Immunomodulation to treat atherosclerotic disease

1. Immunomodulation to treat atherosclerotic disease G. Pasterkamp, UMCU, Utrecht, the Netherlands

2. Atherosclerosis and inflammation Hansson GK N Engl J Med 2005;352:1685-95

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

4. Zhou X et al. Circulation 2000

5. 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

6. Anti CD40L in LDL-R mouse. Mach F et al, Nature 1998 CD154 -/-/ ApoE -/- Lutgens et al Nature Med 1999

7. Immunomodulation and atherosclerosis • Active immunization • LDL modified epitopes

8. Palinski et al. PNAS 1995, LDL-R rabbit MDA =malondialdehyde-LDL

9. Hypercholesterolemic rabbit, Ameli S et al. ATVB 1996

10. Immunomodulation and atherosclerosis • Active immunization • LDL modified epitopes • Passive immunisation • IgG • Antibodies Against Aldehyde-Modified Apolipoprotein B-100 Peptide Sequences

11. ivIg reduces fatty streak formation in apo E KO mice Nicoletti A et al J Clin Invest 1998

12. 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..

13. 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

14. Immunomodulation and atherosclerosis • Induction of tolerance • Influencing Toll Like Receptor Responsiveness.

15. 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

16. NODs: intracellular proteins involved in inflammation Toll-like receptor pathway TLR1 or TLR6 TLR2 MyD88 TIRAP/Mal IRAK TRAF6 NEMO/IKK IKK IKKβ NF- B NF- B Inflammatory cytokines

17. 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)

18. Pretreatment with LPS results in impaired infarct size in animal experimental model Eising et al Cardiovasc Res 1996

19. cTnT release in a model of myocardial ischemia of the LAD in the anesthetized rat Zacharowski et al ATVB 1999

20. Liuzzo et al Circulation 2001 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

21. 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

22. 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

23. TLR2 response after 5000 ng/ml Pam3Cys 600 400 p < 0.01 TNF-α concentration (pg/ml) 200 0 before PTCA 2h after PTCA

24. TLR4 response after 100 ng/ml LPS 5000 4000 p < 0.01 3000 TNF-α concentration (pg/ml) 2000 1000 0 before n=80 after n=80

25. TLR2 response (Pam3Cys)

26. TLR4 response (LPS)

27. TLR2 expression

28. TLR4 expression

29. 5000 4000 3000 TNF-α concentration (pg/ml) 2000 1000 0 before, n=13 after, n=13 TLR4 response without dilatation

30. 600 400 TNF-α concentration (pg/ml) * 200 0 before, n=13 after, n=13 TLR2 response without dilatation

31. TLR2 response without dilatation

32. 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

33. percentage diameter stenosis in relation to TLR4 response 6000 S 5000 4000 3000 TNF-α concentration after 10 ng/ml LPS A 2000 A A A 1000 A 0 51-70% 71-90% 91-99% 100% percentage diameter stenosis

34. 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.

35. 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

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

37. 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