Efavirenz increases lipid content and regulates the inflammatory response in hepatic cells

A Blas-García1, C Hernández-Sáez2, D Ortiz-Masiá3, LJ Gómez-Sucerquia1, N Apostolova1, JV Esplugues1,2. 1Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia 46010, Spain, 2Fundación de Investigación Hospital Universitario Doctor Peset, Valencia 46017, Spain, 3CIBERehd, Valencia 46010, Spain

Introduction. The non-nucleoside reverse transcriptase inhibitor Efavirenz (EFV) has recently been associated with changes in lipid and body fat composition leading to enhanced levels of plasma fatty acids. In addition, we have previously reported that this compound promoted an accumulation of neutral lipids in hepatic cells, primarily in lipid droplets. However, the impact of the EFV-induced alterations on hepatic lipid metabolism and liver physiology has not been extensively studied.

Aims. To study the effects of EFV on lipid metabolism and inflammatory pathways in hepatocytes and the involvement of specific mediators and transcription factors in these effects.

Methods. Non-HIV-infected Hep3B cells were treated with clinically-used concentrations of EFV (10 and 25μM) for different times. Protein and mRNA expression of different genes involved in lipid metabolism in the liver was quantified by Western blot and real-time RT-PCR. In addition, a real time RT-PCR Array (RT2ProfilerTM PCR Array: Human Stress and ToxicityFinderTM, SABiosciences) was performed in order to study inflammation and immune response-related gene expression. The results of the array were validated by Real-time PCR. Activation of NF-κB was confirmed by Western blot. An electrophoretic mobility shift assay (EMSA) was performed to determine the binding of NF-κB to the promoters of some of the genes whose expression was found to be up-regulated. Cytokine and chemokine secretion was evaluated in culture supernatant samples using an immunoassay kit. Data (n≥3) were analyzed using one-way ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 (vs control).

Results. Enhanced intracellular lipid content was correlated with an induction of the mRNA expression of the fatty acid transporter CD36 as well as several lipid droplet proteins (adipophilin, S3-12). In addition, expression of the nuclear receptors PPARγ and LXRα was increased in EFV-treated cells. On the other hand, the real time RT-PCR Array showed that EFV significantly induced mRNA expression of the inflammatory mediator PAI-1. Furthermore, EFV significantly and dose-dependently decreased IκBα protein levels, increasing NF-κB translocation to the nucleus. EMSA assay demonstrated that trans-activation of PAI-1 was mediated by interaction of NF-κB with a consensus sequence located within the PAI-1 promoter. Nevertheless, EFV also significantly reduced the production and secretion of IL-8 and IP-10, chemokines involved in the progression of liver injury.

Conclusion. Due to its inhibitory effect on ETC activity generating ROS, or to the toxic effects resulting from intracellular fatty acid accumulation, EFV promotes NF-κB translocation to the nucleus, leading to the induction of PAI-1 expression. High levels of this protein have been related to pro-inflammatory and pro-fibrogenic actions that affect the phenotype of inflammatory liver diseases. Interestingly, EFV also decreases the production and secretion of IL-8 and IP-10 and, thus, has a dual role on the regulation of the inflammatory response, probably due to the anti-inflammatory effect of PPARγ. Considering the prolonged clinical use of this compound, these effects could easily accumulate and increase the liver toxicity induced by other pro-inflammatory stimuli, such as other antiretroviral drugs, co-infections (hepatitis B and/or C) or the HIV infection itself.