Gastroenterology

Gastroenterology

Volume 143, Issue 5, November 2012, Pages 1277-1287.e4
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
Tissue Inhibitor of Metalloproteinase-3 Regulates Inflammation in Human and Mouse Intestine

https://doi.org/10.1053/j.gastro.2012.07.016Get rights and content

Background & Aims

Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice.

Methods

We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohn's disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-β1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3–knockout (TIMP-3-KO), and transgenic (TIMP-3–Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3–KO T cells.

Results

Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-β1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-β1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3–KO mice developed severe colitis after administration of TNBS, whereas TIMP-3–Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3–KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells.

Conclusions

TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-β1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.

Section snippets

Mucosal Samples

Mucosal biopsy specimens were taken from involved areas of 30 patients with active CD (median age, 35 y, range, 25–55 y). In 6 of the 30 CD patients, paired biopsy samples were taken from endoscopically inflamed and uninflamed mucosa. Eleven CD patients were receiving corticosteroids, and the remaining patients were on mesalazine. Resected intestinal tissue was taken from 8 patients with severe disease poorly responsive to medical treatment and used to isolate lamina propria mononuclear cells

Down-regulation of TIMP-3 Expression in CD

TIMP-3 transcripts were reduced in CD as compared with UC and normal samples (Figure 1A). TIMP-3 RNA expression did not differ between UC and controls (Figure 1A). In CD, decreased TIMP-3 RNA expression was restricted to areas of active inflammation (Figure 1A), and the same biopsy specimens showed enhanced α-secretase activity (Figure 1B). Moreover, we analyzed TACE and ADAM-15 in the same intestinal samples used to evaluate TIMP-3. Data from these experiments show that TACE and ADAM-15

Discussion

This study set out to determine if TIMP-3 is a determinant of human and mouse gut inflammation. We first showed that TIMP-3 expression was decreased in CD mucosa, particularly in T cells, CD68+ cells, and also in epithelial cells. CD samples consistently showed enhanced α-secretase activity, suggesting that the improved α-secretase activity present in CD gut mucosa is the result of a reduced expression of TIMP-3. There was no difference in TIMP-3 expression and α-secretase activity between UC

Acknowledgments

The authors thank Rama Khokha for kindly providing the tissue inhibitor of metalloproteinases-3–deficient mice.

GM and TTM are both co-senior authors of the study.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by Fondazione Umberto Di Mario Onlus (Rome, Italy), Giuliani Spa (Milan, Italy), funding for the Opportunity for Drug Discovery consortium under Grant Agreement 202020 of the Seventh Research Framework Programme of the European Union; and by Telethon GGP08065, Fondazione Roma 2008, Juvenile Diabetes Research Foundation RRG 1-2007-665, FP7-Health-241913, The role of intestinal microflora in non-alcoholic fatty liver disease, and the Ministry of Health RF 2007.

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