Metallothionein - University of Connecticut

Report
Anti-Metallothionein
Therapeutics
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)
Outline
Metallothionein (MT) overview
 The 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

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
metallothionein
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
CXC
CX3C
CC
C
cysteine motifs
MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA
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;16:991-1009
Expression profiles: MT1 and MT2 are ubiquitous
MT3 predominantly expressed in the brain
MT4 predominantly expressed in squamous cell epithelium
Induction of MT Gene Transcription
inflammatory agents
Ca
ionophore
GC
TNF
IL-6
IL-1
H2O2
IFN
[Ca]
cAMP
CalmodulinPK
GC-R
ISRE
-800
GRE
-300
AP2
BLE
DAG
ROS
PKA
PKC
AP1
MRE
phorbal
metal
ester
cations
SP1
MBP
TRE GC MRE TATA
+1
Structural MT gene: three exons interrupted by two introns
Chromosome 8 (mouse) and Chromosome 16 (human)
All of these inducers are immunomodulatory
GRE
1000
Syntenic relationships
between
metallothionein gene
clusters in humans and
mice
mouse
human
A summary of metallothionein functions
Intracellular functions
decreases toxic effects of heavy metals
acts as a free radical scavenger, regulates cellular redox potential
serves as a reservoir for essential heavy metals
regulates NF-kB, Sp-1 transcription factor activity
Extracellular functions
Redistribution of metal cations within body
Interactions with membrane bound receptors
Reports 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.
Metallothionein: an extracellular pool
MT has been found in
serum, urine, pancreatic
acini, liver sinusoids,
glomeruli, etc.
Secretome P analysis
http://www.cbs.dtu.dk/services/SecretomeP/
MT1A_HUMAN” predictions
NN-score
Odds
Weighted
0.835
4.229
0.008
Non-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 inflammation
Wild type and congenic
mutant pups
Mutant adult
Metallothionein is detectable on the surface of viable
motheaten splenocytes
_____
_____
_____
_____
+/mev
mev/mev
+/mev
mev/mev
(unstained)
(unstained)
(UC1MT-FITC)
(UC1MT-FITC)
UC1MT-FITC binding to splenocytes from mev/mev and +/mev
mice
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.
Exogenous extracellular metallothioneinmediated humoral immunosuppression in vivo
mOD/min
80
60
40
ova
ova/mt
20
0
10
12
14
16
days
18
20
22
Collect serum
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.
Monoclonal anti-metallothionein Ab (clone UC1MT) enhances
the humoral response to OVA immunization
anti OVA response (mOD/min)
300
OVA
OVA w/ UC1MT
OVA w/ Ig Control
250
200
150
100
50
0
0
14
18
21
25
32
35
43
days
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)
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
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
Measuring chemotaxis:
ECIS/taxis electrode design
~
Contact Pads
Cell well
Target electrode (~5x10-4 cm2)
Wiring
Chamber
Large
Chemoattractant
Well
Electrode
2
(~0.12 cm )
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.
Single ECIS chamber: side view
Cell Well
Diffusing
chemoattractant from
well
Agarose matrix
Migrating cells
La rg e
E l e c tro de
Ta rg e t
E l e c tro de
To ECIS
Instrumentation
ECIS/taxis- automated measurement of
dictyostelium folate chemotaxis
Migrating cells
Impedance
measurements
Target electrode
Diffusing
chemoattractant
Metallothionein induces leukocyte chemotaxis
Metallothionein and SDF-1a evoke a chemotactic response in Jurkat T cells
Both cholera toxin and pertussis toxin block the MT-mediated
chemotactic response (suggesting a GCPR-type receptor target)
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
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):
55.156 K
MT4
55.180 K 55.200 K
MT3
MT2A
MT1L
MT1E
MT1K MT1J MT1A MTM MT1C
MT1D
2000 bases
MT1B MT1F
MT1G MT1H MT1I
MT1X
MT functions relevant in IBD.
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 Laukens
http://dx.doi.org/10.1155/2009/729172
Metallothioneins in clinical samples of IBD:
Crohn’s Disease/Ulcerative Colitis
Anouk Waeytens, Martine De Vos, and Debby Laukens
http://dx.doi.org/10.1155/2009/729172
Mouse Models
Available Congenic strains of C57BL/6J
Wild Type Control (MT-WT) – C57BL/6J
 MT 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?
MT knockout and wild type mice in DSS-colitis
Dextran sulphate sodium-induced colitis - ACUTE
0
1
2
3
4
5
6
4% DSS
7
8
9
10
11
12
13
14
H2O
Dextran sulphate sodium-induced colitis - CHRONIC
0
1
4% DSS
2
3
4
5
H2O
6
7
8
9
10
11
12
13
4% DSS
x3
14
….
MT knockout mice are favored during DSS-colitis
ACUTE COLITIS
MT knockout mice show reduced leukocyte infiltration
P=0.06
MT knockout mice develop a less severe
phenotype during DSS-colitis
CHRONIC COLITIS
Anti-MT antibody therapy in DSS- and TNBScolitis
DSS-colitis
0
1
2
3
4
5
6
7
8
9
randomize
10
11
12
13
samples
4% DSS
H2O
100 mg UC1MT or IgG i.p.
TNBS-colitis
0
1
Randomize
TNBS IR
2
3
Days
samples
100 mg UC1MT
or IgG i.p.
14 Days
UC1MT in acute DSS-colitis
UC1MT in acute TNBS-colitis
What is the site of action of the UC1MT
antibody?
Approach: small animal imaging
Small animal imaging - µSPECT-CT
Indium 111
DOTA
Monoclonal UC1MT
injection
4 control mice
4 colitis mice,
day 7
4 colitis mice,
day 14
µSPECT-CT and autoradiography
2 days later
Healthy
Inflammation
Colon
SPECT/CT data:
kidney
Quantifying radioactivity in
the colon
Autoradiography of colon
section
Prox
Mid
Dist
Healing
Genetic deletion of MT and antibody-mediated
MT inhibition
dampens experimental colitis,
characterized by reduced leukocyte infiltration
UC1MT antibody binds the inflamed colon
during colitis
Cellular release of MT?
MT release from stressed/damaged HT29 cells
HT29 cells
CELL DEATH
 TNF/IFN
 Staurosporine
APOPTOSIS
 Freeze/thawing
NECROSIS
PRO-INFLAMMATORY STIMULI
 LPS
 H2O2
 TNF
Does the supernatant contain
bioactive
MT?
Metallothioneins are released from
necrotic HT29 cells
6 kDa
LPS
H2O2
TNF
2µM
stauro
10µM
stauro
INF
Freeze/thawing
Will endogenous, released MT attract leukocytes?
Boyden chamber migration assay
500.000 blood isolated leukocytes
+ anti-MT antibody
(100 μg/ml UC1MT)
MT containing conditioned medium
Endogenous released MT acts as potent chemokine
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
Metallothioneins act as danger signals in the
gut
‘Find-me’ signals
DAMPs
•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
•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 function as chemotactic danger signals
and represent a novel target to dampen inflammation by
reducing leukocyte infiltration in mice models for
inflammatory bowel diseases
Pending patent: P10/099: The use of antagonists
targeting metallothionein to treat intestinal inflammation
MT expression in human IBD?
Ileal MT expression
Paneth cell
Colonic MT expression
Healthy control
Colonic Crohn’s Disease
Ulcerative Colitis
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
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
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 Gratingcoupled 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
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

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