Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2020 Apr 30;15(4):e0231761. doi: 10.1371/journal.pone.0231761

Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation

Louie Mar A Gangcuangco 1, Brooks I Mitchell 1, Chathura Siriwardhana 1, Lindsay B Kohorn 1, Glen M Chew 1, Scott Bowler 1, Kalpana J Kallianpur 1, Dominic C Chow 1, Lishomwa C Ndhlovu 1, Mariana Gerschenson 1, Cecilia M Shikuma 1,*
Editor: Qigui Yu2
PMCID: PMC7192478  PMID: 32353005

Abstract

Background

Cellular immunometabolism among people living with HIV (PLWH) on antiretroviral therapy (ART) remains under investigated. We assessed the relationships between mitochondrial oxidative phosphorylation (OXPHOS) in peripheral blood mononuclear cells (PBMCs) and blood parameters associated with HIV immune dysregulation.

Methods

PLWH ≥40 years old and on stable ART ≥3 months were enrolled (N = 149). OXPHOS complex I (CI, NADH dehydrogenase) and complex IV (CIV, cytochrome c oxidase) protein levels in PBMCs were quantified using immunoassays. Monocyte subsets and markers of T-cell activation, senescence, and exhaustion were measured on PBMC by flow cytometry. Plasma inflammatory mediators were quantified using a multiplex assay. HIV-uninfected group (N = 44) of similar age, gender, and ethnicity had available OXPHOS levels.

Results

PLWH had a median age of 51 years. Majority were male (88.6%), Caucasian (57.7%), and with undetectable plasma HIV RNA <50 copies/mL (84.6%). Median CI level was lower in PLWH compared with the HIV-seronegative group (65.5 vs 155.0 optical density/μg protein x 103, p <0.0001). There was no significant difference in median CIV levels. Lower OXPHOS levels correlated with lower CD4% and CD4/CD8 ratio. On multivariable linear regression adjusted for age, current use of zidovudine/didanosine, and HIV RNA (detectable versus undetectable), lower OXPHOS levels were significantly associated with higher MPO, SAA, SAP, and sVCAM, and higher frequencies of intermediate (CD14++CD16+) monocytes and TIGIT+TIM3+ CD4 T-cell (p<0.01).

Conclusion

CI PBMC protein levels were decreased in PLWH on ART. Decreased OXPHOS correlated with disease severity and inflammation. Further studies on the relationship between immunometabolism and immune dysregulation in HIV are warranted.

Background

Mitochondria are considered “powerhouses” of eukaryotic cells. They are ubiquitous organelles whose primary function is to produce energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). Enzyme complexes embedded in the inner mitochondrial membrane facilitate electron cascade that eventually generate ATP. These enzyme complexes include complex I (nicotinamide adenine dinucleotide [NADH]: ubiquinone oxidoreductase, CI), complex II (succinate dehydrogenase), complex III (ubiquinol-cytochrome c oxidoreductase or cytochrome bc1), complex IV (cytochrome c oxidase, CIV), and complex V (ATP synthase) [1].

CI serves as the entry-point for majority of the electrons in the respiratory chain. As electrons cascade through CI to CIV, hydrogen ions are pumped into the intermembrane space, creating a proton-motive force across the inner mitochondrial membrane, which is then used by Complex V to generate ATP from adenosine diphosphate and inorganic phosphate. The components of the respiratory chain are multi-subunit complexes composed of up to 92 different structural proteins encoded by both maternally-derived mitochondrial DNA (mtDNA) and nuclear genes [2, 3].

Mutations in mitochondrial or nuclear DNA can impair cellular respiration. Cells and tissues with high-energy demand, such as the brain, nerves, retina, skeletal and cardiac muscle are especially vulnerable to defects in the electron transport chain [2]. Mutations in the components of the respiratory chain are associated with diseases such as Parkinson’s and Huntington’s disease, seizures, hypotonia, ophthalmoplegia, stroke-like episodes, muscle weakness, and cardiomyopathy [4]. Lower cellular respiration in peripheral blood mononuclear cells (PBMCs) was also reported among patients with chronic fatigue syndrome [5].

People living with HIV (PLWH) are at increased risk of mitochondrial dysfunction as a result of older mitochondrial-toxic antiretrovirals, direct viral toxicity, chronic inflammation, and concurrent comorbidities [6]. Older nucleoside reverse transcriptase inhibitors (NRTIs) are known to cause depletion of mitochondrial DNA via inhibition of the mitochondrial-specific DNA polymerase-γ. HIV replication has been associated with altered mtDNA transcription and reduced activity of mitochondrial respiratory complexes [7]. Protease inhibitors cause mitochondrial damage by increasing oxidative stress and reducing mitochondrial function [8].

The role of cellular immunometabolism in HIV remains under investigated. Utilizing banked specimens from a cohort of chronically HIV-infected adults on stable ART, we examined the relationships between cellular bioenergetics as determined by mitochondrial OXPHOS proteins in PBMCs and various plasma pro-inflammatory biomarkers, circulating monocyte subpopulations, and T-cell immune phenotypes.

Materials and methods

Participant recruitment

Mitochondrial OXPHOS parameters were assessed cross-sectionally from the Hawaii Aging with HIV Cardiovascular Disease cohort consisting of PLWH ≥40 years old, and on stable ART for ≥3 months. Participants were recruited between the years 2009 and 2012. PLWH with active malignancy, acute infection, or AIDS-defining illness at the time of enrollment were excluded. A cohort of HIV-seronegative individuals were recruited as a comparator group. IRB approval was obtained from the University of Hawaii Human Studies Program. All participants provided written informed consent. All banked specimens and data collected from participants were anonymized and de-identified prior to analysis.

Mitochondrial assessments

Quantitation immunoassays (Abcam, PLC, Cambridge, MA) were performed to quantify OXPHOS CI and CIV protein levels in viable PBMCs, as previously reported [9]. Cell viability was between 90–95%, determined using AOPI (acridine orange/propidium iodide). Each vial of viable PBMCs was thawed and washed in 0.5 ml of phosphate-buffered saline (PBS) twice before addition of 0.5 ml of ice-cold extraction buffer [1.5% lauryl maltoside, 25 mM HEPES (pH 7.4), 100 mM NaCl, plus protease inhibitors (Sigma, P-8340)]. Samples were mixed gently and kept on ice for 20 min, and then they were spun in a microcentrifuge at 16,400 rpm at 4°C for 20 min to remove insoluble cell debris. The supernatant, an extract of detergent-solubilized cellular proteins, was then assayed with the OXPHOS immunoassays. All samples were loaded on the immunoassays with equal amounts of total cell protein using an amount previously established with control samples to generate signals within the linear range of the assay. Therefore, the resulting signal was directly proportional to the amount of OXPHOS protein or enzyme activity in the sample. Quantitation of the signal was done by densitometric scanning with a Hamamatsu ICA-1000 reader.

