metallothionein and Colitis

Inflammatory bowel diseases (IBDs) are a group of chronic, relapsing, immune-mediated disorders of the intestine, including Crohn's disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in their pathogenesis. Metallothioneins (MTs) are a family of small proteins with a high and conserved cysteine content that are rapidly upregulated in response to an inflammatory stimulus. Herein, we review the current knowledge regarding the expression and potential role of MTs in IBD. MTs exert a central position in zinc homeostasis, modulate the activation of the transcription factor nuclear factor (NF)-kappaB, and serve as antioxidants. In addition, MTs could be involved in IBD through their antiapoptotic effects or through specific immunomodulating extracellular effects. Reports on MT expression in IBD are contradictory but clearly demonstrate a deviant MT expression supporting the idea that these aberrations in IBD require further clarification.

Topics: Animals; Colitis; Disease Models, Animal; Humans; Inflammatory Bowel Diseases; Metallothionein; Models, Biological

Inflammatory bowel disease (IBD) is an inflammatory response relative chronic disease in the intestinal tract. Our previous study demonstrated melatonin exerts an improvement effect on stress related IBD. The present study was further performed to clarify the mechanism of melatonin in dextran sodium sulfate (DSS)-induced colitis in mice.. We successfully established a DSS-induced colitis mouse model and hydrogen peroxide (H. Melatonin supplementation normalized the colitis, oxidative stress, mitochondria dysfunction, apoptosis and inflammation response, including the increase of intestinal permeability, histological score and the level of IL-1β, TNF-α, iNOS, NLRP3, MDA, Bax, Caspase3, Cytochrome C and Caspase9, as well as the reduction of body weight, colon length, Card9, IFN-γ, IL-10, T-AOC, Calpain1, Mfn2, VDAC1, RORα and SIRT1 proteins in DSS-treated mice. However, the improvement effects of melatonin were blocked by MT2 antagonist 4P-PDOT, PI3K antagonist LY294002, AKT antagonist GSK690693 and Nrf2 antagonist ML385, while mimicked by P65 antagonist PDTC in H. Melatonin-mediated MT2 activated PI3K/AKT/Nrf2/RORα/SIRT1 pathway and suppressed NF-κB pathway, ultimately improved DSS-induced colitis, which provides evidence for melatonin as an efficient therapy against oxidative stress associated IBD.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Colitis; Dextran Sulfate; Epithelial Cells; Hydrogen Peroxide; Male; Melatonin; Metallothionein; Mice, Inbred ICR; NF-E2-Related Factor 2; NF-kappa B; Nuclear Receptor Subfamily 1, Group F, Member 1; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Sirtuin 1

Although it is known that zinc has several beneficial roles in the context of gut inflammation, the underlying mechanisms have not been extensively characterized. Zinc (Zn) is known to be the primary physiological inducer of the expression of the metallothionein (MT) superfamily of small stress-responsive proteins. The expression of MTs in various tissues is induced or enhanced (including the gastrointestinal tract (GIT)) by a variety of stimuli, including infection and inflammation. However, the MTs' exact role in inflammation is still subject to debate. In order to establish whether or not MTs are the sole vectors in the Zn-based modulation of intestinal inflammation, we used transcriptomic and metagenomic approaches to assess the potential effect of dietary Zn, the mechanisms underlying the MTs' beneficial effects, and the induction of previously unidentified mediators. We found that the expression of endogenous MTs in the mouse GIT was stimulated by an optimized dietary supplementation with Zn. The protective effects of dietary supplementation with Zn were then evaluated in mouse models of chemically induced colitis. The potential contribution of MTs and other pathways was explored via transcriptomic analyses of the ileum and colon in Zn-treated mice. The microbiota's role was also assessed via fecal 16S rRNA sequencing. We found that high-dose dietary supplementation with Zn induced the expression of MT-encoding genes in the colon of healthy mice. We next demonstrated that the Zn diet significantly protected mice in the two models of induced colitis. When comparing Zn-treated and control mice, various genes were found to be differentially expressed in the colon and the ileum. Finally, we found that Zn supplementation did not modify the overall structure of the fecal microbiota, with the exception of (i) a significant increase in endogenous Clostridiaceae, and (ii) some subtle but specific changes at the family and genus levels. Our results emphasize the beneficial effects of excess dietary Zn on the prevention of colitis and inflammatory events in mouse models. The main underlying mechanisms were driven by the multifaceted roles of MTs and the other potential molecular mediators highlighted by our transcriptomic analyses although we cannot rule out contributions by other factors from the host and/or the microbiota.

