Kelesidis T, Kendall MA, Yang OO, Currier JS for the NWCS 332/A5078 Team

1. The Interplay of the Osteoprotegerin/RANKL Axis and Dysfunctional HDL in HIV-Infected AdultsAIDS Clinical Trials Group Study NWCS 332/A5078 Kelesidis T, Kendall MA, Yang OO, Currier JS for the NWCS 332/A5078 Team Abstract THAB0201, IAS 2012, July 26th, 2012

2. Oxidized Lipids as a link between oxidative stress and the RANKL-OPG pathway Oxidative stress T cells RANKL OPG Graham et al Clin Immunol 2009; Graham et al J Bone Miner Res 2010; Zhang et al Diabetes Metab Res Rev 2010; 26(6):455-463). Abstract THAB0201, IAS 2012, July 26th, 2012

3. HIV infection as a model for systemic oxidative stress: Possible cross-talk between HIV infection and the RANKL-OPG axis HIV infection T cell activation B cells CD4 T cells CD8 T cells RANKL TRAIL OPG INFLAMMATION: Proinflammatory cytokines and chemokines ART HIV infection Abstract THAB0201, IAS 2012, July 26th, 2012 • Antivir Ther 2011; 16:1063; AIDS 2005; 19:1240; AIDS 2007; 21:1129; J Med Virol 2007; 79:1446-1454

4. Dysfunctional HDL in the intersection of HIV infection and the RANKL-OPG axis • We have previously shown that HIV-infected subjects with suppressed viremia have dysfunctional HDL and that oxidized HDL is dysfunctional HDL Kelesidis et al JLR 2011, Lipids Health Dis 2011, Lipids Health Dis 2012 • Limited data exist regarding the relationship between dysfunctional HDL (ox-HDL) and the osteoprotegerin (OPG)/receptor activator of the NF-kB ligand (RANKL) in HIV infection. Abstract THAB0201, IAS 2012, July 26th, 2012

5. Hypothesis/Study Aims We hypothesized that increased serum levels of RANKL/OPG are independently associated with lipid levels and functional (redox) properties of HDL in HIV infected subjects RANKL Lipid levels (TC, HDL, LDL) OPG HDL redox activity (HDLox) RANKL/OPG Abstract THAB0201, IAS 2012, July 26th, 2012

6. Methods: Oxidation rate of a fluorescent probe (DOR) as an vitro biochemical measure of HDL redox activity Kelesidis et al J Lipid Res 2011 52(12):2341-51 Abstract THAB0201, IAS 2012, July 26th, 2012

7. Design • Matched by: • Cardiovascular risk factors • Age within 5 years • Race • Sex • Blood pressure status • Smoking status • Menopausal status Design: We used a subgroup of stored serum samples (n=91) from a previously completed prospective matched 3-year study (ACTG 5078) (Currier J et al AIDS 2007, 21:1137-1145) HIV-infected (HIV RNA < 500 copies/mL (n=55) HIV-uninfected subjects (n=36) Non-PI n=26 (47%) PI n=29 (53%) Abstract THAB0201, IAS 2012, July 26th, 2012

8. Results (1) : Metabolic Abnormalities HIV vs Control Abstract THAB0201, IAS 2012, July 26th, 2012

9. Results (2): HIV infected subjects had significantly lower RANKL and RANK/OPG compared to uninfected subjects Abstract THAB0201, IAS 2012, July 26th, 2012

10. Results (3) Baseline levels of the rate of oxidation of the fluorescent probe did not significantly differ between HIV infected and uninfected subjects Abstract THAB0201, IAS 2012, July 26th, 2012

11. Results (4) Serum levels of RANKL-OPG axis are independently associated with baseline levels of lipids and Dysfunctional HDL in HIV-1 infection In multivariate analyses within the HIV-infected (but NOT within uninfected) subjects ↑HDL ≥35 mg/dl  ΔRANKL  ΔOPG ↑Τotal cholesterol ≥ 200 mg/dl For each 10 units > average baseline RANKL/OPG  DOR by 0.81 (0.16, 1.47) x 104 FU/min HDL redox activity Abstract THAB0201, IAS 2012, July 26th, 2012

12. Results (5) The RANKL/OPG NFkB pathway is associated with changes in levels of lipids in HIV-infected subjects HIV Non HIV Abstract THAB0201, IAS 2012, July 26th, 2012

13. Results (6) The RANKL/OPG NFkB pathway is associated with HDL redox activity in HIV-infected subjects Non HIV HIV HDL redox activity (FU/min) HDL redox activity (FU/min) RANKL (pg/ml) RANKL/OPG ratio RANKL/OPG ratio RANKL (pg/ml) Abstract THAB0201, IAS 2012, July 26th, 2012

14. Summary • HIV-infected subjects on ART had significantly lower levels of total serum RANKL and RANKL/OPG ratio compared to HIV-uninfected subjects at both baseline and after 2-3 years of follow-up. • Higher baseline levels of HDL and total cholesterol were significantly associated with higher ΔRANKL and ΔOPG in HIV infected but not in uninfected subjects. • Functional properties of HDL were significantly associated with the serum RANKL/OPG ratio in HIV infected but not in uninfected subjects. Abstract THAB0201, IAS 2012, July 26th, 2012

15. Conclusions • RANKL/OPG axis may be aberrantly regulated in HIV-infected compared to -uninfected adults and is independently associated with baseline levels of cholesterol and changes in functional properties of HDL in HIV-infected subjects. • The interplay between T lymphocytes, lipids may explain our findings. • These in vivo data are consistent with previous in vitro results that have shown that oxidized HDL may affect the NF-kB pathway(Clin Immunol 2009; 133(2):265-275; J Bone Miner Res 2010; 25(11):2460-2469; Diabetes Metab Res Rev 2010; 26(6):455-463). • Further studies need to establish the relationship between lipids, T cells, RANKL-OPG axis and HDL function in HIV infection. Abstract THAB0201, IAS 2012, July 26th, 2012

16. Acknowledgements Statistician: Michelle Kendall, M.S., HSPH Investigators: Judith Currier, M.D., M.Sc., UCLA Otto Yang, MD, UCLA Howard Hodis, MD USC Special Thanks to the Study Participants