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)

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

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

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)

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

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

Syntenic relationships between metallothionein gene clusters in humans and mice

mouse human

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.

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.

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

UC1MT-FITC binding to splenocytes from mev/mev and +/mev mice

Metallothionein is detectable on the surface of viable motheaten splenocytes

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 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

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)

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

~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.

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

ECIS/taxis- automated measurement of dictyostelium folate chemotaxis

Migrating cells

Diffusing chemoattractant

Impedance measurements

Target electrode

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

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):

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

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.

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

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

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

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 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.

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

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

kidney

Colon

Mid

Dist

Prox

Inte

nsity

sca

le

SPECT/CT data:

Quantifying radioactivity in the colon

Autoradiography of colon section

Healthy Inflammation 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 colitisCellular release of MT?

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

Metallothioneins are released from necrotic HT29 cells

LPS H2O2 TNF 2µM stauro

10µM stauro

INF Freeze/thawing

6 kDa

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

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

‘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

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

MT expression in human IBD?

Ileal MT expression

Paneth cell

Colonic MT expression

Ulcerative ColitisColonic Crohn’s Disease

Healthy control

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

6.

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

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