1. Inflammation and atherosclerosis Immune cells dominate early lesions Immune effector molecules accelerate lesion progression Activation of inflammation elicits acute coronary syndromes Atherosclerosis is an inflammatory disease in which immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial wall

2. The numbers CAD causes 38% of all deaths in North America Most common cause of death in men under 65 y.o. and second most common cause of women Dispite advances in control of hypercholesterolemia (statins),CVD expected to be the main cause of death globally over the next 15 years due to rapidly increasing prevalence of obesity and diabetes

3. Pathogenesis of coronary artery thrombosis

4. Cellular players in atherogenesis and plaque rupture

5. The early lesion: modified lipoproteins and foam cell formation

6. Role of macrophages MCSF induces monocytes entering lesion to differentiate into macrophages Macrophage differentiation associated with TLRs and scavenger receptors MCSF/apoE KO shows reduced atherosclerosis Scavenger receptors bind bacterial endotoxins, apoptotic cells and oxLDL SR/apoE KOs show reduced atherosclerosis

7. The macrophage as an inflammatory mediator

8. Toll-like GENES IL-1R TNF-R IFN-  R IRF NF  B MAP KINASE JAK/STAT Inflammation, Anti-Microbial Response, Apoptosis, …

9. TLR receptors and atherosclerosis 10 family members Recognize pathogen associated molecular patterns (e.g. LPS, dsRNA) as well as oxLDL, HSP60 etc. Initiate signaling cascades leading to production of inflammatory cytokines, proteases, reactive oxygen species In addition to macrophages, expressed by dendritic cells, mast cells, endothelial cells

10. Macrophage-T cell interactions in atherosclerosis

11. T cells and atherosclerosis Immune cells patrol tissues in search of antigen T cell infiltrate is common feature of atherosclerotic lesions Predominantly CD4+ cells, recognize antigen/MHC II CD4+ T cells reactive to oxLDL, HSP60, bacterial products detected in human lesions NK cells present in early lesions, recognize lipid antigens NK activation increases athero in apoE KO mice

12. T cell responses Th1 response activates macrophages and functions in the defense against intracellular pathogens Th2 response elicits allergic inflammation Atherosclerotic lesions contains cytokines that promote Th1 responses Activated Th1 effector cells in lesions produce macrophage activating cytokine IFN  IFN  improves efficiency of ag presentation and augments synthesis of TNF  and IL-1 IFN , TNF  and IL-1 in turn stimulate production of many other inflammatory mediators apoE mice lacking IFN  or downstream mediators such as IL-18 or T-bet show reduced athero

13. Anti-inflammatory factors and atherosclerosis Anti-inflammatory factors such as TGF  and IL-10 are protective IL-10 KO increases athero in mice and exacerbates thrombosis Abrogation of TGF signaling in T cells leads to large unstable atherosclerotic lesions

14. Immune cells and plaque rupture Preferentially occurs where fibrous cap is thin Active immune cells are abundant at site of rupture Immune cells produce inflammatory molecules and proteolytic enzymes that weaken cap, activate cells in the core and transform stable plaque into vulnerable, leading to plaque rupture MMPs likely to play important roles

15. Systemic inflammatory markers in atherosclerosis

16. Systemic indicators of inflammation Inflammatory process in lesions may lead to increased plasma levels of cytokines and acute phase proteins CRP and IL-6 are elevated in patients with unstable angina and MI Levels correlate with prognosis

17. TLR-3/4 IRF-3 NEMO IKK-  IKK  TANK TBK-1IKK i I  NF-  MyD88 “Independent” MyD88 “Dependent” Antiviral Response Inflammatory Response

18. MyD88/apoE KO mice show reduced athero

19. MyD88/apoE Ko mice show reduced chemokine expression

20. MyD88/apoE KO macs show reduced chemokine expression and recruitment in response to inflammatory cytokines

21. Unlikely to be caused by single organism Diverse pathogens have been detected within lesions Bacteria and viruses accelerate athero in murine models Links between infection and atherosclerosis

22. Potential mechanisms Stimulation of inflammatory cytokines Alteration of adhesion molecule expression ? Lipid metabolism ?

23. RXR LXR INFLAMMATION CHOLESTEROL EFFLUX CE oxLDLoxysterol cholesterol CD36  PLTP  ApoE/CII  ABCAI  LPL  iNOS  IL-6  IL-1   COX2  MMP9 TOLL4 IL-1R TNFR Inflammatory stimuli stimuli LPS, IL-1 , TNF  ABCA1 MACROPHAGE

24. 197870 136610 0 50000 100000 150000 200000 250000 300000 p < 0.005 Lesion area (  M 2 /section) + GW3965high fat LDLR-/- Aortic Sections 0 p < 0.005 50000 100000 150000 200000 250000 70621 134470 GW3965vehicle Lesion area (  M 2 /section) ApoE-/- Aortic Sections Synthetic LXR agonist reduces atherosclerosis inLDLR-/- and apoE-/- mice Joseph et al. PNAS 2002

25. C57Bl6BMT LXR  -/- BMT apoE -/- BMT Aortic Lesion Coverage (%) 0.02.55.07.5 * * Loss of bone marrow LXR expression accelerates athero Tangirala et al. PNAS 2002

26. Bacterial or viral infection blocks expression of LXR target genes in macrophages ctrl GW3965T1317E. ColiE. Coli + GW E. Coli + T ABCA1 apoE 36B4 FAS SREBP-1c iNOS ctrl GW3965T1317Infl AInfl A + GWInfl A + T ABCA1 ABCG1 MX-1 FAS IFN-  36B4 +/- E. coli +/- Influenza A Castrillo et al., Molecular Cell 2003 Antonio Castrillo, Sean Joseph

27. TLR3 and TLR4 ligands inhibit cholesterol efflux from macrophages Ctrl LTA Poly IC Lipid A TNF  apoAI-dependent efflux (%) 0 2 4 6 8 10 12 ** * * 14 Ctrl LTA Poly IC Lipid A TNF  Ctrl LXR ligand Cholesterol efflux

28. ABCA1 1 3 5 apoE 1 2 3 ABCG1 1 3 5 IFN-  100 300 500 IP-10 Spleen * * * * 10 30 50 mRNA ABCA1 apoE IFN-  1 3 5 7 200 300 400 100 IP-10 Vehicle Poly IC ABCG1 1 5 9 13 17 1 9 3 5 7 0 1 3 2 4 11 Aorta 19 * * * mRNA Vehicle Poly IC Vehicle Poly IC Vehicle Poly IC vehicle Poly IC Activation of TLR3 inhibits LXR signaling in vivo GW3965 Both

29. Foam cell formation bacteri a IRF-3 virus cholesterol efflux C C C C C C ABCA1 TLR-3/4 ABCA1, ABCG1, apoE LXRRXR TLR-LXR crosstalk: potential implications for atherosclerosis  Foam cell formation other signals TANK TBK-1 IKK i ?

31. Immunization with pneumococus induces oxLDL-specific IgM

32. Immunization with pneumococus induces oxLDL-specific IgM s. pneumo s. pneumo + oxLDL block s. pneumo + s. pneumo block E06 (ox PL)

33. Plasma from mice immunized with pneumococus inhibits macrophage binding of oxLDL

34. Immunization with pneumococus reduces atherosclerosis in LDLR-/- mice