performed in accordance with the Declaration of Helsinki. All subjects provided written informed consent before participation. PBMC Isolation The processing of blood samples is described in detail in the Methods S1. Briefly, PBMCs were isolated by FicollH gradient centrifugation (UNI-SEP MAXI+, Novamed) according to the manufacturer’s instructions. Leucocyte formulae were evaluated by May-Gru¨ nwald-Giemsa staining of cytospin samples. Cell viability was .97% (Methods S1, Figure S1). DYm Measurement DYm was determined using the red/green fluorescence intensity ratio of 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazol-car- bocyanine iodide probe (JC-1, Life Technologies). PBMCs were stained with 0.5 mM probe for 15 minutes at 37uC, 5% CO2. Pre- incubation with 10 mM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma) for 20 minutes was used as a positive control for mitochondrial membrane depolarization. Cells incubated without JC-1 probe were used as auto-fluorescence negative control cells. Monocytes were labelled using CD14-PC5 antibody (Beckman Coulter Inc) according to the manufacturer’s instructions. Residual red blood cells, if any, were hypotonically lysed using a solution of 100 mM EDTA, 150 mM NH4Cl, 1 mM KHCO3, pH 7.4. HIV-1 was inactivated by fixation for 30 minutes in ice-cooled 0.5% paraformaldehyde solution. As previously described [28], we confirmed that fixation induced no changes in the fluorescence pattern. Red and green mean fluorescence intensity and standard deviation (MFI 6 SD) were measured by flow cytometry (FC500, Beckman Coulter Inc) on an average of 3,000 CD14+ monocytes and 15,000 lymphocytes identified according to forward/side scatter (FSC/SSC). No CD14+ cells were observed in the lymphocyte population after gating. All data were analysed using FlowJoH software (Tree Star Inc). ROS Production ROS production was detected using 5-(and-6)-chloromethyl- 29,79-dichlorodihydro-fluorescein diacetate acetyl ester probe (CM-H2DCFDA, Life Technologies). PBMCs were incubated with 5 mM probe for 30 minutes at 37uC, 5% CO2. Co-incubation of CM-H2DCFDA with 50 ng/ml of phorbol 12-myristate 13- acetate (PMA, LC Laboratories) was used as a positive control. Cells incubated without CM-H2DCFDA probe were used as auto- fluorescence negative control cells. Staining of CD14-monocytes, lysis of residual red blood cells (if any), fixation and flow cytometry analysis were performed as described above. Analysis of the Mitochondrial Network The mitochondrial network was stained using MitotrackerH Red CMXRos probe (Life Technologies). PBMCs were stained with 25 nM probe for 15 minutes at 37uC, 5% CO2. After fixation for 15 minutes at room temperature (RT) with 4% paraformaldehyde solution, cytospin samples of PBMCs were prepared at 23 g for 5 minutes and nuclei were labelled with DAPI (0.1 mg/ml, Sigma) for 10 minutes at RT. Slides were mounted using FluorSaveTM reagent (Merck). A single confocal slide (Leica SP5, LAS 6000 AF Leica software, Leica Microsystems) was recorded at 1006magnification from 30 lymphocytes and 30 monocytes identified according to their nuclear size and shape after DAPI staining. Three individuals from each of the 5 groups (uninfected control, ART naive, 3 ART combinations) were selected for analysis. The individuals were chosen on the basis of possessing maximal (44 to 71), minimal (15 to 22) and medium (31 to 37) ROS values in high-ROS producing HIV and ART Effects on PBMC Mitochondria lymphocytes from flow cytometry data. DYm was confirmed as being inversely correlated with ROS values in the total lymphocyte population (Figure S2). Image analysis was performed using Image J 1.44 d software (http://rsbweb.nih.gov/ij/index.html). Three mi- tochondrial network stereological parameters (volume density, fragmentation and branching) were calculated for lymphocytes and monocytes using a specific macro designed by Le´on Espinosa (available on request). Figure S3 shows the macro steps and the corresponding results for 4 different monocytes. Western Blotting of Mitochondrial Proteins Total PBMC proteins from 160 cohort participants were extracted and analysed by a standard western blotting procedure, as detailed in Methods S1. Statistical Analysis Statistical analysis was conducted using SAS software, version 9.2 (SAS Institute Inc). DYm, ROS, mitochondrial network and quantitative protein parameters were compared between groups by analysis of variance or Kruskall-Wallis test. Pairwise compar- isons were conducted using the Bonferroni method for multiple comparisons. Relationships between parameters were assessed by Pearson’s correlation coefficient. XLSTATH software (AddinSoft) was used for box plot drawings and discriminant analysis, whereas comparison between groups used Wilks’ Lambda (Rao’s approx- imation) or Roy’s greatest root tests [29]. Percentage changes were calculated using mean values. p values ,0.05 were considered as statistically significant. Results Identification of Subpopulations of Lymphocytes and Monocytes by Flow Cytometric Measurements of Two Mitochondrial Functional Parameters: ROS Production and Inner Membrane Potential DYm Evidence for high- and low-ROS producing cells in all patients (monocytes), or in 72–77% of patients according to HIV-1 and ART status (lymphocytes) is shown in representative zebra plots and histograms of CD14+ monocytes (Figures 1B, 1C) or lymphocytes (Figures 1E, 1F). High-ROS cells significantly produced (p,0.0001) 8 to 10 times (monocytes) or 1.5 to 2 times (lymphocytes) more ROS than low-ROS cells. Among the five participant groups, no significant differences in the percentage of high-ROS producing monocytes (from 66 to 76%) or lymphocytes (from 48 to 55%) were observed. Further characterisation of these two lymphocyte subpopulations has been achieved in two small groups of control subjects, age- and sex-matched with HIV-1 infected and treated patients (CD4+ cell count .500/mm3; viral load ,40 copies/ml), out of the ANRS EP45 cohort. Regardless of HIV-1 status, four lymphocytes subtypes, CD3+, CD3+ CD4+, CD3+ CD8+ and CD32 CD19+, exhibited similar patterns of ROS production within the total lymphocyte population. This indicated that these cells were subdivided into both high- and low-ROS cells, whereas CD32 CD192 CD16+ CD56+ NK cells were always low-ROS producing cells (Figure S4). HIV-1 infected CD4+ lymphocytes presented the same pattern as CD8+ and CD19+ uninfected cells. Measurements of DYm by flow cytometry after incubation with the DYm inhibitor, CCCP, clearly identified two subpopulations of lymphocytes (Figures 2F). These two subpopulations were separated by quadrant markers and were considered as high-DYm lymphocytes with functional mitochondria (top right quadrant), and low-DYm lymphocytes containing weakly functional mito- chondria (bottom right quadrant). CCCP markedly decreased PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41129