HIV and ART Effects on PBMC Mitochondria 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. doi:10.1371/journal.pone.0041129.g005 proteins [5], miRNAs [6], immunosenescence [30] and the innate immune response [3]. Western blot analysis showed a minor 29 kDa CoxIV-4 band in 21% of ART naive patients, which may have represented mono- ubiquitined CoxIV-4. Confirmation by co-immunoprecipitation experiments could not be performed because of the large amount of protein required. Mitochondrial protein mono- or poly- ubiquitinylation [31] may be the first step of mitophagy [32] or of protein proteasomal destruction [33]. Some E3 ubiquitin ligases have been localized in the OMM [34]. No variations in the amounts of the mitochondrial proteins, porin, CoxIV-2 and CoxIV-4, relative to GAPDH, were observed in PBMCs from ART naive patients compared to control subjects. Because protein assays were performed on total PBMC lysates, no distinction could be made between lymphocytes or monocytes, or between cell subgroups defined by their ROS production and DYm. Thus, the variations between the different cell types may be masked. Nonetheless, proteome analysis in a cohort with similar clinical characteristics to ours showed that, compared to healthy donors, PBMCs from ART naive patients exhibited decreased levels of some Complex I (3), IV (5a) and ATP synthase (alpha) subunits but not of CoxIV-2 or CoxIV-4 [35]. Therefore, changes in subunits other than the two studied may result in abnormalities of ROS-producing complexes I and III [36,37], and result in increased ROS production [35]. Mitochondrial functional investigations were performed on lymphocytes and moncytes separately. Lymphocyte FSC/SSC gate included various lymphocyte subpopulations such as T lymphocytes (< 80% of blood lymphocytes; CD3+ CD4+ and CD3+ CD8+ < 40% each), B lymphocytes (CD32 CD19+ < 4%) and NK cells (CD32 CD192CD16+ CD56+ < 3%) as shown by a ancillary study we performed. Our lymphocyte FSC/SSC gate may also contain dendritic cells present in blood (0.3 - 0.5% of PBMC) [38]. Flow cytometry identified two lymphocyte and CD14+ mono- cyte subgroups characterized by either high- or low-ROS production. While high- and low-ROS producing monocytes have been described previously [39,40], the identification of lympho- cytes that produce either high or low amounts of ROS is novel. The ancillary study, we performed, further characterised lympho- cyte subtypes through their ROS production. T lymphocytes and B lymphocytes exhibited the same ROS pattern as the whole FSC/SSC-gated lymphocyte population. In contrast, NK cells were a pure population of low-ROS cells only. ROS production may therefore be another parameter that differentiates NK cells from T and B lymphocytes. Indeed, ROS are known to be physiological messengers in T and B cells [41]. IMM permeability, which is measured by DYm, and ROS production are two related parameters [12]. DYm measurements also indicated the presence of two statistically different groups of lymphocytes, as described for lymphocytes [24] and PBMCs [42]. As discussed previously, there were no relationships between ROS and the DYm subgroups. Discriminant analysis combining ROS and DYm measurements better emphasized the mitochondrial differential effects of HIV-1 infection on the two peripheral blood cell types than comparison of single parameters. While lymphocyte mitochondria were statistically different in ART naive patients compared to control subjects, monocyte mitochondria showed no differences. HIV infection has previously been shown to induce general oxidant stress associated with a decrease in both lymphocyte and blood serum oxidant defence systems [43]. In addition, a decreased DYm was previously reported in PBMCs from ART naive patients exhibiting a slightly lower CD4+ cell count than our patients [44,45]. This decrease could be linked to HIV-1-encoded Vpr through its binding to mitochondrial perme- ability transition pore components [46]. Significant correlations between functional parameters and viral load further underlined the effect of HIV-1 infection on lymphocyte mitochondria. Numerous events may result in mitochondrial impairment in both infected cells and uninfected bystander cells through various signaling events. Indeed, ROS production by NADPH oxidase has been observed in uninfected bystander CD4+ lymphocytes after binding of the HIV-1 protein gp41 to a plasma membrane receptor [47]. Through the secretion of cytokines [48] or of viral proteins [49], infected macrophages or CD4+ T lymphocytes modified various functions of neighboring non-infected cells [50]. Additional interactions between infected and bystander T cells involved exocytosis and uptake of HIV-encoded Nef-bearing exosomes [51,52]. Exosomes are also known to carry miRNAs, some of which are encoded by the HIV genome and are suspected to modifiy gene expression in recipient cells [53]. ‘‘Tunneling nano- tubes’’ were also reported to carry viruses and viral proteins between infected macrophages and B lymphocytes [54]. Decreased ATP supply due to increased ROS production and lowered DYm have been reported to result in mitochondrial network fragmentation, mitophagy, or cell apoptosis [13]. Morphological quantitative analysis of mitochondrial networks in lymphocytes from our ART naive patients argued against these events. These cells exhibited an increase in mitochondrial volume density and branch- ing, whereas mitochondrial network fragmentation did not change. These observations exclude both mitophagy and subsequent lymphocyte apoptosis, and may rather reflect a primary cell response to oxidative stress [55] and/or to HIV-1 proteins [5], in addition to the triggering of innate immunity [56,57]. In summary, HIV-1 infection in ART naive patients with a controlled CD4+ cell count and viral load resulted in significant alterations in four of the lymphocyte mitochondrial parameters (ROS production, DYm, network volume density, branching), without irreversible damage that may lead to mitophagy and/or apoptosis. In contrast to lymphocytes and previously published ROS data [58], mitochondria from monocytes did not exibit changes in functional or morphological parameters. Our results also con- trasted with data from peripheral blood monocytes infected in vitro with HIV-1 and cultured for 7 days [59]. Nevertheless, monocyte sensitivity to HIV-1 infection was detected through correlation of one of the functional parameters (DYm) and viral load in ART naive patients. Regarding the central role of ROS production via NADPH oxidase in phagocytic cells as a defence mechanism PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41129