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Anti-MetallothioneinTherapeutics
opportunities for the treatment of inflammatory bowel
diseases
Martine De Vos, Debby Laukens and Lindsey Devisscher
(University of Gent, Ghent, Belgium)and
Michael Lynes (University of Connecticut, Storrs, CT, USA)
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Outline
Metallothionein (MT) overviewThe role of mammalian MT on
immune functions◦Focus on extracellular MT
The presence of MT in inflammatory bowel disease and the consequences of MT manipulations
Future directions and opportunities
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metallothionein
STRESSORS INITIATE HOMEOSTATIC RESPONSES, AND CAN INDUCE A
SPECTRUM OF PROTEINS
Heat shock proteins glucose regulated
proteins FKBP cyclophilins
acute phase proteins some cytokines histone 2B ubiquitin glucocorticoids
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Metallothionein: an unusual biochemistry
• Small (6-7 kDa), heat stable molecule
• About 61 amino acids• 20/61 are cysteines• 4-11 molecules of
heavy metal divalent cation per molecule of MT
• no aromatic or histidine residues, no disulfide linkages
• No signal peptide
MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA
CXC CX3C CC C cysteine motifs
Crystal structure of Cd5, Zn2-MT2 (based on Robbins, A.H, et al. PDB structure 4MT2)
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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;16:991-1009
Expression profiles: MT1 and MT2 are ubiquitousMT3 predominantly expressed in the brainMT4 predominantly expressed in squamous cell epithelium
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Induction of MT Gene Transcription
ISRE GRE BLE MRE TRE GC MRE TATA+1
IFN
Ca iono-phore
TNF IL-6 IL-1
phorbalester metal
cations
GC
GC-R
DAG
PKC
cAMP
PKA
[Ca]
Calmodulin-PK
MBPAP2 SP1AP1
-300-800
H2O2
ROS
1000
GRE
inflammatory agents
Structural MT gene: three exons interrupted by two intronsChromosome 8 (mouse) and Chromosome 16 (human)
All of these inducers are immunomodulatory
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Syntenic relationships between metallothionein gene clusters in humans and mice
mouse human
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A summary of metallothionein functions
Intracellular functionsdecreases toxic effects of heavy metals
acts as a free radical scavenger, regulates cellular redox potential
serves as a reservoir for essential heavy metalsregulates NF-kB, Sp-1 transcription factor activity
Extracellular functionsRedistribution of metal cations within body
Interactions with membrane bound receptorsReports of an astrocyte receptor
Interactions 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.
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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 0.835 4.229
0.008Non-classically secreted proteins should obtain an NN-score
exceeding the normal threshold of 0.5.
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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 inflammation
Wild type and congenic mutant pups
Mutant adult
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UC1MT-FITC binding to splenocytes from mev/mev and +/mev mice
Metallothionein is detectable on the surface of viable motheaten splenocytes
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Divalent heavy metal cations (Zn or Cd) induce Metallothionein in T cells
Jurkat-T cells (1x106 cells/ml) were cultured in 24-well plates in RPMI-1640 supplemented with 20 µM Cd, 100 µM 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.
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Exogenous extracellular metallothionein-mediated humoral immunosuppression in vivo
22201816141210
20
40
60
80
ovaova/mt
days
mO
D/m
in
Mice 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 serum
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Monoclonal anti-metallothionein Ab (clone UC1MT) enhances the humoral response to OVA immunization
0
50
100
150
200
250
300
0 14 18 21 25 32 35 43days
an
ti O
VA
resp
on
se (
mO
D/m
in)
OVA OVA w/ UC1MTOVA w/ Ig Control
BALB/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)
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How might this work?Intracellular MT is critical both as
a metal reservoir, as an antioxidant and as a transcription factor regulator
Extracellular 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 antibody
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Sequence 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 receptor
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Measuring chemotaxis: ECIS/taxis electrode design
~Cell well
Target electrode (~5x10-4 cm2)
LargeElectrode (~0.12 cm2)
Chemoattractant Well
Wiring
Chamber
Contact Pads
Circuit: 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.
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Single ECIS chamber: side view
Cell Well
La rg eEle c tro de
Ta rg e tEle c tro de
To ECISInstrumentation
Diffusing chemoattractant from well
Agarose matrix
Migrating cells
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ECIS/taxis- automated measurement of dictyostelium folate chemotaxis
Migrating cells
Diffusing chemoattractant
Impedance measurements
Target electrode
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Both cholera toxin and pertussis toxin block the MT-mediated chemotactic response (suggesting a GCPR-type receptor target)
Metallothionein induces leukocyte chemotaxis
Metallothionein and SDF-1a evoke a chemotactic response in Jurkat T cells
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Summary thus far:
•Chronic inflammation can be associated with MT expression•MT can bind to lymphocyte surfaces, and lymphocytes can also make MT•MT 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
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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
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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/10.1155/2009/729172
MT functions relevant in IBD.