Mitochondrial DNA-specific 8-oxo-2’-deoxyguanosine (mt-specific 8-oxo-dG) was measured by the Gene Specific Repair Assay [10, 11], which identifies the frequency of oxidative changes in guanine nucleotides within the mitochondrial DNA–16 kb molecule. Quantitation of the break frequency of mt-specific 8-oxo-dG was based on comparing the undigested and digested mitochondrial DNA break frequencies calculated using the Poisson distribution, with units reported in break frequency (BF). Mt-specific 8-oxo-dG BF was available only among PLWH.

Immune parameters in persons living with HIV

Multiparametric flow cytometry was performed on cryopreserved PBMCs to measure the frequencies of monocyte phenotypes [classical (CD14++CD16−), intermediate (CD14++CD16+), non-classical (CD14low/+CD16++)], and T-cells expressing activation (HLA-DR and CD38), exhaustion (PD-1,TIM-3 and TIGIT), and senescence (CD57+) markers, as previously described [12, 13]. Data was acquired on a custom 4-laser BD LSR Fortessa Cell Analyzer and all compensation and gating analyses were performed in FlowJo analytical software. Plasma samples were assayed for target inflammatory markers, using high-sensitivity Milliplex assays (Human CVD panels, EMD Millipore, Billerica, MA). Plasma soluble inflammatory markers included C-reactive protein (CRP), interferon gamma (IFN-y), interleukins (IL-1β, IL-6, IL-8, IL-10), monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), serum amyloid A (SAA), serum amyloid P (SAP), soluble adhesion molecules (sE selectin, sICAM, sVCAM), tumor necrosis factor-alpha (TNF-α), tissue plasminogen activator inhibitor-1 (tPAI-1), and vascular endothelial growth factor (VEGF).

Statistical analyses

Median CI and CIV protein levels were compared between HIV-positive patients and seronegative controls using independent samples Mann-Whitney U test. The correlation of various immune parameters with CI and CIV were assessed using Spearman’s correlation (rho, r). Immunologic parameters that were found to correlate with CI and CIV at the level of significance of p<0.01 were further analysed using linear regression analyses. Immune parameters were log-transformed to improve normal distribution of values. Multiple linear regression models were adjusted for age, current use of zidovudine or didanosine, and undetectable plasma HIV RNA. Differences in the median levels of various immune parameters were compared among patients with detectable versus undetectable mt-specific 8-oxo-dG using independent samples Mann-Whitney U test. Statistical analyses were performed using the IBM SPSS statistics version 25.0 (Armonk, NY).

Results

The demographic and immunologic parameters of the 149 PLWH are summarized in Table 1. The median age was 51 years. Majority were male, Caucasian, and had undetectable plasma HIV RNA <50 copies/mL. Median current CD4 count was 505 cells/μL. Compared with PLWH, the HIV-seronegative group (N = 44) had similar age (51.0 years in PLWH vs 54.2 years in HIV-seronegatives, p = 0.36), gender (88.6% vs 81.8% male, p = 0.25), and ethnicity (57.7% vs 61.4% Caucasian, p = 0.67).

Table 1. Demographic and immunologic parameters of persons living with HIV (N = 149).

Demographic Parameters
Age, years 51.0 (46.0–57.5)
Male, n(%) 132 (88.6%)
Caucasian ethnicity, n(%) 86 (57.7%)
Immunologic Parameters
Undetectable HIV RNA < 50 copies/ml, n(%) 126 (84.6%)
Nadir CD4 T cell count, cells/ μL 150 (39, 266)
CD4 T cell count, cells/μL 505.0 (345.0–660.5)
CD4 T cell % 29.0% (21.0% - 36.5%)
Activated CD8 T cell % (CD8+ HLA-DR CD38+) 10.5% (7.6% - 17.0%)
Negative checkpoint receptors (%)
PD1+ CD4 T cell 41.1 (30.3–48.3)
TIGIT+ CD4 T cell 22.6 (16.4–28.3)
TIM3+ CD4 T cell 15.7 (11.9–19.2)
TIGIT+PD1+ CD4 T cell 15.8 (11.0–20.9)
TIM3+PD1+ CD4 T cell 6.49 (4.1–9.2)
TIGIT+TIM3+ CD4 T cell 3.5 (2.6–5.2)
PD1+ CD8 T cell 36.3 (25.7–47.3)
TIGIT+ CD8 T cell 44 (36.4–59.4)
TIM3+ CD8 T cell 21.9 (18.1–28.0)
TIGIT+PD1+ CD8 T cell 24.4 (17.7–32.0)
TIM3+PD1+ CD8 T cell 5.36 (3.7–7.7)
TIGIT+TIM3+ CD8 T cell 8.64 (6.1–12.9)
Senescent markers (%)
CD57+CD28- CD8 T cell 17.3 (12.7–24.4)
CD57+CD28- CD4 T cell 4.16 (1.1–9.9)
Monocyte subsets (%)
Classical (CD14++CD16−) 75.9 (70.4–81.9)
Intermediate (CD14++CD16+) 1.6 (0.6–4.2)
Non-classical (CD14low/+CD16++) 6.0 (4.0–9.1)
Plasma soluble biomarkers
CRP, ng/mL 10,886.3 (3,584.0–46,060.0)
IFN-γ, pg/mL 0.7 (0.4–1.3)
IL-1β, pg/mL 0.3 (0.3–0.3)
IL-6, pg/mL 1.6 (0.9–2.5)
IL-8, pg/mL 3.5 (2.7–4.4)
IL-10, pg/mL 2.1 (0.8–5.0)
MCP-1, pg/mL 147.0 (115.0–179.1)
MMP-9, ng/mL 55.4 (37.3–87.0)
MPO, ng/mL 16.4 (11.4–23.0)
SAA, ng/mL 14,000 (3,933.2–46,224.0)
SAP, ng/mL 88,660.3 (51,867.1–181,582.0)
sE-selectin, ng/mL 33.6 (23.1–48.0)
sICAM-1, ng/mL 140.1 (110.0–172.0)
sVCAM-1, ng/mL 1168.5 (931.0–1335.4)
TNF-α, pg/mL 3.1 (1.8–4.5)
tPAI-1, ng/mL 86.1 (68.0–113.4)
VEGF, pg/mL 24.4 (13.4–49.5)
Mitochondrial assessments
Complex I, optical density/ug x 103 65.5 (48.0–87.0)
Complex IV, optical density/ug x 103 49.2 (37.3–64.3)
Presence of mitochondria-specific 8-Oxo-2'-deoxyguanosine (n, %) break frequency* 85 (53.1%)
Open in a new tab

