HIV and ART Effects on PBMC Mitochondria Figure 2. Determination of mitochondrial inner membrane potential (DYm) in lymphocytes and monocytes. JC-1 is a cationic carbocyanine probe that exibits a potential-dependent accumulation in mitochondria as either a monomer at low concentrations (green fluorescence) or as aggregates at higher concentrations (red fluorescence) [28]. Therefore, the red/green fluorescence intensity ratio illustrates DYm. (A to C) Monocytes. (A) CD14+/SSC monocyte gating. (B) Representative zebra plot based on side scatter and DYm. (C) Representative zebra plot based on side scatter and DYm after inhibition of DYm with CCCP (control experiment). (D to F) Lymphocytes. (D) FSC/SSC lymphocyte gating. (E) Representative zebra plot based on side scatter and DYm. Two lymphocyte subpopulations were distinguished (low-DYm, bottom right quadrants; high-DYm, top right quadrants). The top and bottom right quadrants indicate the DYm mean values (cell percentages). The percentages of low- DYm lymphocytes were: Control: 14.768.2%; ART naive: 12.5664.4% (statistically different from control); 2NRTI+1PI/r: 17.068.4%; 2NRTI+1NNRTI: 15.367.3%; 3NRTI: 16.365.1%. (F) Representative zebra plot based on side scatter and DYm after inhibition of DYm with CCCP (control experiment). doi:10.1371/journal.pone.0041129.g002 Among the 4 monocyte mitochondrial functional parameters (Figure 3F to H), changes were observed regarding the ratio of PMA-stimulated to basal ROS production by high-ROS cells (Figure 3G). Two Mitochondrial Functional Parameters in Lymphocytes, but Only One in Monocytes, were Correlated with Viral Load ROS production by high-ROS lymphocytes positively correlat- ed with the viral load (Figure 4A) while no correlation was found in monocytes (Figure 4D). The DYm of high-DYm lymphocytes decreased with viral load (Figure 4B), whereas the percentage of low-DYm lymphocytes simultaneously increased (Figure 4C). A similar correlation was found between monocyte DYm and viral load (Figure 4E). Discriminant Analysis Combining Three Morphological Parameters Revealed Differential Mitochondrial Network Responses to HIV-1 Infection and to ART in Lymphocytes and Monocytes Morphological parameters were measured by stereological analysis of confocal photomicrographs after image analysis processing (Figure S3). Lymphocyte mitochondrial networks from control subjects were statistically different to those from ART naive patients (p = 0.002) and those from each ART group (2NRTI+1NNRTI: p = 0.027; 2NRTI+1PI/r: p = 0.011; 3NRTI: p = 0.009). However, no statistical difference was observed by comparison of ART naive patients with each ART group, or by comparison of the three ART groups, as shown by the close superposition of circles representing each patient group (Figure 5A). In the ART naive patients, changes were mainly observed in volume density (Figure 5B) and branching (Figure 5D), but not fragmentation (Figure 5C). The ART combinations reversed the network volume density but failed to recover network branching. In contrast, the same parameters either in isolation (Figures 5F to 5H), or in combination through discriminant analysis (Figure 5E), were similar in monocytes from ART naive patients and control subjects (p = 0.344). Interestingly, each ART popula- tion was statistically different from both control subjects (2NRTI+1NNRTI: p = 0.0002; 2NRTI+1PI/r and 3NRTI: p,0.0001) and ART naive patients (2NRTI+1NNRTI: p = 0.039; 2NRTI+1PI/r: p = 0.002; 3NRTI: p = 0.0001), as shown by the ART-induced increases in network volume density and branching. The three ART groups were not statistically different (p = 0.084). Demonstration of Qualitative, but not Quantitative, Changes in Three PBMC Mitochondrial Proteins by Western Blotting The amounts of porin (a mitochondrial protein loading control), CoxIV-2 (encoded by the mitochondrial genome) and CoxIV-4 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41129