Metallothionein and oxidative stress responses moderators: M Lynes & L Hightower

Anti-MetallothioneinTherapeuticsopportunities for the treatment of inflammatory bowel diseasesMartine De Vos, Debby Laukens and Lindsey Devisscher (University of Gent, Ghent, Belgium)andMichael Lynes (University of Connecticut, Storrs, CT, USA)

OutlineMetallothionein (MT) overviewThe role of mammalian MT on immune functionsFocus on extracellular MTThe presence of MT in inflammatory bowel disease and the consequences of MT manipulationsFuture directions and opportunitiesmetallothioneinStressors initiate homeostatic responses, and can induce a spectrum of proteinsHeat shock proteinsglucose regulated proteinsFKBPcyclophilinsacute phase proteinssome cytokineshistone 2BubiquitinglucocorticoidsMetallothionein: an unusual biochemistrySmall (6-7 kDa), heat stable moleculeAbout 61 amino acids20/61 are cysteines4-11 molecules of heavy metal divalent cation per molecule of MTno aromatic or histidine residues, no disulfide linkagesNo signal peptideMDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA

CXCCX3CCCCcysteine motifs Crystal structure of Cd5, Zn2-MT2 (based on Robbins, A.H, et al. PDB structure 4MT2)

Highly homologous isoforms of Mammalian MT

Palacios O, Atrian S, Capdevila M. Zn- and Cu-thioneins:a functional classification for metallothioneins. J Biol Inorg Chem 2011;16:991-1009Expression profiles: MT1 and MT2 are ubiquitousMT3 predominantly expressed in the brainMT4 predominantly expressed in squamous cell epitheliumInduction of MT Gene Transcription ISRE GRE BLE MRE TRE GC MRE TATA+1IFN Ca

iono-phoreTNFIL-6IL-1phorbalestermetalcationsGCGC-RDAGPKCcAMPPKA[Ca]Calmodulin-PKMBPAP2SP1AP1-300-800H2O2ROS1000GREinflammatory agentsStructural MT gene: three exons interrupted by two intronsChromosome 8 (mouse) and Chromosome 16 (human)All of these inducers are immunomodulatory

Syntenic relationships between metallothionein gene clusters in humans and micemousehumanA summary of metallothionein functionsIntracellular functionsdecreases toxic effects of heavy metalsacts as a free radical scavenger, regulates cellular redox potentialserves as a reservoir for essential heavy metalsregulates NF-kB, Sp-1 transcription factor activity Extracellular functionsRedistribution of metal cations within bodyInteractions with membrane bound receptorsReports of an astrocyte receptorInteractions with megalin (surface molecule on kidney cells)

Hypothesis: Metallothionein that is synthesized as a result of stress can alter the capacity of the immune system, and manipulation of metallothionein can influence adaptive and innate immune activities and immune-related diseases.Metallothionein: an extracellular pool

MT has been found in serum, urine, pancreatic acini, liver sinusoids, glomeruli, etc.Secretome P analysishttp://www.cbs.dtu.dk/services/SecretomeP/MT1A_HUMAN predictionsNN-score Odds Weighted 0.8354.229 0.008Non-classically secreted proteins should obtain an NN-score exceeding the normal threshold of 0.5.Targeted disruption of Mt1 and Mt2 genes decreases Ptpn6me-v lifespan

50% survival

Ptpn6me-v or viable motheaten is a mutation in a cytosolic protein tyrosine phosphatase negative regulator of immune function that causes congenital inflammationWild type and congenic mutant pupsMutant adult

UC1MT-FITC binding to splenocytes from mev/mev and +/mev miceMetallothionein is detectable on the surface of viable motheaten splenocytes

Divalent heavy metal cations (Zn or Cd) induce Metallothionein in T cellsJurkat-T cells (1x106 cells/ml) were cultured in 24-well plates in RPMI-1640 supplemented with 20 M Cd, 100 M Zn, or vehicle control for 6 hours. After incubation, cells were fixed. Cells were then treated with UC1MT (IgG1) or isotype-matched MOPC21 and then stained with goat-anti-mouse IgG-FITC. Cells were mounted using Invitrogen ProLong Gold and analyzed using a Leica SP2 spectral confocal microscope.Exogenous extracellular metallothionein-mediated humoral immunosuppression in vivo 2220181614121020406080ovaova/mtdaysmOD/minMice were injected with 200 ug OVA with or without the addition of 120 ug MT on day 0 and day 10. Samples obtained on the days indicated were used in ELISA to determine the anti-OVA activity. Results are representative of three independent experiments and are reported as the average of triplicates + s.d.0

