HIV and ART Effects on PBMC Mitochondria Figure 3. Mitochondrial functional parameters: differential effects of HIV-1 infection and ART on lymphocytes and monocytes. (A, E) Discriminant analysis using ROS and DYm 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: DYm of high-DYm lymphocytes. cLow: DYm of low-DYm lymphocytes (see Figure 3D). cLow%: Percentage of low-DYm lymphocytes. dHigh: Basal to CCCP DYm ratio of high-DYm lymphocytes. dLow: Basal to CCCP DYm ratio of low-DYm lymphocytes. dLow%: CCCP to Basal ratio of low-DYm 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: DYm of monocytes (see Figure 3H). d: Basal to CCCP DYm 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. doi:10.1371/journal.pone.0041129.g003 (encoded by the nuclear genome) purified from PBMCs were analysed relative to GAPDH in all cohort participants (Figure S5A). No statistically significant variation in the amounts of the three proteins was observed by either discriminant analysis combining the measurements of all three proteins (Figure S5B), or by comparing the amounts of the individual proteins (Figure S5C to E). Differences in the CoxIV-4 to CoxIV-2 ratio were also not observed (not shown). A 29 kDa band corresponding to CoxIV-4 was detected in 21% of the ART naive patients, 30% of the 2NRTI+1NNRTI patients, and 36% of the 3NRTI patients. However, this band was detected in only 1 of 38 PBMC homogenates from control subjects, and in 1 of 28 2NRTI+1PI/r patients (Figure S5F). Co-labelling with two specific antibodies suggested that this band may correspond to monoubiquitinylated CoxIV-4 (not shown). Finally, bands corre- sponding to a defect in the cleavage of the CoxIV-4 mitochondrial targeting signal in PBMCs from patients treated 2NRTI+1PI/r were not observed (Figure S5G). with Discussion The goal of the ANRS EP45 ‘‘Aging’’ study is to analyse, in a large cohort of HIV-1 infected and uninfected subjects, the impact of HIV-1 infection and of antiretroviral regimen on mitochondrial parameters. HIV-1 Infection Clearly Impacted Lymphocyte Mitochondria While Monocyte Mitochondria Appeared to be Less Sentitive PBMC mitochondrial parameters from ART naive patients compared to control subjects were used to delineate the consequences of HIV-infection, including the effects of viral 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) DYm of high-DYm lymphocytes (r = 20.417, p = 0.007, n = 40). (C) Percentage of low-DYm 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) DYm of monocytes (r = 20.455, p = 0.003, n = 40). doi:10.1371/journal.pone.0041129.g004 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41129