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 damageare released. Metallothioneins (MTs) are such proteins who have been pointed forward as extracellular signal regulating the immune response.Weherein exploredtriggers releasing MTs from intestinal epithelial cells (IECs) and investigated their role asdanger signal during experimental colitis.
Methods:The release of MTs following inflammatory triggers and different cell death inducers from colonic epithelial HT29 cellswas 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 assaywas 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 vivousing 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- and2,4,6-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 ofUC1MT 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 infiltratein 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.