Values shown are median (quartile 1, quartile 3), unless otherwise specified.

*Patients with available mt-specific 8-oxo-dG (N = 147)

Median CI protein level was significantly lower in PLWH compared with the HIV-seronegative group (65.5 vs 155.0 optical density/μg of protein x 103, p < 0.0001; Fig 1). In contrast, there was no significant difference in median CIV protein levels (49.2 vs 46.0 optical density/μg of protein x 103, p = 0.20).

Fig 1. Comparison of mitochondrial complex I and complex IV protein levels in peripheral blood mononuclear cells between HIV+ and seronegative group.

Fig 1

Open in a new tab

Among PLWH, lower CI protein levels correlated with lower CD4 count (r = 0.19, p = 0.02), CD4% (r = 0.18, p = 0.02), and CD4/CD8 ratio (r = 0.18, p = 0.03) (Table 2). Similarly, lower CIV protein levels correlated with lower CD4% (r = 0.18, p = 0.02) and CD4/CD8 ratio (r = 0.18, p = 0.03).

Table 2. Spearman’s correlation between mitochondrial oxidative phosphorylation protein levels in peripheral blood mononuclear cells and various immunologic parameters.

Complex I (optical density (OD)/μg of protein×103) Complex IV (OD/μg of protein×103)
Absolute CD4 count 0.19 (p = 0.02) 0.13 (p = 0.13)
CD4 percent 0.18 (p = 0.02) 0.18 (p = 0.02)
Absolute CD8 count -0.06 (p = 0.50) -0.10 (p = 0.25)
CD8 percent -0.16 (p = 0.06) -0.14 (p = 0.09)
CD4/CD8 ratio 0.18 (p = 0.03) 0.18 (p = 0.03)
CD38+HLA-DR+ CD8 T cell -0.36 (p = 0.70) 0.02 (p = 0.85)
Plasma soluble biomarkers
CRP -0.01 (p = 0.93) 0.15 (p = 0.20)
IFN-y -0.08 (p = 0.37) -0.09 (p = 0.33)
IL-1β -0.36 (p<0.001) -0.42 (p<0.001)
IL-6 -0.11 (p = 0.22) -0.05 (p = 0.56)
IL-8 -0.07 (p = 0.43) -0.04 (p = 0.65)
IL-10 -0.02 (p = 0.81) 0.01 (p = 0.89)
MCP-1 -0.23 (p = 0.008) -0.21 (p = 0.02)
MMP-9 -0.18 (p = 0.03) -0.11 (p = 0.23)
MPO -0.33 (p<0.001) -0.30 (p = 0.001)
SAA -0.37 (p<0.001) -0.37 (p<0.001)
SAP -0.43 (p<0.001) -0.47 (p<0.001)
sE-Selectin -0.19 (p = 0.03) -0.20 (p = 0.02)
sICAM -0.12 (p = 0.18) -0.10 (p = 0.24)
sVCAM -0.27 (p = 0.002) -0.32 (p<0.001)
TNF-a -0.13 (p = 0.13) -0.08 (p = 0.35)
tPAI-1 -0.11 (p = 0.20) -0.03 (p = 0.71)
VEGF -0.04 (p = 0.61) 0.02 (p = 0.77)
Monocyte subsets (%)
Classical (CD14++CD16−) -0.19 (p = 0.03) -0.15 (p = 0.08)
Intermediate (CD14++CD16+) -0.28 (p = 0.001) -0.27 (p = 0.002)
Non-classical (CD14low/+CD16++) -0.09 (p = 0.32) -0.15 (p = 0.08)
Negative checkpoint receptors (%)
PD1+ CD4 T-cell -0.28 (p = 0.08) -0.22 (p = 0.17)
TIGIT+ CD4+ T-cell -0.33 (p = 0.03) -0.35 (p = 0.02)
TIM3+ CD4 T-cell -0.07 (p = 0.67) -0.09 (p = 0.57)
PD1+ TIGIT+ CD4+ T-cell -0.35 (p = 0.03) -0.36 (p = 0.02)
PD1+ TIM3+ CD4 T-cell -0.31 (p = 0.05) -0.29 (p = 0.06)
TIGIT+ TIM3+ CD4 T-cell -0.40 (p = 0.009) -0.42 (p = 0.006)
TIGIT+ CD8 T-cell -0.06 (p = 0.70) -0.11 (p = 0.49)
PD1+ CD8 T-cell -0.22 (p = 0.16) -0.20 (p = 0.22)
TIM3+ CD8+ T-cell -0.04 (p = 0.80) -0.21 (p = 0.18)
PD1+ TIGIT+ CD8 T-cell -0.14 (p = 0.39) -0.16 (p = 0.31)
TIGIT+ TIM3+ CD8 T-cell -0.16 (p = 0.31) -0.32 (p = 0.04)
PD1+TIM3+ CD8 T-cell -0.20 (p = 0.21) -0.34 (p = 0.03)
Senescence markers
CD57+CD28- CD8 T cell -0.15 (p = 0.33) -0.17 (p = 0.29)
CD57+CD28- CD4 T cell 0.16 (p = 0.32) 0.34 (p = 0.03)
Open in a new tab

On multivariable linear regression analyses, lower CD4% and lower CD4/CD8 ratio remained significantly associated with lower CI and CIV protein levels (Table 3). OXPHOS parameters did not correlate with absolute CD8 count, CD8%, or percentage of activated CD38+HLA-DR+ CD8 T-cell.

