Fig 2: MMP-7 neutralizing antibody mitigates colonic inflammation in rodent models of colitis. For acute model mimicking ulcerative colitis, C57BL/6 mice were given 3% DSS in drinking water for 7 days. MMP-7 neutralizing antibody was administered once daily by i.p. injection. For chronic model mimicking Crohn’s disease, Sprague Dawley rats were treated with intrarectal injection of TNBS (50 mg/kg) 4 times, with intermittent 3-week intervals. MMP-7 neutralizing antibody was i.p. administered on Mondays, Wednesday, and Fridays for a total of 12 weeks. Animals in the control groups received regular drinking water (for acute model) or saline (for chronic model) as well as i.p. injection of respective IgG in saline. Colon tissue was fixed or snap-frozen after permeability assays. Inflammatory cells were evaluated by two independent pathologists. (A) In vivo colonic permeability of DSS mice measured with FITC-dextran at day 7. (B) MPO activities in the DSS mouse colon tissue. (C) RT-qPCR analysis of interleukin 1β (Il1b) mRNA levels in the DSS mouse colon tissue. (D) Numbers of neutrophils stained with H&E in the DSS mouse colons. (E) Numbers of mast cells stained with toluidine blue in the DSS mouse colons. (F) Numbers of eosinophils stained with H&E in the DSS mouse colons. n=4. *p<0.01 vs control (Ctl) group. #p<0.01 vs DSS group. (G) Dosing protocol (top) and H&E staining of the TNBS rat colons (bottom). Black arrow, severely damaged mucosa. (H) Histological colitis score of the rats. (I) Colon lengths of the TNBS rats. (J) In vivo colonic permeability of the TNBS rats. (K) MPO activities in the TNBS rat colons. (L) Il1b mRNA levels in the TNBS rat colons quantitated by RT-qPCR. (M) Immunofluorescence staining of Claudin-7 in colon Swiss roll sections. Nuclear counterstain was done with DAPI. (N) Quantification of Claudin-7 staining intensity, ranging from absent (0) to highest (3). n=5. *p<0.01 vs control (Ctl) group. # p<0.01 vs TNBS group.

Fig 3: MMP-7 knockout prevents DSS-induced suppression of Claudin-7 in the colon. C57BL/6 and Mmp7 -/- mice received 3% DSS in drinking water for 7 days as shown in Figure 4 . Full thickness colon tissue was fixed in formalin for immunohistochemical analysis or snap-frozen in liquid nitrogen for immunoblot analysis. (A) Representative images of immunofluorescence staining for MMP-7 in the colons of C57BL/6 and Mmp7 -/- mice treated with or without DSS. (B) Quantification of MMP-7 staining intensity. (C) Representative images of WB for indicated proteins in the colon. (D–F) Protein expression levels determined by densitometry. Arbitrary optical density units of the targeting proteins were normalized vs β-actin and presented as fold change. n=5. *p<0.01 vs WT control. NS, not significant.

Fig 4: Differential mRNA expression of junction proteins in the colons of wild type and Mmp7 -/- mice treated with 3% DSS. Colitis was induced with 3% DSS in drinking water in both C57BL/6 and Mmp7 -/- mice for 7 days as shown in Figure 4 . RT-qPCR was performed to determine mRNA levels of Ctnnb1 (A), Cdh1 (B), Cldn1 (C), Cldn7 (D), Cldn13 (E), Cldn15 (F), Ocln (G), Tjp1 (H), Cldn8 (I), Muc2 (J), Muc3 (K), and Muc4 (L). n=4 or 5. *p<0.05 vs wild type control. **p<0.05 vs wild type DSS.

Fig 5: MMP-7 is up-regulated by inflammatory mediators in epithelial cells, smooth muscle cells, and macrophages of the colon. (A) RT-qPCR analysis of MMP7 mRNA levels in human FHC (white bars), rat colonic smooth muscle (RCSMC, black bars), and human THP-1 (gray bars) cells treated with 10 ng/mL indicated cytokine or LPS. RT-qPCR assays also determined the MMP7 mRNA levels in the FHC cells treated with serial doses of IL-1β (B), TNFα (C), or LPS (D), in RCSMC treated with IL-1β (E), IL-4 (F), or IL-13 (G), and in THP-1-derived macrophages treated with IL-1β (H), TNFα (I), or LPS (J) for 24 hours. Time course experiments were performed to evaluate temporal changes of MMP7 mRNA expression in FHC cells treated with 10 ng/ml IL-1β (K) or 100 ng/ml TNFα (L), in RCSMC treated with 10 ng/ml IL-1β (M), 100 ng/ml IL-4 (N), or 100 ng/ml IL-13 (O), and in THP-1 cells treated with 10 ng/ml IL-1β (P), 10 ng/ml TNFα (Q), or 100 ng/ml LPS (R). n=3 or 4. *p<0.01.