Introduction Since 1996, antiretroviral therapy (ART) has increased life expectancy in HIV-infected patients who exhibit aging-related diseases [1]. The ANRS EP45 ‘‘Aging’’ study investigates the cellular mechanisms in peripheral blood mononuclear cells (PBMCs) that lead to aging in treated or untreated (naive) HIV- 1 infected patients. This paper is focused on mitochondria, the main energy- producing factories in the cell, which are known to be involved in ROS production [2], and also in antiviral innate immune defense [3] and aging [4]. Moreover, direct targeting of mitochondria either by proteins [5] and miRNAs [6] encoded by HIV, or by ART [7,8], is thought to trigger apoptosis [9]. The production of ATP by the respiratory chain involves multiheteromeric enzymatic complexes located in the inner mitochondrial membrane (IMM). Protons are pumped from the mitochondrial matrix to the intermembrane space to establish an electrochemical gradient that results in the IMM potential (DYm) required for ATP synthesis. More than 90% of the oxygen in tissues is consumed by mitochondria, and between 1 and 2–5% of the oxygen is transformed into reactive oxygen species (ROS) as respiratory chain by-products [10]. At low concentrations, ROS can function as signaling molecules [11]. However, at high concentrations, ROS may cause damage to cellular components even though the cell possesses sophisticated antioxidant defense systems [12]. Overproduction of ROS may therefore directly decrease DYm and lead to a lowered ATP supply, and may also cause mitochondrial network fragmentation and subsequent mitochondrial autophagy (mitophagy), cell apoptosis or cell senescence [13]. Mitochondrial network dynamics, cell apoptosis and autophagy exhibit close reciprocal relationships with innate antiviral signaling and mitochondrial morphological or functional (ROS production, DYm and ATP supply) parameters. These events are coordinated by common mitochondrial or cytosolic partner proteins that are regulated by post-translational modifications [14]. Mitochondria form a dynamic reticulum that is continuously remodeled by balanced fission and fusion (or ‘‘kiss and run’’) events controlled by two sets of outer (OMM) and inner (IMM) mitochondrial membrane specific proteins [15]. Fission events often generate uneven daughter mitochondria, with the fusion- competent mitochondria exhibiting a higher DYm. Fusion- incompetent mitochondria are characterized by a low DYm due to the accumulation of ROS-damaged molecules and mutated mtDNA [16], and are targeted for degradation by mitophagy [17]. Molecular partners that link ROS overproduction, DYm decrease, mitochondrial fission and mitophagy through the sequential recruitment and interaction of cytosolic proteins, OMM GTPases, IMM GTPases and oxidative phosphorylation (OXPHOS) complexes, have been implicated in the pathogenesis of Parkinson’s disease [18]. ROS [3] and DYm [19] also regulate the innate immune response triggered by cytosolic RNA helicases of the RLR (RIG-1- like receptors) family, via activation of the MAVS (Mitochondrial Antiviral Signaling) protein [20]. HIV escapes from antiviral signaling and innate immune responses through RIG-1 lysosomal degradation induced by HIV protease [21]. HIV-encoded proteins or miRNAs trigger mitochondrial- mediated apoptosis, which may explain the progressive decline in CD4+ T cells in infected patients [9,22]. Apoptosis has been shown to be triggered by ROS overproduction [23], DYm lowering [24] or network disruption [25]. HIV and ART Effects on PBMC Mitochondria ART mainly targets two steps of the HIV lifecycle. Nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) block reverse transcrip- tion, whereas ritonavir-boosted protease inhibitors (PI/r) prevent the cleavage of HIV-encoded gag-pol proteins. A combination of molecules from these two groups currently represents the most common HIV infection treatment. However, ART with primarily first generation NRTIs can cause mitochondrial toxicity through mitochondrial DNA polymerase c inhibition [26], which may contribute to patient aging [27]. Only limited data are available regarding toxicity due to NNRTIs and PIs [7]. We used PBMCs as an easily available cell type targeted by both HIV-1 and ART. As a direct or indirect response of HIV-1 infection, flow cytometry demonstrated that ROS production and mitochondrial DYm were altered in lymphocytes but not in monocytes from ART naive patients. ART partially rescued these lymphocyte parameters but induced slight mitochondrial changes in monocytes. Mitochondrial network morphology parameters (volume, fragmentation and branching) observed by confocal microscopy in these two cell types confirmed the functional mitochondrial results. Network fragmentation was stable regard- less of subject status, and an increase in network volume and branching argued against mitophagy or mitochondria-linked apoptosis related to ROS and DYm changes. No modifications in the amounts of mitochondrial proteins were observed in PBMCs. Even in patients considered to be clinically stable, the changes in PBMCs reported here, together with alteration of mitochondrial functional and morphological parameters in other tissues, may contribute to aging in HIV-1 infected patients. Methods ANRS EP45 Study & Participant Characteristics The ANRS EP45 ‘‘Aging’’ study is a cross-sectional and longitudinal (3 years) multicentre study. Patients were recruited by three specialised AIDS centres in France at Marseilles (the main coordination centre), Nice and Montpellier, and control subjects were recruited by CIC-UPCET (Pharmacologie Clinique & Evaluations The´rapeutiques, Timone Hospital) in Marseilles. Forty-nine HIV-1 patients infected for at least 24 months but who had not received ART (ART naive), and eighty-one HIV-1 infected patients in the first line of ART for at least 12 months, were enrolled. According to their ART combination, patients were distributed into three subgroups: patients treated with 2NRTIs and PI/r; patients treated with 2NRTIs and 1NNRTI; and patients treated with 3NRTIs. Forty-nine seronegative control subjects were age- and sex-matched with the ART naive patients. The basic demographic, clinical and biological parameters of the study participants, together with their ART combinations, are detailed in Tables S1 and S2. A complete clinical and biological description of the cohort will be detailed in another report (Faucher et al., in preparation). All experiments described below were performed blinded with respect to subject status (naive or treated HIV patients, control subjects). We report here the mitochondrial data measured at baseline. Ethics Statement The protocol was approved by the French Health Products Safety Agency Regulatory Authority (AFSSAPS, Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´) and Marseille’s Ethical Committee (Comite´ de Protection des Personnes Sud Me´diterrane´e I). The study was registered on the ClinicalTrials.gov web site (Identifier: NCT01038999, see supporting Protocol S1) and PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41129