Table 3. Linear regression analyses of immunologic parameters associated with mitochondrial complex I and complex IV levels.

Univariable Multivariable*
Complex I Complex IV Complex I Complex IV
Absolute CD4 count 0.19 (p = 0.020) 0.13 (p = 0.109) 0.21 (p = 0.014) 0.14 (p = 0.098)
CD4 percent 0.20 (p = 0.017) 0.18 (p = 0.028) 0.21 (p = 0.015) 0.19 (p = 0.026)
Absolute CD8 count -0.05 (p = 0.578) -0.08 (p = 0.318) -0.03 (p = 0.700) -0.08 (p = 0.342)
CD8 percent -0.15 (p = 0.064) -0.10 (p = 0.204) -0.16 (p = 0.069) -0.11 (p = 0.209)
CD4/CD8 ratio 0.22 (p = 0.008) 0.19 (p = 0.023) 0.23 (p = 0.008) 0.20 (p = 0.022)
IL-1β 0.09 (p = 0.298) -0.03 (p = 0.766) 0.06 (p = 0.543) -0.06 (p = 0.508)
MCP-1 -0.21 (p = 0.017) -0.15 (p = 0.087) -0.24 (p = 0.008) -0.17 (p = 0.059)
MPO -0.24 (p = 0.006) -0.30 (p = 0.001) -0.25 (p = 0.005) -0.31 (p<0.001)
SAA -0.38 (p<0.001) -0.34 (p<0.001) -0.42 (p<0.001) -0.37 (p<0.001)
SAP -0.40 (p<0.001) -0.44 (p<0.001) -0.42 (p<0.001) -0.45 (p<0.001)
sVCAM -0.23 (p = 0.009) -0.32 (p<0.001) -0.25 (p = 0.006) -0.33 (p<0.001)
Intermediate (CD14++CD16+) monocyte % -0.25 (p = 0.003) -0.23 (p = 0.007) -0.26 (p = 0.002) -0.23 (p = 0.007)
TIGIT+TIM3+ CD4 T-cell % -0.42 (p = 0.006) -0.49 (p = 0.001) -0.42 (p = 0.014) -0.52 (p = 0.002)
Open in a new tab

Presented are β-coefficient and p-values.

*Separate multivariable linear regression analyses were performed for each immunologic parameter, adjusted for age, current use of didanosine/zidovudine, and undetectable HIV RNA. Plasma soluble inflammatory markers were log-transformed.

The most frequently used ART at the time of enrollment were: tenofovir disoproxil fumarate (72.5%), emtricitabine (65.6%), efavirenz (42.5%), ritonavir (39.4%), lamivudine (23.1%), and atazanavir (17.5%). Of the older NRTIs, 7.5% were on zidovudine, 1.9% were on didanosine, and none on stavudine or zalcitabine. No participant was on an integrase strand transfer inhibitor. Current didanosine use was associated with significantly lower median CI (23.5 vs 66.5 optical density/μg of protein, p = 0.03) and CIV (21.9 vs 49.5 optical density/μg of protein, p = 0.05) protein levels. There were no significant differences in mt-specific 8-oxo-dG BF by current ART use.

OXPHOS protein levels in PBMC correlated inversely with several markers of inflammation. Lower CI strongly correlated with higher IL-1β (r = -0.36, p<0.001), MCP-1 (r = -0.23, p = 0.008), MPO (r = -0.33, p<0.001), SAA (r = -0.37, p<0.001), SAP (r = -0.43, p<0.001), and sVCAM (r = -0.27, p = 0.002). Additionally, lower CI correlated with higher frequencies of intermediate monocytes (r = -0.28, p = 0.001) and dual-expressing TIGIT+TIM3+ CD4 T-cells (r = -0.40, p = 0.009). Similar correlations were found with PBMC CIV levels (Table 2).

On multivariable linear regression (Table 3), lower CI remained significantly associated with higher MCP-1, MPO, SAA, SAP, sVCAM, intermediate monocyte, and dual TIGIT+TIM3+ CD4 T-cell frequencies. CIV remained negatively associated with MPO, SAA, SAP, sVCAM, intermediate monocytes, and dual TIGIT+TIM3+ CD4 T-cell frequencies. Other NCR-bearing T-cells correlated with OXPHOS protein levels at p<0.05 but did not reach the threshold of significance of p<0.01 (Table 2). Further analyzing these NCR-bearing T-cells using multivariable linear regression, lower CI was associated with higher frequencies of TIGIT+ CD4 T-cell (β = -0.35, p = 0.039) and PD1+TIGIT+ CD4 T-cell (β = -0.35, p = 0.037). Similarly, lower CIV was associated with higher frequencies of TIGIT+ CD4 T-cell (β = -0.38, p = 0.022), PD1+TIGIT+ CD4 T-cell (β = -0.41, p = 0.016), and PD1+TIM3+ CD4 T-cell (β = -0.37, p = 0.040).

The presence of mt-specific 8-oxo-dG BF was significantly associated with higher CI protein levels (69.48 vs 57.18 optical density/ug x 103, p = 0.069), CIV protein levels (53.08 vs 43.33 optical density/ug x 103, p = 0.004), and IL-6 (1.78 vs 1.17 pg/ml, p = 0.008), but with lower frequencies of dual TIGIT+TIM3+ CD8 T-cell (7.99% vs 12.90%, p = 0.019). No associations were found between mt-specific 8-oxo-dG BF and other immune parameters, such as CD4 percent, CD4/CD8 ratio, soluble plasma biomarkers, monocyte phenotypes, and NCR-bearing CD4 T cells, which correlated with CI and CIV.

Discussion

In this cohort of older PLWH on stable ART, we found that mitochondrial CI protein levels were significantly decreased compared to HIV-seronegative persons. Lower CI and CIV protein levels in PBMCs correlated with disease severity among PLWH, as assessed by CD4 percent and CD4/CD8 ratio. Decreased PBMC CI and CIV protein levels were associated with higher plasma inflammatory markers and increased frequencies of intermediate monocytes and TIGIT+TIM3+ CD4 T-cells.

