Download Metallothionein, an emerging danger signal during experimental colitis

Metallothionein as extracellular danger signal: a novel target to treat intestinal inflammation?
Authors:
Lindsey Devisscher1, Pieter Hindryckx1, Kim Olievier1, Harald Peeters1, M. Lynes2, C. Cuvelier3,
Martine De Vos1 and Debby Laukens1
Affiliation:
1Department
of Gastroenterology, Ghent University, De Pintelaan 185, 1K12IE, B-9000 Ghent,
Belgium
2Department
of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125
3Department
of Pathology, Ghent University, De Pintelaan 185, 1K12IE, B-9000 Ghent, Belgium
BACKGROUND: Danger signals have been postulated as regulators of gut mucosal immunity. During
intestinal inflammation, the epithelium is compromised and signals alerting adjacent cells of tissue
damage are released. Metallothioneins (MTs) are such proteins who have been pointed forward as
extracellular signal regulating the immune response. We herein explored triggers releasing MTs from
intestinal epithelial cells (IECs) and investigated their role as danger signal during experimental colitis.
METHODS: The release of MTs following inflammatory triggers and different cell death inducers from
colonic epithelial HT29 cells was analysed. Supernatant was analysed for MT levels using western blot
and for lactate dehydrogenase activity (LDH). Cell lysates were analysed for caspase-3 activity using a
DEVD-assay. A Boyden transwell migration assay was used to evaluate the chemotactic potential of
released MT and the capacity of monoclonal anti-MT antibody (100µg/ml UC1MT ) to abolish this. The
role of MT as chemo-attractant was explored in vivo using dextran sulphate sodium (DSS)-induced
colitis in MT knockout (MT-/-), transgenic (MT+/+) and wild type mice (WT). Additionally, UC1MT
antibody treatment (4 mg/kg IP) was tested in DSS- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)induced colitis. Intestinal inflammation was assessed in all experiments conform standards
procedures.
RESULTS: Cellular release of MT was detected after necrotic cell death, characterized by positive
LDH activity in cell supernatant, and was not dependent on caspase-3 activation. Leukocyte migration
towards MT-containing supernatant was demonstrated, whereas the addition of UC1MT antibody was
able to overcome this chemo-attraction (p<0.05). MT-/- mice showed a significant higher survival rate
compared to MT+/+ mice (90% versus 52%) following DSS application, which was associated with
reduced signs of colitis (p<0.05). Histological evaluation showed significant less inflammatory infiltrate
in MT-/- mice compared to WT and MT+/+ mice, confirmed by lower myeloperoxidase levels for MT-/mice. UC1MT antibody treatment in DSS- and TNBS-induced colitis confirmed these results with
reduced signs of inflammation, a lower influx of leukocytes and less F4/80 positive macrophages in
UC1MT treated mice.
CONCLUSION: MTs act as chemotactic danger signal released upon IEC death in vitro. Targeting
their function during experimental colitis may represent a novel therapeutic strategy to reduce cellular
infiltration and subsequently intestinal inflammation.