Background:  Abacavir (ABC) may be associated with a small increased risk of myocardial infarction in HIV⁺ adults, possibly related to cytokine-mediated inflammation. We recently reported that ABC up-regulates pro-inflammatory cytokine mRNA transcripatients in treated individuals. Based on these findings, we hypothesized that ABC could stimulate Toll-like receptor (TLR) signaling, as has been demonstrated for similar guanine nucleoside analogs.

Methods:  We transfected with a secreted alkaline phosphatase (SEAP) reporter 293 cells expressing various TLR under the transcriptional control of the NF-κB consensus sequence. Cells were stimulated with 100 µM ABC for 16 hours, after which SEAP expression was quantified via 650-nm absorption. Subsequently, 293 TLR-8 cells were stimulated with various concentrations of ABC, zidovudine, lamivudine, or tenofovir ranging from 100 to 1000 µM, and NF-κB activity determined. Luciferase-based reporter arrays that employ a 96-well plate format were used to determine the activity of 45 transcription factors after ABC stimulation. We transfected the 293-TLR-8 cells for 16 hours, then treated them with either 250 µM ABC or the equivalent volume of dimethyl sulphoxide (DMSO) for 6 hours, after which luciferase levels were quantified. Statistical significance was determined by Mann-Whitney test across multiple replicates.

Results:  TLR8⁺ cells demonstrated a >2-fold increase in NF-κB activation compared to unstimulated controls after ABC stimulation (p = 0.02). No difference was detected in 293 cells expressing TLR-2, -3, -5, -7, or -9. A dose-response curve identified NF-κB up-regulation between 1.7-fold (100 µM) and 25.7-fold (1000 µM) in cells treated with ABC, but not those treated with other NRTI. After 6 hours of ABC exposure, 7 transcription factors had a significant change in activity:  CREB (2.3-fold, p = 0.03), CCAAT-binding factor (CBF) (2.4-fold, p = 0.02), GATA-binding factor (1.8-fold; p = 0.03), hypoxia-inducible factor-1 (HIF-1) (1.7-fold, p = 0.047), serum response factor (SRF) (3.0-fold, p = 0.04), RBP-Jk (3.0-fold, p = 0.0005), and STAT3 (–1.5-fold, p = 0.01).

Conclusions:  Despite associations between ABC and myocardial infarctions, no molecular mechanism has been described. Our data show that ABC can stimulate an intracellular inflammatory response via TLR-8. Given that the intracellular concentration of carbovir triphosphate, the ABC metabolite, is between 50 and 500 µM in lymphocytes, our findings are within the physiological range expected in ABC-treated individuals.