Topics: Animals; Colitis; Colon; Dietary Supplements; Feces; Female; Gastrointestinal Microbiome; Ileum; Inflammation; Metallothionein; Mice; Mice, Inbred BALB C; Transcriptome; Zinc

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antibodies; Apoptosis; Biopsy; Case-Control Studies; Chemotaxis, Leukocyte; Chronic Disease; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Female; HT29 Cells; Humans; Macrophages; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Necrosis; Severity of Illness Index; Signal Transduction; Time Factors; Trinitrobenzenesulfonic Acid; Young Adult

Metallothioneins (MTs) are a family of cysteine-rich low molecular-weight proteins that can act as reactive oxygen species scavengers. Although it is known that the induction of MT expression suppresses various inflammatory disorders, the role of MTs in intestinal inflammation remains unclear. In this study, we investigated the effects of dextran sulfate sodium (DSS) administration in mice with targeted deletions of the MT-I/II genes. Acute colitis was induced by 2% DSS in male MT-I/II double knockout (MT-null) and C57BL/6 (wild-type) mice. The disease activity index (DAI) was determined on a daily basis for each animal, and consisted of a calculated score based on changes in body weight, stool consistency and intestinal bleeding. Histology, colon length, myeloperoxidase (MPO) activity and colonic mRNA expression and the concentration of inflammatory cytokines were evaluated by real-time-PCR and enzyme-linked immunosorbent assay (ELISA). The localization of MTs and macrophages was determined by immunohistological and immunofluorescence staining. To investigate the role of MTs in macrophages, peritoneal macrophages were isolated and their responses to lipopolysaccharide were measured. Following DSS administration, the DAI score increased in a time-dependent manner and was significantly enhanced in the MT-I/II knockout mice. Colonic MPO activity levels and inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-17] production increased following DSS administration, and these increases were significantly enhanced in the MT-I/II knockout mice compared with the wild-type mice. MT-positive cells were detected in the lamina propria and submucosal layer by immunohistochemical and immunofluorescence staining, and were mainly co-localized in F4/80-positive macrophages. The production of inflammatory cytokines (TNF-α, IFN-γ and IL-17) from isolated peritoneal macrophages increased following lipopolysaccharide stimulation, and these increases were significantly enhanced in the macrophages obtained from the MT-I/II knockout mice. These data indicate that MTs play an important role in the prevention of colonic mucosal inflammation in a mouse model of DSS-induced colitis, thus suggesting that endogenous MTs play a protective role against intestinal inflammation.

Topics: Animals; Body Weight; Colitis; Colon; Cytokines; Dextran Sulfate; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Immunohistochemistry; Inflammation; Inflammation Mediators; Intestinal Mucosa; Lipopolysaccharides; Macrophages, Peritoneal; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; RNA, Messenger

A positive-feedback mechanism between hypoxia-inducible factor 1 alpha (HIF-1α) and metallothioneins (MTs) has been identified in different diseases. Both proteins have been independently proposed in the pathogenesis of inflammatory bowel disease (IBD); however, their relation has never been studied in the gut. In this study, we investigated the interaction between HIF-1α and MTs in colonic epithelial cells and during experimental colitis. Dimethyloxalylglycine (DMOG) was used to subject colonocytes to hydroxylase inhibition and HIF-1α stabilization in three experimental models (in vitro, in vivo and ex vivo). Small interfering RNA targeting HIF-1α (siRNA-HIF) and MT (siRNA-MT) together with zinc-mediated MT induction were used to study the interaction between HIF-1α and MT in HT29 cells. Acute colitis was induced in C57BL/6 mice using dextran sulphate sodium. MT expression and HIF-1α protein levels were measured using quantitative real-time PCR and ELISA, respectively. Vascular endothelial growth factor (VEGF) expression was quantified as an indirect measure of HIF-1 transcriptional activity. DMOG down-regulated MT expression in HT29 cells, in freshly isolated human colonocytes and in colonocytes isolated from mice treated with DMOG (p<0.05). SiRNA-HIF-treated cells displayed significant higher basal MT levels (p<0.05) and an attenuated MT down-regulation after DMOG treatment. In turn, HIF-1α stabilization was significantly lower in zinc-treated control cells, displaying high MT levels, compared to siRNA-MT cells treated with DMOG (p<0.05). In the course of experimental colitis, MT and VEGF mRNA expression were inversely related. MTs were induced in the acute phase and down-regulated during recovery. Opposing results were observed for VEGF expression levels (p<0.05). The present study underscores the inverse relation between HIF-1α and MT expression in colonocytes and during experimental colitis. The manipulation of MTs may represent a novel therapeutic strategy for patients suffering from IBD.

