against infection [60], it is of interest to note that in resting monocytes, mitochondria use more oxygen (70%) than plasma membrane enzymes (30%) [61]. Our study of ART naive patients who were considered stable according to their clinical parameters, highlighted that HIV-1 infection did not affect monocyte mitochondria as much as it directly or indirectly impaired lymphocyte mitochondria, leading to minor oxidant stress known to favour viral replication, immune dysfunction and disease progression [62,63]. ART Partially Rescued HIV-1 Infection-induced Mitochondrial Abnormalities in Lymphocytes but Led to Mitochondrial Changes in Monocytes Three different ART combinations did not modify the amounts of porin, CoxIV-2 and CoxIV-4, or the CoxIV-4/ CoxIV-2 ratio, as previously reported [64,65]. Nonetheless, conflicting data on the mtDNA content (and coxIV-2 mRNA or protein) in PBMCs have been reported in treated patients from cohorts exhibiting heterogeneous characteristics (e.g. CD4+ cell count, viral load.) [66,67,68]. In skeletal muscle, NRTIs have been shown to increase the frequency of the mtDNA D4977 ‘‘common deletion’’, leading to a CoxIV complex biosynthesis defect and enzymatic deficiency [27]. The lack of CoxIV protein abnormalities in our study could be explained by the large difference in lifespan between PBMCs and skeletal muscle, and/or by the fact that PBMC mitochondria may be less sensitive to ART side-effects than other uninfected tissues (e.g. skeletal muscle, adipose tissue), as shown for mtDNA [69,70]. In addition, a PI-containing regimen did not inhibit mitochondrial protease(s) involved in the cleavage of the mitochondrial targeting signal in nuclear-encoded CoxIV-4 isoforms 1 or 2 [71], because we did not observe a 20 kDa band that corresponded to uncleaved proteins. Thus, the biosynthesis and maturation of PBMC proteins encoded either by nuclear or mitochondrial genomes from our cohort patients were not impaired. The CoxIV-4 29 kDa band observed in PBMCs from some ART naive patients was also detected in PBMCs from some patients under 2NRTI+1NNRTI and 3NRTI. If this band represented mono-ubiquitined CoxIV-4, the lack of its detection in PBMCs from patients treated with a PI/r-containing ART combination could argue against the fact that PI inhibits proteasomal activity, as postulated from in vitro studies [72,73]. Discriminant analyses integrating functional and morpholog- ical mitochondrial parameters revealed another discrepancy between lymphocytes and monocytes in response to ART. While ART regimens partially improved lymphocyte mitochon- drial parameters, none of the ART combinations fully restored mitochondrial changes induced by HIV-1 infection. In mono- cytes, the mitochondrial parameters remained unchanged in ART naive patients whereas the three ART regimens resulted in slight (functional) or significant (morphological) modifications. Previous studies have shown either ART-induced DYm recovery [24,44] or impairment [74] in lymphocytes or PBMCs. Heterogeneous data regarding mitochondrial networks were also reported. Under the NRTI regimen, mitochondrial mass/ volume has been shown to decrease in PBMCs [75], to be unchanged in PBMCs [76] or CD4+ T cells [77], and to be increased in CD8+ T cells [78]. However, NRTI induced a mitochondrial mass/volume increase in cultured human adipocytes [79], in a cultured hepatocyte cell line [80], and in subcutaneous adipocytes from patient biopsies [81]. None of the three first line ART regimens appeared to be more toxic towards PBMC mitochondria than the others. Moreover, the majority of 2NRTI+1PI/r and 2NRTI+1NNRTI patients shared HIV and ART Effects on PBMC Mitochondria the same NRTI backbone. Because no statistical difference was found between these two populations using discriminant analysis, our data suggested that boosted Lopinavir (68% of PI) and Efavirenz (72% of NNRTI) did not elicit major mitochondrial effects. The ART regimens in our patient cohort mainly included drugs of the second generation, whose mitochondrial toxicity is reduced in comparison with first generation drugs [82]. As for HIV-1 infection, the increases in volume density and branching parameters induced by ART combinations could be interpreted as responses to mitochondrial stress [55], the result of immunosenes- cence [30], and/or the result of an innate immune response [83,84]. Several Factors could Explain the Mitochondrial Differences between Lymphocytes and Monocytes In contrast to lymphocytes, HIV-1 infects very few blood monocytes (less than 1%) as shown by the lower proviral DNA copy number in monocytes than in CD4+ T cells. Moreover, blood monocytes supported only a very low viral replication rate, as shown by the detection of 2 LTR circles, HIV mRNAs, or changes with time in viral nucleotide sequences. Monocyte migration out of the blood into tissues, followed by differenti- ation into macrophages or dendritic cells, increased the inhibition of viral replication (reviewed in [85,86,87]). HIV- 1 Vpx protein has been demonstrated to relieve replication restriction in monocytes [88] by inducing the proteasomal degradation of SAM domain and HD domain-containing protein 1 (SAMHD1), the HIV-1 restriction factor that is specific for myeloid lineage cells and is not expressed by lymphocytes [89]. Despite their short (< 72 h) blood circulation time [90], monocytes may become infected as bone marrow precursors [91] or later in the blood, and may contribute to the viral reservoir [92] together with bone marrow stem cells [93]. Correlation between monocyte DYm and viral load could argue in favour of monocyte sensitivity to viral replication, thus demonstrating the limit of restriction defence systems. In lymphocytes but not monocytes, the negative side-effects of ART (mitotoxicity) on mitochondria could be balanced by the positive effects of ART, i.e. decreased viral load and replication [94]. ART antiviral activity is less effective towards chronically infected monocytes than lymphocytes for several reasons, one of them being that HIV-related ART targets are present in lower amounts in refractory monocytes [95]. Moreover, the different mitochondrial sensitivities to ART could also be the result of distinct pathways involved in ART intracellular metabolism (e.g. influx, efflux, degradation) within the two cell types [96]. Taken together, these data suggest that mitochondria from blood lymphocytes and monocytes exhibit differential responses to either HIV-1 infection or ART. Whereas mitochondrial ROS and DYm functional parameters exhibited changes, mitochondrial morphological modifications argued against apoptosis [9] or mitophagy [32] induced by either HIV-1 infection or ART regimens. These mitochondrial changes could be related to innate immunity signalling pathways through ROS- and DYm-regulated MAVS [3,19]. Both direct and bystander effects are associated with HIV-1 infection [51] clearly impacted lymphocyte mito- chondria while monocyte mitochondria appeared to be less sentitive. First line ART tended to rescue lymphocyte mitochon- drial parameters altered by viral infection but induced only slight changes in monocytes. Besides the slight alterations reported in our patients, who were considered to be clinically stable, evolution and/or persistent changes overtime in PBMCs and other tissues may contribute to body aging. Indeed, physiological aging is PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41129