Immunomodulation to treat atherosclerotic disease

G. Pasterkamp, UMCU, Utrecht, the Netherlands

Atherosclerosis and inflammation

Hansson GK N Engl J Med 2005;352:1685-95

Immunomodulation and atherosclerosis

• Immunosuppression– Cyclosporin (Emeson et al. Am J path 1993)– Transfer of CD4+ cells in Apo E mice

Zhou X et al. Circulation 2000

Immunomodulation and atherosclerosis

• Immunosuppression– Cyclosporin (Emeson et al. Am J path 1993)– Transfer of CD4+ cells in Apo E mice– Transfer of B2GP-I lymphocytes in LDL-R

mice (George J et al. Circulation 2000)

– Antibodies against CD40 and CD40 L

Anti CD40L in LDL-R mouse.

Mach F et al,

Nature 1998

CD154 -/-/ ApoE -/-

Lutgens et al

Nature Med 1999

Immunomodulation and atherosclerosis

• Active immunization– LDL modified epitopes

Palinski et al. PNAS 1995, LDL-R rabbit

MDA =malondialdehyde-LDL

Hypercholesterolemic rabbit,

Ameli S et al.

ATVB 1996

Immunomodulation and atherosclerosis

• Active immunization– LDL modified epitopes

• Passive immunisation– IgG– Antibodies Against Aldehyde-Modified

Apolipoprotein B-100 Peptide Sequences

ivIg reduces fatty streak formation in apo E KO mice

Nicoletti A et al

J Clin Invest 1998

Antibodies Against Aldehyde-Modified Apolipoprotein B-100

Schiopu et al.

Circulation 2004

Mice were treated with different doses of IEI-E3 (red) or FITC-8 (blue) antibodies. Values on y axis represent oil red O–stained areas as percentage of total descending aorta area..

Immunomodulation and atherosclerosis

• Active immunization– LDL modified epitopes

• Passive immunisation– IgG– Antibodies Against Aldehyde-Modified Apolipoprotein B-100

Peptide Sequences

• Interleukin-12 (IL-12) has been identified as a key inducer of a type 1 T-helper cell cytokine pattern. Blockade of interleukin-12 function by protein vaccination attenuates atherosclerosis. (Hauer et al. Circulation 2005)

• Cytokine network manipulation

Immunomodulation and atherosclerosis

• Induction of tolerance– Influencing Toll Like Receptor Responsiveness.

Toll-like receptors

• Innate immune system

• First line of defense

• Receptors for pathogen-associated patterns

• Family of 10 receptors in human

• Toll-like receptor 2 and 4 most attention in the cardiovascular field

Toll-like receptor pathway

NODs: intracellular proteins involved in inflammation

Inflammatory cytokines

TLR2TLR1

orTLR6

MyD88

TIRAP/Mal

IRAKTRAF6

NEMO/IKKIKK

IKKβ

NF- B

NF- B

Immunomodulation and atherosclerosis

• Induction of tolerance– Influencing Toll Like Receptor Responsiveness.

• Cross-tolerance TLR2 and TLR4

• Surgery influences endotoxin responsiveness (Lemaire et al J Clin Imunology 1998)

Pretreatment with LPS results in impaired infarct size in animal experimental model

Eising et al Cardiovasc Res 1996

cTnT release in a model of myocardial ischemia of the LAD

in the anesthetized rat

Zacharowski et al ATVB 1999

1a- patients with a history of UA and persisting complaints 1b- patients with a history of UA who were free of symptoms 2- patients with chronic angina 3- healthy volunteers

Liuzzo et al

Circulation 2001

Methods

• Patients scheduled for percutaneous coronary intervention (PCI) in the morning included after informed consent was signed

• Bloodsamples drawn immediately after sheath insertion and 2 hours after procedure

• Clinical questionnaires, data from patient file and angiographic data

Results

• 100 patients included

• 20 patients excluded:– 5 patients without 2nd bloodsample – 2 patients with intravenous corticosteroids excluded– In 13 patients no balloon dilatation was performed

due to negative FFR, unpassable lesion or impossibility to visualize coronary lesion

• Flowcytometry of TLR2 and TLR4

TLR2 response after 5000 ng/ml Pam3Cys

0

200

400

600

TNF-α concentration (pg/ml)

p < 0.01

beforePTCA

2h afterPTCA

TLR4 response after 100 ng/ml LPS

0

1000

2000

3000

4000

5000

TNF-α concentration (pg/ml)p < 0.01

beforen=80

aftern=80

TLR2 response (Pam3Cys)

mean TNF-α (±SEM)n=80

stimulatoryligand before after p

Pam3Cys5000 ng/ml

159(±20)

68(±10)

<0,01

Pam3Cys500 ng/ml

68(±9)

32(±5)

<0,01

Pam3Cys50 ng/ml

41(±5)

20(±4)

<0,01

TLR4 response (LPS)

mean TNF-α (±SEM)n=80

stimulatoryligand before after p

LPS 1000ng/ml

2338 (±161)

1957(±147)

<0,01

LPS 100ng/ml

1723(±128)

1271(±112)

<0,01

LPS 10ng/ml

893(±83)

519(±51)

<0,01

TLR2 expression

before after p

TLR2 ongranulocytes

0,73(±0,02)

0,70(±0,02)

0,01

TLR2 onmonocytes

3,30(±0,19)

2,90(±0,12)

<0,01

TLR4 expression

before after p

TLR4 ongranulocytes

0,61(±0,02)

0,59(±0,01)

0,65

TLR4 onmonocytes

3,31(±0,19)

2,87(±0,19)

0,05

TLR4 response without dilatation

0

1000

2000

3000

4000

5000

TNF-α concentration (pg/ml)

before, n=13

after, n=13

TLR2 response without dilatation

0

200

400

600

TNF-α concentration (pg/ml)

*

before, n=13

after, n=13

TLR2 response without dilatation

before after p

TLR2 ongranulocytes

0,82(±0,08)

0,79(±0,08)

0,26

TLR2 onmonocytes

4,16(±0,57)

3,82(±0,52)

0,08

Summary

• Coronary balloon dilatation decreases responsiveness of TLR2 and 4

• Coronary balloon dilatation decreases expression of TLR2 and TLR4 on individual granulocytes and monocytes

• These effects were also evident but less pronounced in patients with less traumatic intervention

TN

F-α

con

cent

ratio

n af

ter

10 n

g/m

l LP

S

percentage diameter stenosis

percentage diameter stenosis in relation to TLR4 response

51-70% 71-90% 91-99% 100%

0

1000

2000

3000

4000

5000

6000

Inhibition of TLR receptor and cytokine signaling-

A unifying theme in ischemic tolerance (Kariko et al J Cerebral Blood flow &Metabolism, 2004)

• Protection against effects (inflammatory responses) of acute ischemia by TLR tolerance induction.

Conclusion

Baseline responsiveness or tolerance of the innate immune system upon ligand stimulation differs among patients.

Understanding the mechanisms of tolerance development of the innate immune system may provide new targets for intervention

Tracey KJ, Nature. 2002 Dec 19-26;420(6917):853-9

The Immune reflex

Cholinergicanti-inflammatory pathway

• ACh prevents release of pro-inflammatory cytokines like TNF in macrophages

• Direct electrical vagal stimulations inhibits TNF synthesis in RES

• Vagotomy exacerbates TNF response to inflammatoy stimuli