HIV proteins induce direct viral toxicity to the mitochondria. HIV-1 trans-activator (Tat) protein has been shown to cause rapid dissipation of the mitochondrial transmembrane potential and inactivates cytochrome c oxidase in mouse liver, heart, and brain [14]. Similarly, gp120 and Tat proteins induce mitochondrial fragmentation in neurons [15]. In our PLWH cohort, mitochondrial OXPHOS protein levels in PBMC, particularly CI, were significantly decreased compared with the seronegative group. This observation is likely due to several factors, such as direct mitochondrial toxicity of HIV, antiretrovirals, comorbidities, and persistent inflammation. Mitochondrial DNA, which is necessary for the synthesis of the components of the OXPHOS system, has also been reported to be significantly decreased in chronic HIV [16].

Persistent inflammation and immune dysregulation have been well documented among PLWH despite the use of suppressive ART [17]. We found that lower PBMC OXPHOS levels correlated with lower CD4/CD8 ratio, higher pro-inflammatory cytokine levels (MCP-1, MPO, SAA, SAP, and sVCAM), and higher percentages of the pro-inflammatory intermediate monocyte subset. These inflammatory mediators are known to induce mitochondrial dysfunction, which increases reactive oxygen species (ROS), leading to a vicious cycle of mitochondrial damage and inflammation [18].

Mitochondrial damage in chronic HIV is associated with increased lipid peroxidation of PBMC membranes [19]. Dysfunction of mitochondria can cause excess production of O2- ions, resulting in a pro-inflammatory state [20]. Modulation of the inflammatory response via redox-sensitive pathways or direct activation of the inflammasome by mitochondria-derived free radicals may result in the production of cytokines and orchestrate a vigorous inflammatory response [21]. Mitochondrial dysfunction also increases cellular responsiveness to pro-inflammatory cytokines through an increase in ROS production [22] and NF-κB activation [23], and has been shown to cause accumulation of cells bearing pro-inflammatory phenotypes in lung tissue [24]. These inflammatory mediators could lead to further mitochondrial dysfunction [25]. The HIV proteins gp120, Tat, Nef, Vpr, and reverse transcriptase have been shown to enhance ROS production and dysregulate oxidative stress pathways [26].

CI levels in PLWH was decreased but CIV levels were similar to HIV-seronegative controls. The decreasing trend in dysfunction as the electrons progress through the respiratory chain has been previously reported. Among ART-naïve subjects, there was a 41% decrease in mitochondrial respiratory chain complex II activity, 38% decrease in complex III, and 19% decrease in CIV compared to seronegative controls [19].

We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels. Interestingly, 8-oxo-dG, did not correlate with other immune parameters that correlated with CI and CIV, such as CD4 percent, monocyte phenotypes, or NCR-bearing CD4 T-cells. Dysfunctional OXPHOS leads to increased ROS generation, which damages mitochondrial DNA, membrane lipids, and proteins [27]. In particular, CI has been identified as a common site of superoxide generation [28, 29]. Our analyses were limited by the lack of mt-specific 8-oxo-dG levels in the HIV-seronegative group. In addition, we assessed OXPHOS protein levels and not function. One possible mechanism for our observation may be that increased ROS production is a consequence of stoichiometric mismatches in the electron transport chain complexes. This may result in increased residence time of electrons on sites of the complexes that mediate electron reduction of O2- resulting in the increased production of H2O2 and superoxide [30]. We further hypothesize that the lack of changes in CIV protein levels in the setting of low CI may be due to the electron transport compensation via Complexes II-IV, which has been previously reported in computer modelling studies and other disease states [31, 32].

We found that higher percentage of TIM3+TIGIT+ CD4 T-cells was strongly associated with lower PBMC OXPHOS levels. TIM-3 and TIGIT are co-inhibitory receptors expressed by functionally exhausted T-cells, exhibiting decreased proliferation and suppressed T-cell responses [33, 34]. Aged T-cells upregulate the expression of co-inhibitory receptors and demonstrate reduction in respiratory metabolism and electron transport chain activity [35]. We have previously reported that TIGIT expression on CD4 T-cells correlates with total HIV DNA and residual immune activation despite suppressive ART [13]. CD4 T-cells expressing TIGIT, as well as those expressing PD-1 or lymphocyte activation gene 3 (LAG-3), have been identified as a major contributor to the pool of inducible HIV genomes [36]. In the SPARTAC study, the expression on CD4 or CD8 T-cells of TIM-3 as well as expression of PD-1 or LAG-3 measured prior to ART predicted the time to viral rebound after treatment interruption [37]. PD-1 signaling has been shown to switch T cell metabolism from glycolysis to fatty acid metabolism. These metabolic reprogramming mediated by PD-1 may lead to mitochondrial depolarization, reduction of mitochondrial biogenesis, and higher rate of ROS production [38]. The effects of TIM-3 and TIGIT on the OXPHOS system warrant further investigation.

Several limitations of our analyses should be mentioned, including the cross-sectional nature of our study. Mt-specific 8-oxo-dG and other immune parameters (cytokines, monocyte subsets, and NCRs) were not available for the HIV-seronegative controls. In our cohort, CI and IV analyses were performed on PBMCs and not on individual cell populations. Although it has been described in the literature that T-cells constitute up to 90% of immune cells in PBMCs [39], the correlations between PBMC CI and CIV protein levels may not accurately reflect OXPHOS levels in T-cells. Furthermore, a recent review has described that various T-cell and monocyte subsets have unique metabolic profiles, with metabolically active effector CD4 T-cells predominantly utilizing glycolysis instead of OXPHOS [40]. Nonetheless, this study recruited a modest number of PLWH and identified key immune correlates of dysfunction important in inflammation and HIV pathogenesis.

In summary, we found that PBMC CI levels were decreased in PLWH on stable ART and correlated with HIV disease severity and inflammation. Decreased PBMC OXPHOS protein levels were associated with higher plasma inflammatory markers and increased frequencies of intermediate monocytes and TIGIT+TIM3+ CD4 T-cells. Further studies are needed to investigate the relationship between CD4 T-cell exhaustion, immunometabolism, and HIV persistence.

