Role of RANKL-RANK/Osteoprotegerin Pathway in Cardiovascular and Bone Disease Associated with HIV Infection

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1 AIDS Theodoros Rev. 2014;16: Kelesidis, et (Supplementary al.: RANKL/RANK/Osteoprotegerin Data) Pathway in HIV Infection (Supplementary Data) Role of RANKL-RANK/Osteoprotegerin Pathway in Cardiovascular and Bone Disease Associated with HIV Infection Theodoros Kelesidis 1, Judith S. Currier 1, Otto O. Yang 1,2 and Todd T. Brown 3 1 Department of Medicine, Division of Infectious Diseases, David Geffen School of Medicine, University of California, Los Angeles, CA, USA; 2 Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA; 3 Department of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University, Baltimore, MD, USA Supplementary Table 1. Parameters that affect levels of RANKL 1,2 Factors/molecules associated with increased RANKL Renal failure Chronic liver disease Immune disorders (e.g. RA) Malignancy Inherited skeletal diseases Postmenopausal osteoporosis 1,25-dihydroxivitamin D3 Parathyroid hormone-hyperparathyroidism Increased T-cell activation Increased osteoclast activation Glucocorticoids- glucocorticoid-induced Osteoporosis Soluble HIV-1 gp120 HIV Vpr ART e.g. PIs INF-γ (through T-cell activation) IL-1 (through effects on osteoblasts, bone marrow stromal cells) IL-6 ( the proliferation of osteoclast progenitors) IL-7 ( bone resorption, T-cell production of RANKL) IL-11 IL-17 TNF-α Prostaglandin E2 M-CSF ( expression of RANK on osteoclast precursors) Lipopolysaccharide CD40L Factors/molecules associated with reduced RANKL Age ART e.g. NRTI Low bone mass Antiresorptive therapy Osteoprotegerin (OPG) INF-γ (through direct inhibitory activity on osteoclast differentiation) ART: antiretroviral therapy; IL: interleukin; M-CSF: macrophage colony-stimulating factor; NRTI: nucleotide reverse transcriptase inhibitor; PI: protease inhibitor; RA: rheumatoid arthritis; RANKL: receptor activator of nuclear factor kappa-b ligand; TNF: tumor necrosis factor. 1

2 AIDS Reviews. 2014;16 Supplementary Table 2. Factors that affect levels of OPG 3,4 Factors/molecules that are associated with increased OPG Factors/molecules that are associated with reduced OPG Age Renal failure Chronic liver disease Antiresorptive therapy Exercise IL-1β IL-4 IL-6 IL-7 IL-11 IL-17 IL-18 TNF-a TNF-b TGF-β ( OPG production by osteoblasts) Bone morphogenic protein-2 Calcium Vitamin D 3 GM-CSF TNF-α, Angiotensin II CD40L Estrogens Platelet-derived growth factor PTH Glucocorticoids Cyclosporine A Prostaglandin E2 Peroxisome proliferators activated receptor-γ IL-10 TRAIL HIV-1 gp120 proteins Protease inhibitors Basic fibroblast growth factor GM-CSF: granulocyte-macrophage colony-stimulating factor; IL: interleukin; PTH: parathyroid hormone; OPG: osteoprotegerin; TGF: transforming growth factor; TNF: tumor necrosis factor; TRAIL: TNF-related apoptosis-inducing ligand. 2

3 Theodoros Kelesidis, et al.: RANKL/RANK/Osteoprotegerin Pathway in HIV Infection (Supplementary Data) Supplementary Table 3. Summary of interplay between RANKL, OPG, and immune system 5-7 Factor Role in the RANKL/OPG system Lymphocytes T-cells B-cells Monocytes/ osteoclasts Dendritic cells Neutrophils Thymocytes RANKL expressed by thymic epithelial cells may be responsible for the development and maturation of RANK-positive lymphocyte. Mice with a disrupted RANKL gene show a lack of all lymph node organogenesis, and impaired thymocyte development. PBMC have been reported to release OPG. RANKL is expressed on both activated CD4 + T-cells and CD8 + T-cells. RANKL may enhance the effects of CD40L on CTL responses by increasing the survival of CD40L-stimulated DCs. T lymphocytes from RANKL-deficient mice show poor induction of the cytokines IFN-γ, IL-2, IL-4, IL-5, and IL-6 upon stimulation with anti-cd3 and anti-cd28. OPG-deficient mice exhibit impaired thymocyte development. RANKL expression in the skin influences the number of regulatory T-cells. RANKL/RANK system may be involved in controlling T-regulatory cell function. B-cells are a significant source of RANKL when activated in vitro in postmenopausal humans in vivo and in inflammatory conditions. RANKL can influence B-cell development. RANKL / and RANK / mice have reduced cellularity of B-cells. B-cells produce OPG, which is regulated, in part, by T-cells through CD40/CD40 ligand (CD40L) costimulation. OPG regulates B lymphocyte development, maturation, and function. OPG-deficient mice exhibit impaired thymocyte development and presented disturbance in B-cell maturation and antibody response. RANKL directly increases osteoclast differentiation. RANKL behaves as a chemotactic factor for monocytes. OPG also can modulate the migration of monocytes. RANKL is an important survival factor for DCs. RANK, the receptor for RANKL, is constitutively expressed on DCs. RANKL/RANK signaling is important in T-cell/DC interactions. Similar to CD40L, conditioning of murine DCs with RANKL has been shown to elicit production of proinflammatory cytokines, IL-1 and IL-6, and T-cell growth and differentiation factors, IL-12 and IL-15. RANK expression is greater on mature DCs or on CD40-ligated DCs. RANKL enhances DC survival in tissues and priming of T-cells. DCs from OPG / mice present antigens (Ag) more efficiently and produce more inflammatory cytokines under soluble RANKL stimulation than do control DCs. RANKL dramatically inhibits DC apoptosis. The decoy receptor for RANKL, OPG, is also expressed in DCs. OPG promotes the adherence of neutrophils to endothelial cells in vitro and in vivo. RANKL/RANK controls the development of AIRE-positive thymic mtecs. RANKL/RANK has an important role in the regulation of central tolerance. Positively selected thymocytes express RANKL and that such RANKL-expressing T-cells expand mtec cellularity required to form the mature adult thymic medulla. CTL: cytotoxic T lymphocyte; DC: dendritic cell; IFN: interferon; IL: interleukin; mtec: medullary epithelial cell; OPG: osteoprotegerin; RANK: receptor activator of nuclear factor kappa-b; RANKL: receptor activator of nuclear factor kappa-b ligand. 3

4 AIDS Reviews. 2014;16 Supplementary Table 4. Studies in which RANKL blood levels were determined Study Comparison groups Matrix Antiretroviral therapy Range of RANKL in HIV patients Results Brown, et al HIV + (n = 331) ART-naive persons None 34 (16-57) (median, IQR pg/ml) No association observed with RANKL or OPG/RANKL and BMD Z score Gazzola, et al Hoy, et al Kelesidis, et al HIV + patients (n = 78) ART-naive (26%) or on stable ART for at least 12 months HIV + subjects randomized to continuous or intermittent ART HIV + subjects (n = 55) and HIV individuals (n = 36) TDF-based backbone: 53% PI-based: 33% NNRTI-based: 37% PI-based: 37% NNRTI-based: 36% NRTI-regimens: 79% Serum PI-based: 60% NNRTI-based: 31% NRTI-only regimens: 57% Hwang, et al HIV + men and 32 healthy controls Serum About 95% of these receiving ART and 53% on a regimen that included PIs LBMD: 5,9 ( ) (median, IQR pg/ml) NBMD: 8.4 ( ) (median, IQR pg/ml) No differences in median RANKL plasma levels between LBMD and NBMD patients (p = 0.08) 8.3 ( ) (median, IQR pg/ml) 3.9 ( ) Increases in RANKL and RANKL:OPG ratio at month 4 predicted increase in hip and spine BMD at month ( ,5) (median, IQR pg/ml) HIV + subjects had significantly (p < 0.01) lower baseline serum levels of RANKL and serum RANKL/ OPG compared to HIV subjects 2.52 ( ) (median, IQR pg/ml) srankl lower in HIV + individuals compared with controls Ross, et al HIV + subjects on ART 41% on NNRTIs and 59% on PIs NR No significant differences in plasma levels of RANKL among HIV + patients with or without use of rosiglitazone Haskelberg, et al Prospective randomized study of 357 HIV + subjects on ART Foca, et al Brown, et al HIV + patients (34.7% with viral load > 100,000 copies/ml) 87 HIV + subjects, 76% males with unsuppressed viremia (TDF n = 44 and non-tdf n = 43) Serum PI-based regimen: 23% TDF/abacavir naïve: 52% at baseline pmol/l No significant differences in serum levels of RANKL among HIV + patients on TDF/FTC versus ABC/3TC 44% TDF/FTC + EFV and 56% TDF/FTC + ATV/r < pg/ml in 86% patients RANKL remained or became undetectable in 87% patients throughout follow-up PI-based: 36% Non-TDF based: 49.4% TDF-based: 50.6% Pre-ART: 0.1 ( ) pg/ml Post-ART: 0.08 ( ) pg/ml In the total cohort, srankl decreased significantly with ART initiation, but the RANKL:OPG ratio remained unchanged Decrease in srankl in the TDF group greater than the non-tdf group (p = 0.03) Ofotokun, et al treatment-naive HIV patients starting ART Serum Lopinavir/r plus TDF/emtricitabine NR RANKL significantly elevated at week 24, explaining the surge in bone resorption Yin, et al HIV and 68 HIV + women Serum PI-based: 46% NNRTI-based: 28% NRTI-only regimens: 7% PI plus NNRTI-based: 3% 55.8 ± 2.6 pg/ml Serum RANKL levels comparable between HIV + and HIV women No significant difference in RANKL serum levels between ART-naive and ART-treated patients Gibellini, et al ART-naive HIV-1 + men No 535 ± 660 pg/ml (range 32-2,989 pg/ml) RANKL significantly (p < 0.01) increased in plasma of ART-naive HIV-1 + patients compared to control group of healthy blood donors RANKL plasma concentrations positively correlated to HIV-1 RNA viral load Mora, et al vertically HIV-infected children and adolescents on long-term ART Serum 3TC/d4T/IDV (9 cases), 3TC/d4T/NFV (8 cases), 3TC/d4T/RTV (10 cases). Changed to 3TC/EFV/ TDF (all 27 cases) after 6 months Konishi HIV + men Serum 8 patients did not receive any ART and 15 were on ART including PI PI regimen: 1.99 ± 0.45 pmol/l Non-PI regimen: 0.39 ± 0.05 pmol/l ART: ± pg/ml Non-ART: ± pg/m Baseline RANKL concentrations of HIV + patients significantly higher than in healthy children (p < ) RANKL concentrations decreased after 6 months, and were no longer different to controls Serum RANKL levels significantly higher in ART-treated patients than in non-art-treated patients. Serum RANKL levels inversely proportional to the BMD of the lumbar spine (r = 0.57; p < 0.01) Seminari HIV + patients Serum Heavily pretreated HIV-infected patients 0.1; pmol/l No differences in RANKL serum concentrations between osteopenic and control group Fakruddin, et al Fakruddin, et al HIV and HIV + women, ART-naive or on various ART regimens 25 HIV and 40 HIV + postmenopausal women, ART-naive or on various HAART regimens Serum NR NR Serum RANKL elevated in all HIV + women, regardless of ART use compared to controls (p = 0.046), but did not differ among the HIV + ART-naive or ART-treated Sera Treatment-naive