Metabolic and mitochondrial dysregulation in CD4+ T cells from HIV-positive women on combination anti-retroviral therapy

Abstract

Background: For optimal functionality, immune cells require a robust and adaptable metabolic program that is fueled by dynamic mitochondrial activity. In this study, we investigate the metabolic alterations occurring in immune cells during HIV infection and antiretroviral therapy by analyzing the uptake of metabolic substrates and mitochondrial phenotypes. By delineating changes in immune cell metabolic programming during HIV, we may identify novel potential therapeutic targets to improve anti-viral immune responses.

Methods: After consent and voluntary participation was confirmed, whole blood was drawn from HIV uninfected women and women with chronic HIV infection on long-term combination antiretroviral therapy (HIV/cART). Peripheral blood mononuclear cells-derived immune cells were directly incubated with different fluorescently tagged metabolites and markers of mitochondrial activity: FITC-2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose), FITC-BODIPY (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid), FITC-MitoTracker Green and APC-MitoTracker Deep Red. The uptake of glucose and fats and the mitochondrial mass and potential were measured using flow cytometry. All values are reported quantitatively as geometric means of fluorescence intensity.

Results: During chronic HIV infection, cellular uptake of glucose increases in HIV+ dendritic cells in particular. CD4+ T cells had the lowest uptake of glucose and fats compared to all other cells regardless of HIV status, while CD8+ T cells took up more fatty acids. Interestingly, despite the lower utilization of glucose and fats in CD4+ T cells, mitochondrial mass increased in HIV+ CD4+ T cells compared to HIV negative CD4+ T-cells. HIV+ CD4+ T cells also had the highest mitochondrial potential.

Conclusions: Significant disparities in the utilization of substrates by leukocytes during chronic HIV/cART exist. Innate immune cells increased utilization of sugars and fats while adaptive immune cells displayed lower glucose and fat utilization despite having a higher mitochondrial activity. Our findings suggest that cART treated HIV-infected CD4+ T cells be dysfunctional or may prefer alternative fuel sources not included in these studies. This underscores the importance of understanding the metabolic effects of HIV treatment on immune function.

Fig 1. Substrate uptake and assessment of mitochondrial phenotype and activity in HIV/cART leukocytes.

(A) Assessment of mitochondrial mass (via MTG staining) in uninfected and chronically infected HIV adaptive and innate immune cells. (B) Glucose uptake (via 2NBDG staining) in uninfected and chronically infected HIV⁺ adaptive and innate immune cells. (C) Fat uptake (via BODIPY staining) in uninfected and chronically infected HIV adaptive and innate immune cells. (D) Assessment of mitochondrial potential (via MTDR staining) in uninfected and chronically infected HIV adaptive and innate immune cells (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

Fig 2. Correlation of mitochondrial mass ■ (via MTG staining) and mitochondrial membrane potential ○ (via MTDR staining) with glucose uptake (via 2NBDG staining) in uninfected and chronically infected HIV adaptive and innate immune cells.

(A) Correlations in uninfected DCs. (B) Correlations in HIV-infected DCs. (C) Correlations in uninfected NK cells. (D) Correlations in HIV infected NK cells. Statistical significance is shown in charts.

Fig 3. Correlation of mitochondrial mass ■ (via MTG staining) with lipid uptake (via BODIPY staining) in uninfected and chronically infected HIV adaptive and innate immune cells.

(A) Correlations in uninfected CD4⁺ T-cells. (B) Correlations in HIV CD4⁺ T-cells. (C) Correlations in HIV infected NK cells. (D) Correlations in HIV infected CD8⁺ T-cells. Statistical significance is shown in charts.

Fig 4. Correlations of substrate uptake with CD4 counts and duration of treatment in HIV⁺ participants.

(A) Correlation of substrate uptake with CD4 counts in chronically infected HIV/cART participants and (B) Correlation of substrate uptake with time on ARVs. Statistical significance shown in charts.