ROS-Induced Mitochondrial Dysfunction in CD4 T Cells from ART-Controlled People Living with HIV

Abstract

We have previously demonstrated mitochondrial dysfunction in aging CD4 T cells from antiretroviral therapy (ART)-controlled people living with HIV (PLWH). However, the underlying mechanisms by which CD4 T cells develop mitochondrial dysfunction in PLWH remain unclear. In this study, we sought to elucidate the mechanism(s) of CD4 T cell mitochondrial compromise in ART-controlled PLWH. We first assessed the levels of reactive oxygen species (ROS), and we observed significantly increased cellular and mitochondrial ROS levels in CD4 T cells from PLWH compared to healthy subjects (HS). Furthermore, we observed a significant reduction in the levels of proteins responsible for antioxidant defense (superoxide dismutase 1, SOD1) and ROS-mediated DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) in CD4 T cells from PLWH. Importantly, CRISPR/Cas9-mediated knockdown of SOD1 or APE1 in CD4 T cells from HS confirmed their roles in maintaining normal mitochondrial respiration via a p53-mediated pathway. Reconstitution of SOD1 or APE1 in CD4 T cells from PLWH successfully rescued mitochondrial function as evidenced by Seahorse analysis. These results indicate that ROS induces mitochondrial dysfunction, leading to premature T cell aging via dysregulation of SOD1 and APE1 during latent HIV infection.

Keywords: PLWH; T cell aging; mitochondrial dysfunction; oxidative stress.

Figure 1

ROS production and SOD1/APE1 protein expression in CD4 T cells of PLWH and HS. (A,B) The MFI of CellROX Green (cellular ROS) or the frequency of MitoSOX+ (mitochondrial ROS) in CD4 T cells from PLWH or HS following 2-day culture. (C) LC-MS identification and label-free quantification (LFQ) of proteins isolated from mitochondria of HS (n = 3) and ART-treated PLWH (n = 3). Mitochondrial proteins were assessed using an LC-MS shotgun approach. CD4 T cells from 3 HS or PLWH were pooled to get enough mitochondria for this analysis. The data are a subset of proteins that are known to play a critical role in mtDNA maintenance and have >2-fold change in HIV samples versus HS, as determined by a standard LFQ method. (D,E) RT-qPCR analysis of mRNA expression level of SOD1 or APE1 in CD4 T cells from PLWH or HS following 2-day culture. mRNA expression of SOD1 or APE1 was normalized to GAPDH. (F) Representative and summary data for Western blot analysis of protein levels of SOD1 or APE1 in CD4 T cells from HS or PLWH normalized to β-Actin.

Figure 2

Mitochondrial functions following SOD1 knockdown in CD4 T cells from HS. (A,B) Representative Western blots and summary data of SOD1 and p53 protein levels following SOD1 KD in CD4 T cells from HS, normalized to β-Actin (n = 8). (C,D) Oxygen consumption rate (OCR) as measured using Seahorse XFp Cell Mito Stress Tests, and summary data for non-mitochondrial, basal, and maximal respiration, proton leak, ATP production, and spare respiratory capacity following SOD1 KD (n = 6). KD—knockdown.

Figure 3

Mitochondrial functions following SOD1 overexpression in CD4 T cells from PLWH. (A,B) Representative Western blots and summary data of SOD1-myc and p53 protein levels following SOD1 OE in CD4 T cells from PLWH normalized to β-Actin (n =10). (C,D) OCR and summary data for non-mitochondrial, basal, and maximal respiration, proton leak, ATP production, and spare respiratory capacity following SOD1 OE (n = 12). OE—overexpression.

Figure 4

Mitochondrial functions following APE1 knockdown in CD4 T cells from HS. (A,B) Representative Western blots and summary data of APE1 (n = 11) and p53 (n = 8) protein levels following APE1 KD in CD4 T cells from HS normalized to β-Actin. (C,D) Oxygen consumption rate (OCR) as measured using Seahorse XFp Cell Mito Stress Tests, and summary data for non-mitochondrial, basal, and maximal respiration, proton leak, ATP production, and spare respiratory capacity following APE1 KD (n = 9). KD—knockdown.

Figure 5

Mitochondrial functions following APE1 overexpression in CD4 T cells from PLWH and model of latent HIV infection induced mitochondrial dysfunction via multi-faceted synergistic mechanisms. (A,B) Representative Western blots and summary data of APE1-myc and p53 protein levels following APE1 OE in CD4 T cells from PLWH normalized to β-Actin (n = 7). (C,D) OCR and summary data for non-mitochondrial, basal, and maximal respiration, proton leak, ATP production, and spare respiratory capacity following APE1 OE (n = 18). OE—overexpression. (E) A model depicting the molecular mechanism linking deregulations of APE1 and SOD1 protein levels and mitochondrial compromise via influencing multiple pathways during latent HIV infection. These deregulations result in mitochondrial failure and reduced OXPHOS, as well as accelerated aging and CD4 T cell dysfunction during latent HIV infection.