Collect serumMonoclonal anti-metallothionein Ab (clone UC1MT) enhances the humoral response to OVA immunization050100150200250300014182125323543daysanti OVA response (mOD/min)OVA OVA w/ UC1MTOVA w/ Ig ControlBALB/cByJ mice were challenged with 200 ug OVA in the presence or absence of UC1MT or isotype control on day 0 and day 10. (similar results were observed whenthe immunogen used was synthetic peptide conjugated to carrier protein)

How might this work?Intracellular MT is critical both as a metal reservoir, as an antioxidant and as a transcription factor regulatorExtracellular MT may interact with membrane receptors and alter immune cell behaviors (e.g. proliferation and cellular trafficking)The extracellular pool is amenable to manipulation with antibodySequence comparison of MT with a chemotactic factor, Ccl17

Amino acids compared at a threshold of 85% similarity are colored grey, boxed amino acids are identical.CCL17 or TARC (thymus and activation regulated chemokine), belongs to the IL8-like chemokine family, and maps close to the MT gene cluster. It induces chemotaxis in T cells and binds CCR4 receptorMeasuring chemotaxis: ECIS/taxis electrode design

~Cell wellTarget electrode (~5x10-4 cm2)

LargeElectrode (~0.12 cm2)Chemoattractant WellWiringChamberContact PadsCircuit: 1 volt AC with 1Mohm resistor applied to each well sequentially every x sec. Resistance at the small electrode dominates the circuit due to its small size relative to the large electrode.

Single ECIS chamber: side view

Cell WellLargeElectrodeTargetElectrode

To ECISInstrumentation

Diffusing chemoattractant from wellAgarose matrixMigrating cellsECIS/taxis- automated measurement of dictyostelium folate chemotaxis

Migrating cellsDiffusing chemoattractantImpedance measurementsTarget electrode

Both cholera toxin and pertussis toxin block the MT-mediated chemotactic response (suggesting a GCPR-type receptor target)Metallothionein induces leukocyte chemotaxisMetallothionein and SDF-1a evoke a chemotactic response in Jurkat T cells

Summary thus far:Chronic inflammation can be associated with MT expressionMT can bind to lymphocyte surfaces, and lymphocytes can also make MTMT has structural features that are shared with chemokines (chemotactic cytokines)Metallothionein can act as a chemotactic agent and may act through G protein coupled receptor(s)Manipulation of MT in mouse models of congenital inflammation changes the course of disease

How might MT relate to inflammatory bowel disease? The MT gene cluster is located at an important locus associated with IBD

(this is the most replicated locus ever found associated with IBD and also contains NOD2). Chromosome 16 (IBD1 locus): 2000 bases MT4 MT3 MT2A MT1L MT1E MT1F MT1G MT1H 55.156 K 55.180 K 55.200 K MT1X MT1K MT1J MT1A MTM MT1B MT1D MT1I MT1C 22

IBD is characterized by the presence of an increased level of ROS in the mucosal intestinal tissue as well as oxidative DNA and protein damage, defective host-microbe interactions, immune cell infiltration, and a disturbed T cell apoptosis. On all of these elements, MTs can have effects. In addition, MTs can have a dual role in enzyme activation through the release or sequestration of zinc. Finally, MTs are reported to regulate the activation of the transcription factor NF- B, which has a key role in inflammatory responses.

Anouk Waeytens,Martine De Vos,and Debby Laukenshttp://dx.doi.org/10.1155/2009/729172MT functions relevant in IBD.

Metallothioneins in clinical samples of IBD:Crohns Disease/Ulcerative ColitisAnouk Waeytens,Martine De Vos,and Debby Laukenshttp://dx.doi.org/10.1155/2009/729172

Mouse ModelsAvailable Congenic strains of C57BL/6JWild Type Control (MT-WT) C57BL/6JMT transgenic (MT-TgN) - Tg(Mt1)174Bri / 174Bri MT transgenic (MT-TgN het) - Tg(Mt1)174Bri / -MT knockout (MT-KO) - Mt1tm1Bri Mt2tm1Bri

What is the role of endogenous MT in experimental colitis?