Supporting information

S1 File

(SAV)

Click here for additional data file. (240.2KB, sav)

Acknowledgments

We thank the patients who participated in this study, as well as the staff of the Hawaii Center for AIDS for coordinating patient recruitment and gathering clinical data.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The study was made possible by the funding and clinical research support from National Institute of Health grants R01HL095135 (CMS), U54MD007584, U54MD007601 and P20GM113134 (MG). Details of the grants may be accessed through: https://report.nih.gov/index.aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Sharma LK, Lu J, Bai Y. Mitochondrial respiratory complex I: structure, function and implication in human diseases. Curr Med Chem. 2009;16(10):1266–77. Epub 2009/04/10. 10.2174/092986709787846578 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Koopman WJ, Distelmaier F, Smeitink JA, Willems PH. OXPHOS mutations and neurodegeneration. The EMBO journal. 2013;32(1):9–29. Epub 2012/11/15. 10.1038/emboj.2012.300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Cogliati S, Lorenzi I, Rigoni G, Caicci F, Soriano ME. Regulation of Mitochondrial Electron Transport Chain Assembly. Journal of molecular biology. 2018;430(24):4849–73. Epub 2018/10/08. 10.1016/j.jmb.2018.09.016 [DOI] [PubMed] [Google Scholar]
  • 4.Szalardy L, Zadori D, Klivenyi P, Toldi J, Vecsei L. Electron Transport Disturbances and Neurodegeneration: From Albert Szent-Gyorgyi's Concept (Szeged) till Novel Approaches to Boost Mitochondrial Bioenergetics. Oxidative medicine and cellular longevity. 2015;2015:498401 Epub 2015/08/25. 10.1155/2015/498401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tomas C, Brown A, Strassheim V, Elson JL, Newton J, Manning P. Cellular bioenergetics is impaired in patients with chronic fatigue syndrome. PLoS ONE. 2017;12(10):e0186802 Epub 2017/10/25. 10.1371/journal.pone.0186802 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Hulgan T, Gerschenson M. HIV and mitochondria: more than just drug toxicity. The Journal of infectious diseases. 2012;205(12):1769–71. Epub 2012/04/06. 10.1093/infdis/jis105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rodriguez-Mora S, Mateos E, Moran M, Martin MA, Lopez JA, Calvo E, et al. Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication. Retrovirology. 2015;12:78 Epub 2015/09/18. 10.1186/s12977-015-0203-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Smith RL, de Boer R, Brul S, Budovskaya Y, van Spek H. Premature and accelerated aging: HIV or HAART? Frontiers in genetics. 2012;3:328 Epub 2013/02/02. 10.3389/fgene.2012.00328 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Shikuma CM, Gerschenson M, Chow D, Libutti DE, Willis JH, Murray J, et al. Mitochondrial oxidative phosphorylation protein levels in peripheral blood mononuclear cells correlate with levels in subcutaneous adipose tissue within samples differing by HIV and lipoatrophy status. AIDS research and human retroviruses. 2008;24(10):1255–62. Epub 2008/10/11. 10.1089/aid.2007.0262 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Kallianpur KJ, Gerschenson M, Mitchell BI, LiButti DE, Umaki TM, Ndhlovu LC, et al. Oxidative mitochondrial DNA damage in peripheral blood mononuclear cells is associated with reduced volumes of hippocampus and subcortical gray matter in chronically HIV-infected patients. Mitochondrion. 2016;28:8–15. Epub 2016/03/01. 10.1016/j.mito.2016.02.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gerschenson M, Kim C, Berzins B, Taiwo B, Libutti DE, Choi J, et al. Mitochondrial function, morphology and metabolic parameters improve after switching from stavudine to a tenofovir-containing regimen. The Journal of antimicrobial chemotherapy. 2009;63(6):1244–50. Epub 2009/03/27. 10.1093/jac/dkp100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mitchell BI, Byron MM, Ng RC, Chow DC, Ndhlovu LC, Shikuma CM. Elevation of Non-Classical (CD14+/lowCD16++) Monocytes Is Associated with Increased Albuminuria and Urine TGF-beta1 in HIV-Infected Individuals on Stable Antiretroviral Therapy. PLoS ONE. 2016;11(4):e0153758 Epub 2016/04/21. 10.1371/journal.pone.0153758 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chew GM, Fujita T, Webb GM, Burwitz BJ, Wu HL, Reed JS, et al. TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection. PLoS pathogens. 2016;12(1):e1005349 Epub 2016/01/08. 10.1371/journal.ppat.1005349 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lecoeur H, Borgne-Sanchez A, Chaloin O, El-Khoury R, Brabant M, Langonne A, et al. HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase. Cell death & disease. 2012;3:e282. Epub 2012/03/16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Teodorof-Diedrich C, Spector SA. Human Immunodeficiency Virus Type 1 gp120 and Tat Induce Mitochondrial Fragmentation and Incomplete Mitophagy in Human Neurons. Journal of virology. 2018;92(22). Epub 2018/08/31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Cote HC, Brumme ZL, Craib KJ, Alexander CS, Wynhoven B, Ting L, et al. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients. The New England journal of medicine. 2002;346(11):811–20. Epub 2002/03/15. 10.1056/NEJMoa012035 [DOI] [PubMed] [Google Scholar]
  • 17.Ahmed D, Roy D, Cassol E. Examining Relationships between Metabolism and Persistent Inflammation in HIV Patients on Antiretroviral Therapy. Mediators of inflammation. 2018;2018:6238978 Epub 2018/10/27. 10.1155/2018/6238978 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.van Horssen J, van Schaik P, Witte M. Inflammation and mitochondrial dysfunction: A vicious circle in neurodegenerative disorders? Neuroscience letters. 2017. Epub 2017/07/03. [DOI] [PubMed] [Google Scholar]