Topics: Amino Acids, Dicarboxylic; Animals; Colitis; Colon; Down-Regulation; HT29 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammatory Bowel Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Models, Animal; RNA, Small Interfering; Vascular Endothelial Growth Factor A; Zinc

To evaluate the protective effects of N(G)-nitro-L-arginine methyl ester (L-NAME) and metallothioneins on excess nitric oxide toxicity in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis.. In this study, 70 rats were assigned to 7 groups of controls, and colitis was induced with 120 mg/kg TNBS, 35 mg/kg L-NAME, and 1 and 2 mg/kg metallothionein 1 (MT1) and metallothionein 2 (MT2), respectively. A day after the administration of TNBS, L-NAME, MT1 and MT2 were given intraperitoneally for 3 days to the experimental groups. After the administration of TNBS, dissections of the rats in the L-NAME, MT1 and MT2 groups were performed at 3-day periods under ether anesthesia, and whole blood, bone marrow and colon were obtained.. On the third day, red and white blood cell values were increased, while platelet and bone marrow granule cells decreased in the L-NAME- and TNBS-induced group. On the third day, all the blood values increased in MT1 (1 and 2 mg/kg) and MT2 (1 and 2 mg/kg) in the TNBS-administered groups. Histologic findings were macroscopic score, ulcer, loss of mucous cells, crypt abscess, inflammatory cyst, mucosa atrophy, edema, vascular dilatation and induced nitric oxide synthetase, which increased in the descending colon in the colitis rats, while it was decreased rats given L-NAME, MT1 and MT2 administration.. The results suggest that MT1 and MT2 are more effective in protecting against the toxic effects of excess nitric oxide as compared with L-NAME in the colitis rats.

Topics: Animals; Blood Cell Count; Bone Marrow; Colitis; Colon; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Intestinal Mucosa; Male; Metallothionein; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Trinitrobenzenesulfonic Acid

We evaluated whether modulation of metallothionein (MT, and subsequent oxidative stress) would influence the development and progression of colitis.. MT-transgenic (MT-TG), MT-knockout (MT-KO), and wild-type (WT) mice were treated with Dextran Sodium Sulfate (DSS) to induce colitis compared to controls (no DSS).. The DSS treated MT-TG and MT-KO mice responded in a manner similar to that of DSS treated WT mice, with all groups developing severe colitis. The colonic MT content in DSS treated animals increased drastically when compared to controls. The colonic and blood levels of the antioxidant, glutathione (GSH), were significantly reduced in DSS treated MT-TG and MT-KO mice. Howevei there was no significant difference in the cysteine levels in these mice. The subeellular level of MT was highest in intestinal crypts loci of MT-TG mice and was not observed in MT-KO mice as well as in the inflammatory lesions.. We conclude that MT does not appear to influence the development or progression of intestinal pathology in the DSS animal model of IBD.

Topics: Animals; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Gene Expression; Humans; Immunohistochemistry; Inflammatory Bowel Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic

Zinc enhances cell protection against infection and injury and the healing processes themselves. We evaluated the effect of zinc supplementation at different doses on a model of experimental colitis in the rat.. Colitis, induced by intra-rectal instillation of dinitrobenzen-sulphonic acid, was assessed at 1 week by examining: general outcome and macroscopic damage, myeloperoxidase activity, mucosal zinc, iron and metallothionein concentrations. Rats received zinc sulphate, 2 mg/kg or 30 mg/kg, twice a day by gavage for 9 days, starting 3 days before the induction of colitis, or intrarectal instillation of zinc (20 mg/kg) once daily starting 8 hours after the induction of colitis and for 6 days thereafter. Zinc-treated rats had less diarrhoea, higher body weight and lower colonic weight than untreated rats but no effect was observed on macroscopic inflammation, adhesions, colonic distension and neutrophil infiltration of the colonic mucosa. Zinc supplementation did not affect mucosal iron and zinc concentrations or plasma zinc levels in colitic rats. Metallothionein synthesis was induced in control rats and to a lesser extent in colitic rats.. Zinc administration induces metallothionein synthesis but has little effect on the short-term course of experimental colitis.

Topics: Administration, Oral; Administration, Rectal; Analysis of Variance; Animals; Colitis; Dietary Supplements; Disease Models, Animal; Dose-Response Relationship, Drug; Intestinal Mucosa; Male; Metallothionein; Peroxidase; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Trace Elements; Zinc