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 ± SD) are indicated as percentages relative to CD14+ monocytes. The percentages of high-ROS monocytes were not statistically different between the groups: Control: 75.8±21.7%; ART naive: 70.7±19.4%; 2NRTI+1PI/r: 71.1±21.1%; 2NRTI+1NNRTI: 71.0±22.8%; 3NRTI: 66.2±23.7%. (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 ± 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: 55.4±12.7%; ART naive: 48.0±14.1%; 2NRTI+1PI/r: 55.1±13.8%; 2NRTI+1NNRTI: 50.1±14.1%; 3NRTI: 47.6±15.0%. ROS production by the low- and high-ROS subpopulations was statistically different in both HIV and ART patients.
Figure 2.
Determination of mitochondrial inner membrane potential (ΔΨm) 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 ΔΨm. (A to C) Monocytes. (A) CD14+/SSC monocyte gating. (B) Representative zebra plot based on side scatter and ΔΨm. (C) Representative zebra plot based on side scatter and ΔΨm after inhibition of ΔΨm with CCCP (control experiment). (D to F) Lymphocytes. (D) FSC/SSC lymphocyte gating. (E) Representative zebra plot based on side scatter and ΔΨm. Two lymphocyte subpopulations were distinguished (low-ΔΨm, bottom right quadrants; high-ΔΨm, top right quadrants). The top and bottom right quadrants indicate the ΔΨm mean values (cell percentages). The percentages of low-ΔΨm lymphocytes were: Control: 14.7±8.2%; ART naive: 12.56±4.4% (statistically different from control); 2NRTI+1PI/r: 17.0±8.4%; 2NRTI+1NNRTI: 15.3±7.3%; 3NRTI: 16.3±5.1%. (F) Representative zebra plot based on side scatter and ΔΨm after inhibition of ΔΨm with CCCP (control experiment).
Figure 3.
Mitochondrial functional parameters: differential effects of HIV-1 infection and ART on lymphocytes and monocytes.
(A, E) Discriminant analysis using ROS and ΔΨm parameters. The five cohort populations are delineated by their 95% confidence circles around the means. The perpendicular axes describe the combined variance of the parameters analysed (lymphocytes: 77.26%, monocytes: 92.25%). The contributions of each parameter to the variances on the X and Y axes are shown in the insets. (A) Lymphocytes. ART naive patients are statistically different to control subjects. ART partially rescues mitochondrial parameter changes induced by HIV infection. Inset: The 8 mitochondrial parameters used were: a: Basal ROS production by high-ROS lymphocytes (see Figure 3B). b: PMA/Basal ratio of ROS production by high-ROS lymphocytes (see Figure 3C). cHigh: ΔΨm of high-ΔΨm lymphocytes. cLow: ΔΨm of low-ΔΨm lymphocytes (see Figure 3D). cLow%: Percentage of low-ΔΨm lymphocytes. dHigh: Basal to CCCP ΔΨm ratio of high-ΔΨm lymphocytes. dLow: Basal to CCCP ΔΨm ratio of low-ΔΨm lymphocytes. dLow%: CCCP to Basal ratio of low-ΔΨm lymphocyte percentage. (E) Monocytes. ART naive patients are close to control subjects whereas ART combinations are more dispersed. Inset: The 4 mitochondrial parameters used were: a: Basal ROS production by high-ROS monocytes (see Figure 3F). b: PMA to Basal ratio of ROS production by high-ROS monocytes (see Figure 3G). c: ΔΨm of monocytes (see Figure 3H). d: Basal to CCCP ΔΨm ratio of monocytes. (B to D; F to H) Box plots (XLSTAT) displaying 1st quartile (Q1), median, mean (displayed by ‘+’), 3rd quartile (Q3) together with both lower and upper limits were calculated as follows: lower limit = Xi such that {Xi – [Q1–1.5 (Q3– Q1)]} is the minimum and Xi ≥ Q1–1.5 (Q3– Q1); upper limit = Yi such that { Yi – [Q3+1.5 (Q3– Q1)]} is the minimum and Yi ≤ Q3+1.5 (Q3– Q1). *p<0,05, **p<0,01.
Figure 4.
Correlation between mitochondrial functional parameters and viral load in cells from ART naive patients.
(A) ROS production by high-ROS lymphocytes (r = 0.355, p = 0.029, n = 38). (B) ΔΨm of high-ΔΨm lymphocytes (r = −0.417, p = 0.007, n = 40). (C) Percentage of low-ΔΨm lymphocytes (r = 0.457, p = 0.003, n = 40). (D) ROS production by high-ROS monocytes (r = 0.164, p = 0.288, n = 44) (E) ΔΨm of monocytes (r = −0.455, p = 0.003, n = 40).
Figure 5.
Mitochondrial morphological parameters: differential effects of HIV-1 infection and ART on lymphocytes and monocytes.
(A, E) Discriminant analysis using three mitochondrial morphological parameters. The five cohort populations are delineated by their 95% confidence circles around the means. The two perpendicular axes describe the combined variance of the parameters analysed (lymphocytes: 90.66%, monocytes: 98.73%). The contributions of each parameter to the variances on the X and Y axes are shown in the insets. Inset: The 3 mitochondrial parameters used were: Vv%: Volume density (lymphocytes: see Figure 5B; monocytes: see Figure 5F). Frag: Fragmentation (lymphocytes: see Figure 5C; monocytes: see Figure 5G). Bran: Branching (lymphocytes: see Figure 5D; monocytes: see Figure 5H). (A) Lymphocytes ART naive patients and control subjects are statistically different. Mitochondrial changes induced by HIV infection are partially reduced by ART. (E) Monocytes ART naive patients exhibit no variations compared to control subjects, while significant differences are observed for the three ART groups. (B to D; F to H) Box plots: *p<0,05, **p<0,01.