  • 19.Miro O, Lopez S, Martinez E, Pedrol E, Milinkovic A, Deig E, et al. Mitochondrial effects of HIV infection on the peripheral blood mononuclear cells of HIV-infected patients who were never treated with antiretrovirals. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2004;39(5):710–6. Epub 2004/09/10. [DOI] [PubMed] [Google Scholar]
  • 20.Strowig T, Henao-Mejia J, Elinav E, Flavell R. Inflammasomes in health and disease. Nature. 2012;481(7381):278–86. 10.1038/nature10759 [DOI] [PubMed] [Google Scholar]
  • 21.Escames G, Lopez LC, Garcia JA, Garcia-Corzo L, Ortiz F, Acuna-Castroviejo D. Mitochondrial DNA and inflammatory diseases. Human genetics. 2012;131(2):161–73. 10.1007/s00439-011-1057-y [DOI] [PubMed] [Google Scholar]
  • 22.Vaamonde-Garcia C, Riveiro-Naveira RR, Valcarcel-Ares MN, Hermida-Carballo L, Blanco FJ, Lopez-Armada MJ. Mitochondrial dysfunction increases inflammatory responsiveness to cytokines in normal human chondrocytes. Arthritis and rheumatism. 2012;64(9):2927–36. 10.1002/art.34508 [DOI] [PubMed] [Google Scholar]
  • 23.Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nature immunology. 2011;12(3):222–30. 10.1038/ni.1980 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Aguilera-Aguirre L, Bacsi A, Saavedra-Molina A, Kurosky A, Sur S, Boldogh I. Mitochondrial dysfunction increases allergic airway inflammation. Journal of immunology. 2009;183(8):5379–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ballinger SW. Mitochondrial dysfunction in cardiovascular disease. Free radical biology & medicine. 2005;38(10):1278–95. [DOI] [PubMed] [Google Scholar]
  • 26.Ivanov AV, Valuev-Elliston VT, Ivanova ON, Kochetkov SN, Starodubova ES, Bartosch B, et al. Oxidative Stress during HIV Infection: Mechanisms and Consequences. Oxidative medicine and cellular longevity. 2016;2016:8910396 Epub 2016/11/11. 10.1155/2016/8910396 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cline SD. Mitochondrial DNA damage and its consequences for mitochondrial gene expression. Biochimica et biophysica acta. 2012;1819(9–10):979–91. Epub 2012/06/26. 10.1016/j.bbagrm.2012.06.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Guo C, Sun L, Chen X, Zhang D. Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural regeneration research. 2013;8(21):2003–14. Epub 2013/07/25. 10.3969/j.issn.1673-5374.2013.21.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Jastroch M, Divakaruni AS, Mookerjee S, Treberg JR, Brand MD. Mitochondrial proton and electron leaks. Essays in biochemistry. 2010;47:53–67. Epub 2010/06/11. 10.1042/bse0470053 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Murphy MP. How mitochondria produce reactive oxygen species. The Biochemical journal. 2009;417(1):1–13. Epub 2008/12/09. 10.1042/BJ20081386 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rontoyanni VG, Nunez Lopez O, Fankhauser GT, Cheema ZF, Rasmussen BB, Porter C. Mitochondrial Bioenergetics in the Metabolic Myopathy Accompanying Peripheral Artery Disease. Frontiers in physiology. 2017;8:141 Epub 2017/03/30. 10.3389/fphys.2017.00141 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zielinski LP, Smith AC, Smith AG, Robinson AJ. Metabolic flexibility of mitochondrial respiratory chain disorders predicted by computer modelling. Mitochondrion. 2016;31:45–55. Epub 2016/10/05. 10.1016/j.mito.2016.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Joller N, Hafler JP, Brynedal B, Kassam N, Spoerl S, Levin SD, et al. Cutting edge: TIGIT has T cell-intrinsic inhibitory functions. J Immunol. 2011;186(3):1338–42. Epub 2011/01/05. 10.4049/jimmunol.1003081 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sakhdari A, Mujib S, Vali B, Yue FY, MacParland S, Clayton K, et al. Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection. PLoS ONE. 2012;7(7):e40146 Epub 2012/07/14. 10.1371/journal.pone.0040146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chao T, Wang H, Ho PC. Mitochondrial Control and Guidance of Cellular Activities of T Cells. Frontiers in immunology. 2017;8:473 Epub 2017/05/10. 10.3389/fimmu.2017.00473 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Fromentin R, Bakeman W, Lawani MB, Khoury G, Hartogensis W, DaFonseca S, et al. CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART. PLoS pathogens. 2016;12(7):e1005761 Epub 2016/07/16. 10.1371/journal.ppat.1005761 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Hurst J, Hoffmann M, Pace M, Williams JP, Thornhill J, Hamlyn E, et al. Immunological biomarkers predict HIV-1 viral rebound after treatment interruption. Nature communications. 2015;6:8495 Epub 2015/10/10. 10.1038/ncomms9495 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Velu V, Shetty RD, Larsson M, Shankar EM. Role of PD-1 co-inhibitory pathway in HIV infection and potential therapeutic options. Retrovirology. 2015;12:14 Epub 2015/03/11. 10.1186/s12977-015-0144-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kleiveland CR. Peripheral Blood Mononuclear Cells In: Verhoeckx K, Cotter P, Lopez-Exposito I, Kleiveland C, Lea T, Mackie A, et al. , editors. The Impact of Food Bioactives on Health: in vitro and ex vivo models. Cham (CH)2015. p. 161–7. [PubMed] [Google Scholar]
  • 40.Palmer CS, Henstridge DC, Yu D, Singh A, Balderson B, Duette G, et al. Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure. J Immunol. 2016;196(11):4437–44. Epub 2016/05/22. 10.4049/jimmunol.1600120 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Qigui Yu

