to I) Mice were infected with C. rodentium for 12 days to induce colitis pathology and received daily intraperitoneal injections of vehicle or a small-molecule PAI-1 inhibitor, MDI-2268, from day 0. (H) Percentage of colon with crypt loss/dropout and (I) representative hematoxylin and eosin image of the transverse colon. Scale bars, 0.5 mm. n = 12 to 13 mice per group from two independent experiments; ***P < 0.001 using a two-tailed unpaired t test with 95% confidence intervals. ns, not statistically significant. Data represent means ± SEM.

  • Fig. 6 PAI-1 inhibits the tPA-dependent cleavage and activation of TGF-β–suppressing epithelial hyperproliferation and colitis.

    (A) Immunoblot analysis of a cell-free in vitro reaction for the conversion of latent/inactive TGF-β to mature/active TGF-β. (B) Primary colon epithelial spheroids were cultured with the indicated protein combinations and proliferation measured by luminescence. n = 3 independent experiments; ***P < 0.001 compared to either mature TGF-β or tPA + latent TGF-β + plasminogen using two-way ANOVA with Tukey’s multiple comparison test. (C and D) Ratio of active TGF-β to total TGF-β in colon homogenates of DSS-treated mice measured using a flow cytometric bead assay. n = 4 to 5 mice per group is shown from one experiment that is representative of two independent experiments; *P < 0.05 using a two-tailed unpaired t test with 95% confidence intervals. (E to G) Mice were administered DSS for 6 days followed by 6 days of recovery; treatment with anti–TGF-β (αTGFβ) or isotype immunoglobulin G (IgG) control was initiated on day −2. (E) Weight loss compared to day 0, (F) colon damage and healing (% healed colon), and (G) IL-6 in colon tissue were quantified. n = 7 to 10 mice per group from two independent experiments; *P < 0.05 using one-way ANOVA with Tukey’s multiple comparison test and **P < 0.01 compared to either Serpine1−/− or Serpine1−/− + isotype IgG control using a two-way ANOVA. Data represent means ± SEM.

  • Fig. 7 SERPINE1 is elevated in active disease and in nonresponders to anti–TNF-α biologic therapy.

    (A) ROC curve analysis on gene sets (GSE38713, GSE16879, GSE36807, and GSE23597) defining specificity and sensitivity of SERPINE1 discrimination between active and nonactive disease in colon biopsies (n = 133; AUC = 0.97). (B) Spearman correlation of SERPINE1 expression and Mayo score (n = 87) from the PURSUIT cohort (NCT00487539) at week 0 before treatment and week 6 after golimumab treatment. (C) ROC curve analysis on colon biopsies from gene set GSE16879 defining SERPINE1 discrimination between responders and nonresponders to infliximab before treatment (AUC = 0.92).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/482/eaat0852/DC1

    Materials and Methods

    Fig. S1. Coagulation pathway in IBD.

    Fig. S2. SERPINE1 links myeloid inflammation and epithelial clusters and is enriched in IBD.

    Fig. S3. SERPINE1 correlation cluster of genes dominated by ECM and cytokine pathways.

    Fig. S4. PAI-1 increases severity of colonic tissue damage.

    Fig. S5. IL-17A drives expression of Plat in colon epithelial cells.

    Fig. S6. Plat/tPA protects against colitis and mucosal damage.

    Fig. S7. A small-molecule PAI-1 inhibitor reduces severity of colitis in a tPA-dependent manner.

    Fig. S8. TGF-β is partly responsible for tPA-mediated protection in colonic inflammation.

    Fig. S9. SERPINE1 distinguishes between active and inactive disease and is elevated in anti–TNF-α nonresponders.

    Table S1. Cohorts analyzed.

    Table S2. Overrepresentation analysis in pathway tools.

    Table S3. Description of CERTIFI cohort used to construct the Bayesian network.

    Table S4. SERPINE1 correlation cluster set of genes determined by r2 global linear regression.

    Table S5. Genes regulated in primary colon epithelial cells by recombinant IL-17A treatment.

    Table S6. Genes that were differentially expressed (P < 0.05, twofold change) between responders and nonresponders to infliximab in biopsies taken before biologic therapy.

    Data file S1. Primary data.

    Reference (79)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Coagulation pathway in IBD.
    • Fig. S2. SERPINE1 links myeloid inflammation and epithelial clusters and is enriched in IBD.
    • Fig. S3. SERPINE1 correlation cluster of genes dominated by ECM and cytokine pathways.
    • Fig. S4. PAI-1 increases severity of colonic tissue damage.
    • Fig. S5. IL-17A drives expression of Plat in colon epithelial cells.
    • Fig. S6. Plat/tPA protects against colitis and mucosal damage.
    • Fig. S7. A small-molecule PAI-1 inhibitor reduces severity of colitis in a tPA-dependent manner.
    • Fig. S8. TGF-β is partly responsible for tPA-mediated protection in colonic inflammation.
    • Fig. S9. SERPINE1 distinguishes between active and inactive disease and is elevated in anti–TNF-α nonresponders.
    • Reference (79)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Cohorts analyzed.
    • Table S2 (Microsoft Excel format). Overrepresentation analysis in pathway tools.
    • Table S3 (Microsoft Excel format). Description of CERTIFI cohort used to construct the Bayesian network.
    • Table S4 (Microsoft Excel format). SERPINE1 correlation cluster set of genes determined by r2 global linear regression.
    • Table S5 (Microsoft Excel format). Genes regulated in primary colon epithelial cells by recombinant IL-17A treatment.
    • Table S6 (Microsoft Excel format). Genes that were differentially expressed (P < 0.05, twofold change) between responders and nonresponders to infliximab in biopsies taken before biologic therapy.
    • Data file S1 (Microsoft Excel format). Primary data.

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