HIV and ART Effects on PBMC Mitochondria Figure 1. Flow cytometric analysis of ROS production revealed two subpopulations of both CD14+ monocytes and lymphocytes. (A to C) Monocytes. (A) CD14+/SSC monocyte gating. (B) Representative zebra plot based on side scatter and ROS production showing low- and high- ROS producing cell subpopulations. (C) Histogram illustrating ROS production as a function of cell count (low-ROS production, gray; high-ROS production, red). ROS production levels (MFI 6 SD) are indicated as percentages relative to CD14+ monocytes. The percentages of high-ROS monocytes were not statistically different between the groups: Control: 75.8621.7%; ART naive: 70.7619.4%; 2NRTI+1PI/r: 71.1621.1%; 2NRTI+1NNRTI: 71.0622.8%; 3NRTI: 66.2623.7%. (D to F) Lymphocytes. (D) FSC/SSC lymphocyte gating. (E) Representative zebra plot based on side scatter and ROS production showing low- and high-ROS producing cell subpopulations. (F) Histogram illustrating ROS production as a function of cell count (low-ROS production, gray; high-ROS production, red). ROS production levels (MFI 6 SD) are indicated as percentages relative to FSC/SSC- selected lymphocytes. The percentages of high-ROS lymphocytes were not statistically different between the groups: Control: 55.4612.7%; ART naive: 48.0614.1%; 2NRTI+1PI/r: 55.1613.8%; 2NRTI+1NNRTI: 50.1614.1%; 3NRTI: 47.6615.0%. ROS production by the low- and high-ROS subpopulations was statistically different in both HIV and ART patients. doi:10.1371/journal.pone.0041129.g001 DYm in both lymphocyte subpopulations and considerably increased the percentage of cells with weakly functional mito- chondria. The same quadrant markers were then applied to lymphocytes under basal conditions. In contrast, low- and high- DYm monocytes were not observed (Figures 2B, 2C). Because the emission spectra of the probes overlapped, simultaneous measurement of ROS and DYm was not performed in lymphocytes or monocytes. Therefore, the relationships, if any, between high- or low-ROS and DYm remain unknown. A negative correlation (p,0.001) was highlighted between DYm and ROS production within the total lymphocyte population (Figure S2), but not by monocytes (not shown). Multiparametric Discriminant Analysis of 8 (Lymphocytes) or 4 (Monocytes) Mitochondrial Functional Parameters Revealed Differential Responses by the Two Cell Types to Either HIV-1 Infection or ART Because changes in low-ROS cells between infected and uninfected subjects were not observed, we focused our investiga- tions on high-ROS cells. Lymphocyte mitochondria ROS- and DYm-related parameters in ART naive patients were statistically different to those in control subjects (p,0.0001, Figure 3A). Mitochondrial changes were partially rescued by certain ART combinations. The 2NRTI+1NNRTI population was not statistically different to ART naive patients (p = 0.181) or control subjects (p = 0.068). In contrast, 2NRTI+1PI/r patients were statistically different to both the ART naive patients (p = 0.0001) and control subjects (p = 0.006). The 3NRTI population, which exhibited the most heterogeneous pattern as shown by their large 95% confidence circle, was statistically different to the ART naive patients (p = 0.005) but not the control subjects (p = 0.081). Among the 8 lymphocyte mitochondrial functional parameters, the changes mainly concerned basal ROS production by high- ROS cells (Figure 3B), the PMA-stimulated to basal ratio (Figure 3C), DYm of low-DYm lymphocytes (Figure 3D), and, to a lesser extent, the percentage of low-DYm cells (not shown). Neither HIV-1 infection nor ART induced statistical differences in DYm values from High-DYm lymphocytes compared to control subjects (data not shown). In contrast to lymphocytes, the 4 monocyte ROS- and DYm- related parameters were unchanged in the ART naive patients compared to control subjects (p = 0.838, Figure 3E). The three ART combinations showed slight but not significant differences to the ART naive or control populations. However, a statistical difference (p = 0.023) was recorded between control subjects and 2NRTI+1NNRTI patients. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41129