Macrophage. Adipocyte. Liver. NF-κB NF B 6. TF NF-κB IL-6 PG COX-2 AP 1. NF-κB AP-1 inos CD-36 AP-1 IL-6. basal MCP-1 ABCA1. IL-8 ET-1 NAPDH oxidase

Transcription

1 2010년순환기춘계통합학술대회 CD137(4-1BB), 1BB), a TNF Receptor Superfamily, Deficiency Reduces Atherosclerosis in Hyperlipidemic Mice Goo Taeg Oh, DVM., PhD Laboratory of Cardiovascular Genomics Division of Molecular Life Sciences Ewha Womans University gootaeg@ewha.ac.kr

2 Atherosclerosis is a Systemic Inflammatory Disease Macrophage Smooth muscle cell Endothelial cell AP 1 NF B 6 NF-κB AP-1 inos TF NF-κB IL-6 SR-A MMP- 9 LXR PG COX-2 ABCA1 ABCG1 ApoE SOD CD-36 Growth & migration NF-κB AP-1 IL-6 indmcp-1 VCAM-1 basal MCP-1 IL-8 ET-1 NAPDH oxidase A-I HDL ABCA1, G1 Cholesterol efflux A-I A-II A-I A-II Apos PL VLDL LPL LDL ABCA1 PPARα LDL-R ApoA-I, apoa-ii FA uptake FA β-oxidation ApoC-III circulating TG VLDL TG 1.TNF-α 2.Resistin 3.Adiponetin Adipocyte SR-BI HL Hepatocyte Liver

3 Cellular Composition of Atherosclerotic Plaque Hansson and Libby, Nat. Rev. Immunol., 2006

4 Recruitment and Activation of Immune Cells in Atherosclerotic Plaques Hansson and Libby, Nat. Rev. Immunol., 2006

5 T cell Mediated Immune Response in Atherosclerosis Co-stimulatory Molecules on T-cell TNFR & Atherosclerosis 1. Reduction of atherosclerosis in mice by inhibition of CD40 signaling. Nature 1998; 394: Requirement for CD154(CD40L) in the progression of atherosclerosis. Nat Med 1999; 5: M.Croft, Nature. Rev. Immunol., 2009

6 4-1BB (CD137) on T cell Surface MKKK MKK SAPK p38 4 1BB TRAF 1,2 12 IKK NF B A positive costimulatory role for 4-1BB on CD4 T cells. - The 4-1BBL augmented primary responses of CD4 T cells in the absence of CD28 signaling, and 4-1BB agonistic Ab enhanced CD4 T cell responses in vitro and in vivo. In contrast, it has been reported that 4-1BB ligation has either no specific role or even a negative role in regulating CD4 T cells BBL-/- mice generated normal CD4 T cell responses after lymphocytic choriomeningitis virus and influenza infection, although CD8 T cell responses were downregulated in those mice. In addition, 4-1BB agonistic Ab treatment unexpectedly led to suppressed pathogenic CD4 T cell responses in several autoimmune disease models, including experimental autoimmune encephalomyelitis, lupus, and collagen-induced arthritis. Therefore, 4-1BB may be involved in atherogenesis through its modulatory effect on T cell activation.

7 The Strong Preclinical Potency of anti-cd137 Treatment 3. Potential mechanisms underlying 2008

8 Effect of 4-1bb Agonistic Antibody on Atherosclerotic Plaque Formation in Ldlr Knock-Out Mice

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10 Expression of CD137 on Human Atherosclerosis Olofsson, P. S. et al. Circulation 2008;117:

11 CD3 and I-Ab immunostaining in atherosclerotic lesions of Apoe-/- mice Olofsson, P. S. et al. Circulation 2008;117:

12 Proposed model for CD137 in atherosclerosis Olofsson, P. S. et al. Circulation 2008;117:

13 Hypothesis 4-1BB ApoE-/- or Ldlr-/- Double Knock-out ROS TRAF 2,3,5 4-1bb-/- NIK P IkK P P IkB NF-kB NF-kB Atherogenic Gene Transcription Found it! Removed it! Proved it!