14 Nov 2019

PONE-D-19-24311

Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation

PLOS ONE

Dear Dr. Louie Gangcuangco,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but need a minor revision to fully meet PLOS ONE’s publication criteria. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================Reviewer #1

 Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed.

1.     What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison.

2.     How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently.

3.     Also, to make the case stronger the authors needs to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes?

4.     What is viability of the PBMC used?

5.     What is status of mitochondrial ROS in these samples as this is one of the important functional parameters?

6.     ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV.

7.     Needs more explanation in the discussion section about decreased complex I and unchanged complex IV. Reviewer #2This is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized.

Some minor concerns are described as followed;

1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper.

2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study.

==============================

We would appreciate receiving your revised manuscript by 02/14/2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Qigui Yu, M.D./Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

1.  When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized.

Some minor concerns are described as followed;

1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper.

2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study.

Reviewer #2: Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed.

1. What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison.

2. How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently.

3. Also, to make the case stronger the authors needs to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes?

4. What is viability of the PBMC used?

5. What is status of mitochondrial ROS in these samples as this is one of the important functional parameters?

6. ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV.

7. Needs more explanation in the discussion section about decreased complex I and unchanged complex IV.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Fahim Syed

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 30;15(4):e0231761. doi: 10.1371/journal.pone.0231761.r002

Author response to Decision Letter 0

14 Feb 2020

February 9, 2020

Qigui Yu, MD, PhD

Academic Editor

PLOS ONE

Re: Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation (PONE-D-19-24311)

Dear Dr. Yu:

We would like to thank you and the Reviewers for their careful review of our manuscript. The reviewers thought the work was ‘interesting and relevant to the overall knowledge in the field’ and ‘innovative and significant’. We have addressed their minor revisions below and in the revised manuscript.

Reviewer 1

Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed.

1. What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison.

Unfortunately, mt-specific 8-oxo-dG and other immune parameters (cytokines, monocyte subsets, and NCRs) were not available for the HIV-seronegative controls. We included this in the limitations section in the Discussion. Table 1 would therefore have several missing cells if an HIV-seronegative column is included. To compare and clarify the demographic characteristics of HIV-seronegatives, the following has been added to the first paragraph of the Results section (lines 158-162):

“Compared with PLHIV, the HIV-seronegative group (N=44) had similar age (51.0 years in PLHIV vs 54.2 years in HIV-seronegatives, p=0.36), gender (88.6% vs 81.8% male, p=0.25), and ethnicity (57.7% vs 61.4% Caucasian, p=0.67).”

2. How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently.

Complex I was measured as described in reference 9: PMCID: PMC2649940. Complex I and IV protein levels were measured and not activity, thus inhibitors were not used (e.g. rotenone for Complex I activity, etc.). Additional text describing the methodology has been added to lines 112-122.

3. Also, to make the case stronger the authors need to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes?

We agree with the reviewer that immunostaining of the PBMCS for Complex I and IV would have been feasible. We did not have enough PBMCs for both FACS and the immunoassays. We measured protein levels, not activity.

4. What is viability of the PBMC used?

The viability of the cells was 90-95%. Cell viability was determined using AOPI (acridine orange/propidium iodide). A statement was included in the Methods section.

5. What is status of mitochondrial ROS in these samples as this is one of the important functional parameters?

We measured mitochondrial ROS by measuring the amount of oxidized guanine in the mtDNA. We added a reference in the Methods section for PMID 19321503, Gerschenson et al.

Mitochondrial 8-oxo-dG damage was assessed using a Gene-Specific Repair Assay. Ten micrograms of PBMC DNA was isolated with a DNeasy Blood and Tissue Kit (Qiagen, Inc.). DNA was then digested with PvuII (New England BioLabs, Inc., Ipswich, MA, USA) overnight to linearize mtDNA. Digested DNA was halved: 5 µg of DNA was treated with human 8-oxoguanine DNA glycosylase (hOGG1) for 1 h at 37°C in a reaction volume of 15 µL and then for 1 h at 65°C for enzyme deactivation, and the remaining 5 µg of DNA was left untreated and stored at 4°C. For analysis, 4 µL of 1× Alkaline Agarose Loading Dye (Boston Bioproducts, MA, USA) was added, and cleaved and non-cleaved products were resolved on a 0.75% alkaline agarose gel. DNA was transferred to nylon (+) membranes using standard Southern blot methodology. Human mitochondrial probes specific for cytochrome b were labelled with digoxigenin-dUTP (Roche) by linear PCR amplification. Primer sequences were: DigFor, GCT ACC TTC ACG CCA A (14 976–15 001); and DigRev, CCG TTT CGT GCA AGA AT (15 357–15 341). Blots were hybridized overnight at 45°C and processed for chemiluminescent detection following Roche protocols. Finally, membranes were developed on a chemilumi imager (Roche) using LumiAnalyst software (Roche). Mitochondrial 8-oxo-dG damage was quantified by calculating break frequencies (BFs) based on the Poisson distribution of DNA treated with the hOGG1 repair enzyme and DNA untreated.

6. ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV.

We hypothesize that the lack of changes in Complex IV protein levels may be due to the electron transport compensation via Complexes II-IV. Text was added in Lines 272 to 275.

7. Needs more explanation in the discussion section about decreased complex I and unchanged complex IV.

See lines 271 and 272. For instance, decreased Complex I activity in skeletal muscle has been associated with normalized Complex IV in human peripheral arterial disease (Pubmed: 11158957).

Reviewer #2

This is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized.

Some minor concerns are described as followed;

1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper.

We concur with the reviewer that it would be useful to have a treatment-naive HIV infected individuals assayed. These PBMCs are unavailable in our current banked specimens, since most PLHIV are treated as soon as they are diagnosed per CDC recommendations (https://www.helpstoptheviruspro.com/start-hiv-treatment/treat-hiv-asap?sitelink=treating+hiv+asap&utm_medium=cpc&utm_campaign=2019_Treatment+-+Broad&utm_content=Strategy&utm_term=%2Bguideline+%2Bhiv&moc=HSTV01&utm_source=bing&gclid=CK_F48GtxecCFTuJxQIdNHcCVg&gclsrc=ds).

2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study.

We agree with the Reviewer that that the Seahorse assays and membrane polarization would have given mechanistic information. We developed the OXPHOS immunoassays (reference 9, 26762648, 26002840) and have used the mitochondrial 8-oxo-guanine assays (PMID 19321503, 2257461, 25574857) as an initial assay to evaluate mitochondrial function. We do not have viable PBMCs to conduct Seahorse experiments and will do so in future studies.

Once again, we thank the reviewers for their valuable feedback. We hope that we have addressed the queries adequately.

Sincerely,

Louie Mar Gangcuangco, MD

Attachment

Submitted filename: Response to Reviewers PlosOne Oxphos and NCRs 12Feb2020.docx

Click here for additional data file. (20.7KB, docx)

Decision Letter 1

Qigui Yu

1 Apr 2020

Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation

PONE-D-19-24311R1

Dear Dr. Gangcuangco,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Qigui Yu, M.D./Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Fahim Syed

Reviewer #2: No

Acceptance letter

Qigui Yu

20 Apr 2020

PONE-D-19-24311R1

Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation

Dear Dr. Gangcuangco:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Qigui Yu

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    (SAV)

    Click here for additional data file. (240.2KB, sav)
    Attachment

    Submitted filename: Response to Reviewers PlosOne Oxphos and NCRs 12Feb2020.docx

    Click here for additional data file. (20.7KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES