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Adipose tissue expression of IL-18 and HIV-associated lipodystrophy
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AIDS: Volume 18(14) 24 September 2004
Lindegaard, Birgittea,b; Hansen, Ann-Brit Ega; Pilegaard, Henrietteb,c; Keller, Pernillea,b; Gerstoft, Jana; Pedersen, Bente Klarlunda,b
aDepartment of Infectious Diseases, bThe Copenhagen Muscle Research Centre, Rigshosptalet and cAugust Krogh Institute, University of Copenhagen, Copenhagen, Denmark.
Summary. IL-18 is an inducer of apoptosis/tissue injury. IL-18 messenger RNA expression was examined in adipose tissue (AT) obtained from HIV patients with lipodystrophy, without lipodystrophy and healthy controls. IL-18 mRNA was expressed in AT at increased levels in lipodystrophy-positive compared with lipodystrophy-negative patients and healthy controls. Higher levels of IL-18 mRNA were found in femoral-gluteal AT compared with abdominal AT, and correlated with limb fat loss. These findings suggest that IL-18 is linked to HIV-associated lipodystrophy.
More than 50% of HIV patients receiving highly active antiretroviral therapy (HAART) develop lipodystrophy, characterized by the subcutaneous loss of adipose tissue (AT) from the limbs, buttocks and face. The pathogenesis of lipodystrophy remains elusive. Subcutaneous AT from HIV patients with lipodystrophy demonstrates adipocyte apoptosis and signs of inflammation with high expressions of TNF-α, IL-6 and IL-8. We recently found increased plasma levels of IL-18 in HIV patients with lipodystrophy. IL-18, a pro-inflammatory cytokine, is believed to be involved in apoptosis and ultimately tissue injury in inflammatory diseases. IL-18 induces TNF-a, and TNF-α also stimulates IL-18 production. IL-18 also induces IL-8, a chemotactic factor for neutrophil infiltration, and the production of IL-6. The circulating level of IL-18 is elevated in obesity, declines with weight loss [12], and is elevated in HIV patients with lipodystrophy. Given that IL-18 is associated with altered AT mass, inflammation and apoptosis, we hypothesized that IL-18 belongs to the new class of AT-derived cytokines, the adipocytokines, and that lipodystrophy and especially limb fat loss is associated with the high AT expression of IL-18.
In a cross-sectional study, we included 17 HIV patients with lipodystrophy, 17 without lipodystrophy and 22 healthy controls without the metabolic syndrome, all men and matched for age. All patients were on stable and effective nucleoside analogue-based HAART with no changes during the preceding 8 weeks. Of the patients with lipodystrophy, 12 were receiving protease inhibitors and nine were receiving non-nucleoside analogues. In those without lipodystrophy, nine patients were receiving protease inhibitors and three were receiving non-nucleoside analogues. Fat and fat-free tissue masses for the whole body, trunk and extremities were measured using dual-energy X-ray absorptiometry.
IL-18 mRNA was measured in subcutaneous abdominal and femoral-gluteal AT biopsies. AT RNA was purified and IL-18 mRNA was measured by real-time polymerase chain reaction. Human IL-18 Taqman probe and primers were amplified using pre-developed assay reagents obtained from Applied Biosystems (Foster City, CA, USA). The gene expression levels were normalized to the housekeeping gene 18S. Cytokines were measured in plasma using enzyme-linked immunosorbent assay.
HIV patients did not differ with regard to the duration of HIV infection, CD4 cell counts and viral loads. The mean duration of therapy was 96 months versus 67 months (P < 0.01, Student's t-test), and the mean body mass index was 21 versus 24 kg/m2 (P < 0.01, analysis of variance; ANOVA) in patients with lipodystrophy and those without lipodystrophy, respectively, but did not differ between lipodystrophy-negative patients and controls.
The dual-energy X-ray absorptiometry scans supported the clinical diagnosis of lipodystrophy. HIV patients were characterized by a reduced total fat mass [mean ± 95% confidence interval (CI) lipodystrophy-positive 9.85 kg; 8.04-11.7; lipodystrophy-negative 14.8 kg; 12.5-17.1; controls 19.9 kg; 17.8-21.9; P < 0.0001, ANOVA], truncal fat mass (lipodystrophy-positive 5.86 kg; 4.46-7.27; lipodystrophy-negative 8.02 kg; 6.43-9.62; controls 10.57 kg; 9.38-11.8, P < 0.0001) and limb fat mass (lipodystrophy-positive 3.15 kg; 2.59-3.72; lipodystrophy-negative 5.88 kg; 5.13-6.64; controls 8.41 kg; 7.55-9.28; P < 0.0001). The limb fat loss in patients with lipodystrophy was more pronounced compared with those without lipodystrophy (P < 0.0001), and the trunk : limb ratio was higher in patients with lipodystrophy compared with those without lipodystrophy and controls (lipodystrophy-positive 1.93; 1.53-2.34; lipodystrophy-negative 1.35; 1.18-1.52; controls 1.26; 1.17-1.36; P < 0.01). All groups had the same lean body mass.
IL-18 mRNA was expressed in subcutaneous AT in all three groups. IL-18 mRNA expression in abdominal subcutaneous AT was elevated in patients with lipodystrophy compared with those without lipodystrophy [geometric mean 1.75 (95% CI 1.10-2.77) versus 0.71 (95% CI 0.49-1.01), P = 0.008, ANOVA]. There was no difference between controls (0.95; 95% CI 0.69-1.32) and lipodystrophy-negative patients. In the study group, there was an overall greater expression of IL-18 mRNA in subcutaneous femoral-gluteal AT (1.39; 95% CI 1.11-1.73) compared with abdominal AT (0.96; 95% CI 0.73-1.26; P = 0.003, paired t-test), which was primarily caused by a difference within lipodystrophy-negative patients (P = 0.013). The mean plasma IL-18 was 416 pg/ml (95% CI 341.9-491.4); 347.0 pg/ml (95% CI 272.4-421.6) and 279.6 pg/ml (95% CI 233.9-325.3) in patients with lipodystrophy, those without lipodystrophy and controls, respectively, being significantly different between lipodystrophy-positive patients and controls (P = 0.005, ANOVA).
High levels of IL-18 mRNA in subcutaneous abdominal AT were associated with a high waist : hip ratio (r s = 0.40; P < 0.01, Spearman's correlation test), a high trunk : limb ratio (r s = 0.35; P < 0.05) and a low percentage limb fat mass (r s = -0.35; P < 0.02). The same trend was seen for plasma IL-18. IL-18 mRNA levels correlated positively with TNF-α mRNA (r s = 0.37; P < 0.02), IL-6 mRNA (r s = 0.26; P < 0.05) and IL-8 mRNA (r s = 0.63, P < 0.0001). Plasma IL-18 correlated positively with TNF-α mRNA (r s = 0.53, P < 0.001) and with plasma IL-6 (r s = 0.61; P < 0.0001), but not with IL-18 mRNA (r s = 0.12; NS).
The present study demonstrated for the first time the expression of IL-18 mRNA in human AT. Furthermore, patients with lipodystrophy had increased IL-18 mRNA levels in AT compared with those without lipodystrophy. The finding that high levels of IL-18 mRNA were associated with limb fat loss supports a previous finding of an association between plasma IL-18 and limb fat loss in another cohort of HIV patients. In patients with lipodystrophy, fat atrophy is most pronounced in the peripheral fat regions. The finding of increased IL-18 mRNA in subcutaneous femoral-gluteal AT compared with abdominal AT, is further in accordance with the hypothesis that IL-18 is involved in AT injury.
HIV patients with lipodystrophy demonstrate apoptosis of subcutaneous adipocytes in lipoatrophic areas. IL-18 induces apoptosis in other cell types, and it is possible that IL-18 in HIV patients can induce adipocyte apoptosis and thereby be mechanistically involved in the lipodystrophy syndrome. Alternatively, increased IL-18 mRNA levels may be a secondary phenomenon to a general inflammation in AT in lipodystrophic patients induced by HAART.
No correlation existed between IL-18 mRNA and circulating cytokines, whereas IL-18 mRNA correlated with the expression of IL-6, TNF-α and IL-8 mRNA in AT. As TNF-α, IL-6 and IL-8 mRNA are downstream targets of IL-18 mRNA, this may indicate that IL-18 is produced in AT and induces TNF-α, IL-8 and IL-6 expression in AT.
In conclusion, given that IL-18 is involved in apoptosis, the present finding that IL-18 is expressed in AT and is associated with lipoatrophy suggests that IL-18 is involved in the pathogenesis of the lipodystrophy syndrome in HAART-treated HIV patients.
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