14 Find it!

15 Expression of 4-1BB in infiltrated T cells in human atheroma a b c d e f 4-1BB (green) CD3 (yellow)

16 Expression of 4-1BBL in Infiltrated Monocyte/Macrophage in Human Atheroma a b c d e f CD68 (green) 4-1BBL (red)

17 4-1BB expression of T cells in Mouse Atherosclerotic Lesio n a b c d ApoE-/- Ldlr -/- Ldlr 4-1bb+/+ -/- Ldlr -/- Ldlr -/- 4-1bb +/+ 4-1bb +/+ 4-1bb +/+ 4-1bb +/+ f e f g h Ldlr -/- Ldlr -/- Ldlr -/- Ldlr -/- 4-1bb +/+ 4-1bb -/- 4-1bb -/- 4-1bb -/- Triple immunostain for nuclei (blue), CD3 (green), 4-1bb (red).

18 4-1BBL Expression of Macrophages in Mouse Atherosclerotic Lesion i Ldlr -/- Ldlr j k -/- l Ldlr -/- 4-1bb +/+ 4-1bb +/+ 4-1bb +/+ Ldlr -/- 4-1bb +/+ m Ldlr -/- Ldlr -/- Ldlr -/- Ldlr n o p -/- 4-1bb +/+ 4-1bb -/- 4-1bb -/- 4-1bb -/- Triple immunostain for nuclei (blue), CD68 (green), 4-1bbl (red)

19 Remove it!

20 Removal of 4-1BB Reduced Atherosclerosis in Normal Diet ApoE Null Mice Plaq que/total % P < P < ale Fem 0 ApoE -/- ApoE -/- ApoE -/- ApoE -/- CD137 +/+ CD137 -/- CD137 +/+ CD137 -/- Female Male Male mm 2 ) ApoE -/ /+ P < 0.05 CD137 ApoE -/- CD137 -/- ApoE -/- CD137 +/+ ApoE -/- CD137 -/- Plaque Le esion Area ( P < ApoE -/- ApoE -/- ApoE -/- ApoE -/- CD137 +/+ CD137 -/- CD137 +/+ CD137 -/- Female Male ApoE -/- CD137 +/+ ApoE -/- CD137 +/+ ApoE -/- CD137 -/- ApoE -/- CD137 -/- Female Male

21 Removal of 4-1BB Reduced Atherosclerosis in High Fat Diet Ldlr Null Mice Plaque e/total % P < P < 0.01 Ldlr -/- CD137 +/ Ldlr -/- Ldlr -/- Ldlr -/- Ldlr -/- CD137 +/+ CD137 -/- CD137 +/+ CD137 -/- Female Male Female e Ma ale Ldlr -/- CD137 -/- Ldlr -/- CD137 +/+ Ldlr -/- CD137 -/- ion Area (mm m 2 ) Plaque Lesi P < Ldlr -/- Ldlr -/- Ldlr -/- Ldlr -/- CD137 +/+ CD137 -/- CD137 +/+ CD137 -/- Female P < Ldlr -/- CD137 +/+ Ldlr -/- CD137 +/+ Male Ldlr -/- / Ldlr -/- / CD137 -/- CD137 -/- Female Male

22 Prove it! Nat. Med., 2002

23 CD4 and IFN- Positive T cells are detected in Human Atheroma Control Merged CD4 4-1BB IFN-γ Merged

24 CD8 and IFN- Positive T cells are Detected in Human Atheroma Control Merged CD8 4-1BB IFN-γ Merged

25 4-1BB expressing CD4 + T cells in Mouse Atherosclerotic Lesion Produce Interferon-γ a b c d a b c e d fe gf Quadruple immunostain for nuclei (blue), 4-1BB (green), CD4 (red), Interferon-γ (violet).

26 4-1BB Expressing CD8 + T cells in Mouse Atherosclerotic Lesion Produce Interferon-γ a b c d a b c a b c e d f e gfg f Quadruple immunostain for nuclei (blue), 4-1BB (green), CD8 (red), Interferon-γ (violet).