or on various HAART regimens (NR) 47.5 ± 26.7 pg/ml Trend in elevation of serum RANKL observed in HIV + patients vs. controls HIV + men on HAART exhibited elevated RANKL serum concentrations compared with non-haart-treated and untreated patients 3TC: lamivudine; ART: antiretroviral therapy; ATV: atazanavir; BMD: bone mineral density; d4t: stavudine; EFV: efavirenz; FTC: emtricitabine; IDV: indinavir; LBMD: low bone mineral density; NBMD: normal bone mineral density; NFV: nelfinavir; NNRTI: nonnucleoside reverse transcriptase inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; OPG: osteoprotegerin; PI: protease inhibitor; RANKL: receptor activator of nuclear factor kappa-b ligand; RTV: ritonavir; TDF: tenofovir disoproxil fumarate. 4

5 Theodoros Kelesidis, et al.: RANKL/RANK/Osteoprotegerin Pathway in HIV Infection (Supplementary Data) Supplementary Table 5. Studies in which osteoprotegerin blood levels were determined Study Comparison groups Matrix Antiretroviral therapy Range of OPG in HIV patients Results Brown, et al HIV + subjects (n = 331) ART-naive persons None 4.2 ( ) (median, IQR pmol/l) Higher OPG associated with higher BMD Z score Gazzola, et al Hoy, et al Kelesidis, et al HIV + patients (n = 78) of both sexes who were naive (26%) or on stable HAART for at least 12 months with mean CD4 cell count 507 cells/mm 3 HIV + subjects randomized to continuous or intermittent ART HIV + subjects (n = 55) and HIV individuals (n = 36) TDF-based backbone: 53% PI-based: 33% NNRTI-based: 37% PI-based: 37% NNRTI-based: 36% NRTI-regimens: 79% Serum PI-based: 60% NNRTI-based 31% NRTI-only regimens: 57% Hwang, et al HIV + men and 32 healthy controls Serum About 95% receiving ART and 53% on a regimen that included PIs LBMD: 0.6 ( ) (median, IQR ng/ml) NBMD: 0.9 ( ) (median, IQR ng/ml) No differences in median OPG plasma levels between LBMD and NBMD patients (p = 0.97) 3.9 ( ) (median, IQR pg/ml) Increases in RANKL:OPG ratio at month 4 predicted increase in hip and spine BMD at month (0.8-1,6) (median, IQR ng/ml) HIV + subjects had similar baseline serum levels of OPG compared to HIV subjects 7.9 ( ) (median, IQR ng/ml) No significant differences in levels of OPG or the srankl:opg ratio between groups Ross, et al HIV + subjects with suppressed viremia 41% on NNRTIs 59% on PIs NR No significant differences in plasma levels of RANKL among HIV + patients with or without rosiglitazone use Haskelberg, et al Prospective randomized study of 357 HIV + subjects on ART Foca, et al Brown, et al Baekken, et al HIV + patients (34.7% with viral load > 100,000 copies/ml) 87 HIV + subjects, 76% with unsuppressed viremia 221 HIV + patients; 148 received HAART and 73 without treatment Serum PI-based regimen: 23% TDF/ABC-naive: 52% at baseline 44% TDF/FTC + EFV and 56% on TDF/FTC + ATV/r PI based: 36% Non-TDF based: 49.4% TDF based: 50.6% pmol/l No significant differences in serum levels of OPG among HIV + patients on TDF/FTC vs. ABC/3TC 0.83 ± 0.41 ng/ml Greater increase in OPG over 48 weeks follow-up in patients with better renal function at baseline Pre-ART: 4.5 ( ) pg/ml Post-ART: 4.1 ( ) pg/ml In the total cohort, OPG decreased significantly with ART initiation and the change in OPG with ART initiation was similar in the two treatment groups Serum Yes (NR) 6 ± 0.2 ng/ml HIV + patients had significantly higher levels of OPG compared with control subjects regardless of ART OPG significantly correlated to ACR in HIV + patients without treatment but not in those on ART Martinez, et al patients (46 ABC/3TC and 34 TDF/FTC) ABC/3TC and TDF/FTC ABC/3TC: 4.1 ( ) pg/ml TDF/FTC: 4.2 ( ) pg/ml ABC as the fixed-dose combination ABC/3TC for 48 weeks did not lead to any significant change in OPG compared with the fixed-dose combination TDF/FTC OPG had significant correlation with Framingham score and markers of endothelial dysfunction such as selectin-p Jang, et al HIV + patients (mean age 48.4 ± 9.1 years) Serum Yes-PI, other antiretrovirals (NR) 1,741.7 ± 1,244.3 pg/ml Concentrations of OPG significantly elevated in patients with high ABIs compared with patients with normal ABIs [mean: 3,088.6 (3,565.9) pg/ml vs. 1,428.9 (713.1) pg/ml, respectively; p = 0.03 Brown, et al HIV + adults on ART Serum 44 TDF-treated: 21 on PI-ATV (20), LPV/r (1) 43 non-tdf treated: AZT (39), ABC (17), ATV (6), LPV/r (4), FPV (1) Chakravarti, et al HIV + individuals (26/29 received ART); 62 age-matched controls Prior to ART: TDF group: 4.5 ± 0.4 pg/ml; Non-TDF: 5 ± 0.5 pg/ml After 6-12 months of ART: TDF group: 4 ± 0.8 pg/ml; Non-TDF: 4.2 ± 0.7 pg/ml OPG decreased with ART initiation prior to and 6-12 months after ART initiation OPG:RANKL ratio remained constant Changes in bone turnover not associated with systemic concentrations of OPG ART including PIs 209 ± 23 pg/ml OPG of HIV + individuals significantly lower than plasma OPG concentrations of 62 agematched controls Gibellini, et al ART-naive HIV-1 + men No 2,120 ± 1,816 pg/ml (range 690-1,126 pg/ml) OPG significantly increased in plasma of ART-naive HIV-1 + patients compared to control group of healthy blood donors No correlation between OPG content and HIV-1 RNA viral load TRAIL and OPG values did not exhibit any correlation with Z-score Mora, et al vertically HIV-infected children and adolescents on long-term ART Serum 3TC/d4T/IDV (9 cases), 3TC/d4T/NFV (8 cases), 3TC/d4T/RTV (10 cases). Changed to 3TC/EFV/ TDF (all 27 cases) after 6 months PI regimen: 8.6 ± 0.7 U/l Non-PI regimen: 6.4 ± 0.4 U/l PI regimen: RANKL/OPG: 0.27 ± 0.07 Non-PI regimen: 0.11 ± 0.03 Seminari, et al HIV + patients Serum Heavily pretreated HIV + patients 5; pmol/l OPG/RANKL ratio: 39 ( ) Dolan, et al HIV + and 63 healthy age-matched female control subjects Serum 93% on ART: 42% PIs, 80% NRTIs, and 27% NNRTIs Ueland, et al HIV + patients Serum 17 patients received ART (12 zidovudine, 1 didanosine and 4 zidovudine in combination with 3TC) Baseline OPG concentrations of HIV + patients significantly higher than in healthy children OPG concentrations decreased after 6 months, and were no longer different to controls At baseline, mean RANKL/OPG ratio of HIV + patients significantly higher than healthy controls OPG serum levels but not RANKL levels or the OPG:RANKL ratio significantly higher in the osteopenic versus control group 4.76 ± 0.23 pmol/l Serum OPG significantly increased in HIV + group compared with control subjects 1,250 ± 400 pg/ml 29 HIV + subjects had 52% increase of serum OPG compared to healthy controls ABC: abacavir; ACR: albumin/creatinine ratio; ART: antiretroviral therapy; BMD: bone mineral density; d4t, stavudine; EFV, efavirenz; FTC: emtricitabine; IDV, indinavir; 3TC, lamivudine; LBMD: low bone mineral density; NBMD: normal bone mineral density; NR: not reported; NFV: nelfinavir; NNRTI: nonnucleoside reverse transcriptase inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; OPG: osteoprotegerin; PI: protease inhibitor; RTV, ritonavir; RANKL: receptor activator of nuclear factor kappa-b ligand; TDF: tenofovir. 5

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