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Metallothioneins in clinical samples of IBD:Crohn’s Disease/Ulcerative Colitis
Anouk Waeytens, Martine De Vos, and Debby Laukenshttp://dx.doi.org/10.1155/2009/729172
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Mouse ModelsAvailable Congenic strains of C57BL/6J
Wild 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
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What is the role of
endogenous MT in
experimental colitis?
4% DSS H2O
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Dextran sulphate sodium-induced colitis - ACUTE
4% DSS H2O
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ….
Dextran sulphate sodium-induced colitis - CHRONIC
4% DSS
x3
MT knockout and wild type mice in DSS-colitis
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MT knockout mice are favored during DSS-colitis
ACUTE COLITIS
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MT knockout mice show reduced leukocyte infiltration
P=0.06
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MT knockout mice develop a less severe phenotype during DSS-colitis
CHRONIC COLITIS
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Anti-MT antibody therapy in DSS- and TNBS-colitis
DSS-colitis
100 mg UC1MT or IgG i.p.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
randomize samples
4% DSS H2O
Days
TNBS-colitis0 1 2 3
RandomizeTNBS IR
samples
Days
100 mg UC1MT or IgG i.p.
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UC1MT in acute DSS-colitis
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UC1MT in acute TNBS-colitis
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What is the site of action of the UC1MT antibody?
Approach: small animal imaging
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Small animal imaging - µSPECT-CT
Monoclonal UC1MT
Indium 111
DOTA
4 control mice
4 colitis mice, day 7
4 colitis mice, day 14
injection
µSPECT-CT and autoradiography 2 days later
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kidney
Colon
Mid
Dist
Prox
Inte
nsity
sca
le
SPECT/CT data:
Quantifying radioactivity in the colon
Autoradiography of colon section
Healthy Inflammation Healing
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Genetic 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?
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MT release from stressed/damaged HT29 cells
HT29 cells
Does the supernatant contain bioactive
MT?
CELL DEATH
TNF/IFN Staurosporine
Freeze/thawing
PRO-INFLAMMATORY STIMULI
LPS H2O2
TNF
APOPTOSIS
NECROSIS
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Metallothioneins are released from necrotic HT29 cells
LPS H2O2 TNF 2µM stauro
10µM stauro
INF Freeze/thawing
6 kDa
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Will endogenous, released MT attract leukocytes?
500.000 blood isolated leukocytes
MT containing conditioned medium
+ anti-MT antibody
(100 μg/ml UC1MT)
Boyden chamber migration assay
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Endogenous released MT acts as potent chemokine
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MTs 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
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‘Find-me’ signals
•Dimer of ribosomal protein S19•Endothelial monocyte-activating polypeptide II•Fragments of human tyrosyl tRNA synthetase•Thrombospondin 1•Soluble IL-6 receptor•Fractalkine•Lysophosphatidylcholine•Sphingosine-1-phosphate•Nucleotides•Lactoferrin•Apoptotic micro-blebs
DAMPs
•High mobility group box 1 protein•Hepatoma-derived growth factor•Calgranulin proteins•Heat-shock proteins•ATP•IL-6 •Uric acid
• Metallothioneins
Kono and Rock 2008, Nature reviews; Peter et al. 2010, Apoptosis
Metallothioneins act as danger signals in the gut
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Metallothioneins 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
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MT expression in human IBD?
Ileal MT expression
Paneth cell
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Colonic MT expression
Ulcerative ColitisColonic Crohn’s Disease
Healthy control
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MTs 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
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Ongoing studies/UGent:
1. Induction and release of MT from macrophages
2. Effect of MT on macrophage polarization
3. LPS response of BM-derived macrophages from
MT-KO and WT mice
4. Anti-MT antibody treatment in T cell transfer –
induced colitis
5. Effect of anti-MT treatment on lymphocyte
proliferation
6.
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Ongoing studies/Uconn:1. Role of MT in management of the intracellular Zn pool and
immune activity
2. Influences of MT in Cd-mediated immunomodulation
3. Bacterial MT analog (SmtA, Pseudomonas aeruginosa) and
its role as virulence factor
4. Collaboration: UC1MT influences on Epidermolysis Bullosa
Aquisita
5. Grating-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
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Next 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 MT
3. determine if MT effects influences the microbiome of IBD mice
II. in human patients
1. determine if MT expression levels (promoter occupancy, propensity to synthesize MT, etc) correlates with disease severity
2. 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