27 4-1BB Mediates IFN- Production in Atherosclerotic Lesion IFN-γ γ Staining (%) P < 0.05 ApoE -/- ApoE -/- 4-1BB +/+ 4-1BB -/- 100 ApoE -/- 100 ApoE -/- /4-1BB -/- Relat ive mrna Expressio on (IFN- ) P < 0.01 ApoE -/- ApoE -/- 4-1BB +/+ 4-1BB -/-

28 Removal of 4-1BB Reduced IFN- Production in Mouse T cell 1 Ldlr -/- positive T cells % of IFN-Υ 0.9 Ldlr -/- 4-1BB -/ ApoE -/- ApoE -/- 4-1BB -/- * * 0.6 * * P < * * * CD3 + CD4 + CD8 +

29 Effect of 4-1BBL Antagonistic Antibody and 4-1BB Agonistic Antibody on IFN- Production in Extracted Splenocyte % of Total Splenocyte CD4/IFN- % of Total Splenocyte CD8/IFN- anti-cd3 1 g/ml + Rat IgG 4 g/ml anti-cd3 1 g/ml + anti-4-1bb 4 g/mll anti-cd3 1 g/ml + Rat IgG 10 g/ml anti-cd3 1 g/ml + anti 4-1BB 10 g/ml % of Total Splenocyte CD4/IFN- CD8/IFN- Rat IgG 20 g/ml oxldl 20 g/ml+ Rat IgG 20 g/ml oxldl 20 g/ml+ anti-4-1bbl 20 g/ml

30 SUMMARY??

31 Multimerization of CD137 by natural ligand (CD137L) and agonist mabs 2008

32 Expression of 4-1BB on Blood Vessel Walls Induces Migration of Monocytes

33 4-1BB is Expressed on the Endothelial Cells at the Site of Plaque Lesion a b c d e f g h Normal vessel (C57BL6/J) Triple immunostain for nuclei (blue), 4-1bb (red), CD31 (green).

34 Endothelial 4-1BB Signaling Enhanced the Production of Pro-inflammatory Molecules 25 Control TNF-a P < Human IgG - 4-1BB Ab - Genotype +/+

35 Removal of 4-1BB Reduced Macrophage Infiltration into Plaque Lesion Number of Migrate ed Cells Control TNF-a P < 0.05 P < 0.05 l. MOMA-2 Staining (%) P < 0.05 ApoE -/- 4-1bb +/+ ApoE -/- 4-1bb -/- 0 TNF BB Ab ApoE -/- 4-1bb +/+ ApoE -/- 4-1bb -/- Genotype +/+ +/+ -/- +/+ +/+ -/-

36 Co-localization of 4-1BB on the T cell and 4-1BBL on the Macrophage Triple immunostain for nuclei (blue), CD8(red), CD68(green). a b c d e f g h Triple immunostain for nuclei (blue), 4-1BB(green) 1BB(green), 4-1BBL (red)

37 Monocyte Produced Atherogenic Factors by the Activation via 4-1BB 4-1BB-Fc induce 4-1BBL Expression What kind of atherogenic factors? Isotype 4-1BB-Fc 2μg/ml 4-1BB-Fc 2μg/ml + oxldl 50μg/ml 4-1BBL BB BBL BB BBL BB MCP-1 Production P < 0.01 P < TNF-α Production P < 0.05 P < MCP P-1 (ng/ml) P < TNF-α α (ng/ml) P < LPS Human IgG 4-1BB-Fc μg 5μg - 1μg 5μg 5 0 LPS Human IgG 4-1BB-Fc μg 5μg - 1μg 5μg

38 Removal of 4-1BB Reduced the Production of TNF- and MCP-1 in vivo 80 P < P < 0.05 Staining (% %) Staining (% %) 35 ApoE -/- CD137 +/+ 30 ApoE -/- CD137 +/+ TNF-a 20 ApoE -/- 0 CD137 -/- 20 ApoE -/- ApoE -/- CD137 +/+ CD137 -/- Folds of Expression MCP-1 ApoE -/- CD137 +/+ ApoE -/- CD137 -/- P < P < 0.05 ApoE -/- CD137 +/+ ApoE -/- CD137 -/- ApoE -/- CD137 -/- 0 MCP-1 TNF-

39 MCP-1, TNF-α Production in 4-1BB-Fc treated Atherosclerotic Plaque MOMA-2 / MCP-1 / Nucleus MOMA-2 / TNF-α / Nucleus DAPI MOMA-2 MCP-1 DAPI MOMA-2 TNF-α

40 CONCLUSION Leukocytes MCP-1, IL-6 TNF-α MCP-1 IFN-γ Receptor signaling Endothelial cells 4-1BB 4-1BBL VCAM-1 ICAM-1 Macrophage Activated T cell Reverse signaling

41 Acknowledgement Collaborators Hyeung-Suk Kim Nobuyo Maeda Oliver Smithies Jeong Euy Park Byung Se Kwon Ewha Womans Univ. Young Dae Yoon Soo Young Lee

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