4% DSS

H2O0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Dextran sulphate sodium-induced colitis - ACUTE4% DSS

H2O0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 . Dextran sulphate sodium-induced colitis - CHRONIC4% DSS

x3MT knockout and wild type mice in DSS-colitisMT-KO and WT mice were used to study the role of MT in experimental colitis. Dextran sulphate sodium (through drinking water) was used to induce acute and chronic colitis. Survival, weight loss and inflammatory parameters were assessed to compare the level of inflammation in MT-KO and WT mice.26MT knockout mice are favored during DSS-colitisACUTE COLITIS

Most strikingly, only 10% of the MT-KO mice died whereas almost 40% of the WT mice died. This higher survival rate in MT-KO mice was associated with less severe colonic inflammation in MT-KO mice as demonstrated by less weight loss, less histologic inflammation and less colon shortening (all parameters to assess experimental colitis).27MT knockout mice show reduced leukocyte infiltration

P=0.06

Histological analyses revealed reduced inflammatory cell infiltrate in MT-KO mice: myeloperoxidase activity is a measure for neutrophil infiltration, F4/80 staining was performed on colon sections to assess macrophage infiltration. 28MT knockout mice develop a less severe phenotype during DSS-colitisCHRONIC COLITIS

Chronic DSS-induced colitis confirmed the benefecial results for MT-KO mice with a higher weight preservation and less colon shortening: the latter suggests less structural damage and remodelling in MT-KO mice during chronic colitis.29Anti-MT antibody therapy in DSS- and TNBS-colitisDSS-colitis100 mg UC1MT or IgG i.p. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14randomizesamples4% DSSH2ODaysTNBS-colitis0 1 2 3RandomizeTNBS IRsamplesDays100 mg UC1MT or IgG i.p.

To extrapolate the results of MT-KO mice to a more clinical setting, we tested the monoclonal anti-MT antibody (UC1MT) in 2 acute colitis models, DSS- and TNBS-induced colitis. 30UC1MT in acute DSS-colitis

Antibody treated mice showed a higher survival rate in DSS-induced colitis and again less inflammatory cell infiltrate was observed for the anti-MT antibody treated mice (less macrophage infiltration).31UC1MT in acute TNBS-colitis

The TNBS-induced colitis confirmed the previous results with a trend towards less weight loss for the antibody treated mice and again less macrophage infiltration.32What is the site of action of the UC1MT antibody?

Approach: small animal imagingTo be sure/to investigate the action site of the antibody, small animal imaging was used.33

Small animal imaging - SPECT-CT Monoclonal UC1MTIndium 111DOTA4 control mice 4 colitis mice, day 7 4 colitis mice, day 14 injectionSPECT-CT and autoradiography 2 days laterThe anti-MT antibody was radioactive labeled with indium and injected in mice without colitis, in mice during acute DSS-induced colitis and in mice during recovery of DSS-induced colitis. The mice were scanned for activity 2 days after tracer injection (this ensures a proper wash out of the antibody from the blood through the liver and the kidneys and gives a specific binding on the images)34kidney

Colon

MidDist

Prox

Intensity scale

SPECT/CT data:Quantifying radioactivity in the colonAutoradiography of colon sectionHealthy InflammationHealingAt the top: increased colonic activity during colitis (compared to healthy mice) with a decreased activity during healing of DSS-induced colitis. In the middle: activity measured in the colon was normalized against total body activity (to normalize for injected dose of activity) and the results show a significant increase of activity in the colol during colitis an a deacrease during recovery (but activity during recovery was still higher than compared to colonic activity in healthy mice). At the bottom: prox, mid and distal colon sections were analysed for activity (autoradiography:colonic sections were exposed to a film in an autoradiogaphic cassette and imaged after 15 min.): this showed that the antibody binds the whole colon and again showed an increased activity during colitis and a decrease at the time of healing. Conclusion: the a temporary colonic binding of the antibody during colitis: points to a potential for its therapeutuc application35Genetic deletion of MT and antibody-mediated MT inhibition dampens experimental colitis, characterized by reduced leukocyte infiltration

UC1MT antibody binds the inflamed colon during colitisCellular release of MT?Antibodies act outsite the cell: MT extracellular?36MT release from stressed/damaged HT29 cells

HT29 cellsDoes the supernatant contain bioactive MT?CELL DEATH

TNF/IFNStaurosporine

Freeze/thawing

PRO-INFLAMMATORY STIMULI

LPSH2O2TNF

APOPTOSISNECROSISRelease of MT from HT29 cells following different triggers. HT29 cells: adenocarcinoma celline. We focused on epithelial cells since the epithelium is highly compromised in IBD patients.37Metallothioneins are released from necrotic HT29 cells

LPSH2O2TNF2M stauro10M stauroINFFreeze/thawing

6 kDaMTs are released from necrotic HT29 cells.38Will endogenous, released MT attract leukocytes?

500.000 blood isolated leukocytesMT containing conditioned medium+ anti-MT antibody (100 g/ml UC1MT)Boyden chamber migration assay39Endogenous released MT acts as potent chemokine

40MTs are released from necrotic intestinal epithelial cells

Released MTs acts as potent chemokine in vitro

This chemotactic function can be blocked in vitro by monoclonal therapy

41

Find-me signals

Dimer of ribosomal protein S19Endothelial monocyte-activating polypeptide IIFragments of human tyrosyl tRNA synthetaseThrombospondin 1Soluble IL-6 receptorFractalkineLysophosphatidylcholineSphingosine-1-phosphateNucleotidesLactoferrinApoptotic micro-blebs

DAMPs

High mobility group box 1 proteinHepatoma-derived growth factorCalgranulin proteinsHeat-shock proteinsATPIL-6 Uric acid

MetallothioneinsKono and Rock 2008, Nature reviews; Peter et al. 2010, Apoptosis Metallothioneins act as danger signals in the gut42Metallothioneins function as chemotactic danger signals and represent a novel target to dampen inflammation by reducing leukocyte infiltration in mice models for inflammatory bowel diseasesPending patent: P10/099: The use of antagonists targeting metallothionein to treat intestinal inflammation MT expression in human IBD?Ileal MT expression

Paneth cellImmunofluorescent staining for MT in healthy ileum: MTs are mainly expressed in the epithelium (paneth cells=specialized epithelial cells in charge of production of antimicrobial peptides, defensines..)44Colonic MT expression

Ulcerative ColitisColonic Crohns Disease

Healthy control Immunohistochemical staining for MTs in the colon of healthy, colonic Crohns disease and Ulcerative Colitis patient. MTs are mainly expressed in the epithelium of (healthy colon). During colitis there is a shift in MT immunoreactivity from mainly epithelial to the inflammatory infiltrate: human colitis is characterized by an increase in MT positive inflammatory cells.45MTs are mainly expressed in the colonic epithelium

MT immunoreactivity shifts from mainly epithelial to the inflammatory infiltrate during colitis

Positive correlation between the severity of colitis and lamina propria MT immunoreactivity

No correlation between epithelial MT immunoreactivity and the grade of colitis but MT expression is absent in highly necrotic regions

Ongoing studies/UGent:

Induction and release of MT from macrophages Effect of MT on macrophage polarizationLPS response of BM-derived macrophages from MT-KO and WT mice Anti-MT antibody treatment in T cell transfer induced colitisEffect of anti-MT treatment on lymphocyte proliferation

We are going to investigate 1) if macrophages are also a source for extracellular MT, 2) if MT influences macrophage polarization (M1: pro-inflammatory M; M2: regulatory M; 3) a higher dose of the anti-MT antibody will be tested in another experimental IBD colitis model (chronic colitis induced by transfer of CD4+, CD25- T-cells into SCID mice) which is driven by a Th-response and mimics colonic CD.47Ongoing studies/Uconn:Role of MT in management of the intracellular Zn pool and immune activityInfluences of MT in Cd-mediated immunomodulationBacterial MT analog (SmtA, Pseudomonas aeruginosa) and its role as virulence factorCollaboration: UC1MT influences on Epidermolysis Bullosa AquisitaGrating-coupled Surface Plasmon Resonance (GCSPR) and Grating-coupled Surface Plasmon Coupled Fluorescence (GCSPCE) microarrays and the detection of (a) toxins and toxicants, (b) polymicrobial infections, (c) functional T cell phenotypes in T1D and (d) biomarker signatures of post traumatic stress disorders

We are going to investigate 1) if macrophages are also a source for extracellular MT, 2) if MT influences macrophage polarization (M1: pro-inflammatory M; M2: regulatory M; 3) a higher dose of the anti-MT antibody will be tested in another experimental IBD colitis model (chronic colitis induced by transfer of CD4+, CD25- T-cells into SCID mice) which is driven by a Th-response and mimics colonic CD.48Next steps:I. in animal model(s)1. identification of MT-specific or MT-selective receptors (presumptive G-protein coupled receptors for chemotaxis response)2. determine cellular signaling cascades altered by MT3. determine if MT effects influences the microbiome of IBD mice

II. in human patients1. determine if MT expression levels (promoter occupancy, propensity to synthesize MT, etc) correlates with disease severity2. map the distribution of MT within the IBD wound sites (hypothesis that MT levels in the most severely damaged tissue is down due to the ROS-mediated destruction of MT antigenicity)3. characterize the effect of extracellular MT on released cytokines and leukocyte proliferation in situ