IKAROS expression drives the aberrant metabolic phenotype of macrophages in chronic HIV infection

Abstract

The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by “locking” PLWH monocytes in a pro-inflammatory and activated phenotype.

1. Introduction

Despite access to combined antiretroviral therapy (cART), people living with HIV (PLWH) are at higher risk of developing chronic inflammation and acquiring HIV-defining and non-HIV-defining illnesses, such as secondary infections, cardiovascular disorders, metabolic disorders, and cancer.

Circulating monocytes are highly adaptive cells that possess specific functions beyond being precursors for tissue macrophages [1]. Epigenetic and metabolic reprogramming underlie the highly adaptive nature of these cells, but whether and how pathological conditions, or treatment-associated complications, such as inflammation or cART, could challenge the plasticity of these cells, remain to be determined.

Depending on the type of stimulus and length of exposure, monocytes/macrophages can reach a refractory functional state (tolerance) [2], characterized by the inability of these cells to produce pro-inflammatory cytokines [3], or sustained hyper-responsiveness (trained immunity), characterized by an increased production of pro-inflammatory cytokines [4–7]. A balanced feedback mechanism between these two states of activation is critical for the proper function of monocytes. The transcription factor and chromatin remodeler IKAROS (IKZF1) is known for playing a major role in lymphopoiesis [8, 9]. In macrophages, IKAROS has been shown to be involved in chromatin regulation and transcriptional changes that occur during LPS stimulation [10]. Our previous study demonstrated an imbalance in pro- and anti-inflammatory mechanisms in monocyte-derived-macrophages (MDMs) from PLWH. We found specific alterations in chromatin activation marks of freshly isolated monocytes and increased production of proinflammatory cytokines following lipopolysaccharide (LPS) stimulation in PLWH-MDMs when compared to MDMs from HIV (−) controls [11]. We have further identified a defective expression of the transcription factor IKAROS in PLWH-MDMs as a key factor in mounting a negative feedback response following LPS stimulation [11].

The impact of cART on metabolism has been investigated since antiretroviral therapy was introduced [12–15]. The effect of cART on cellular metabolism has been also investigated in purified immune cells [16–20]. However, the molecular mechanisms underlying the effect of cART on the metabolism of monocytes/MDMs remain largely unknown.

In the present study, we evaluated the metabolic profile of MDMs obtained from PLWH and controls. We found an mTOR-dependent increased mitochondrial respiration and glycolysis in PLWH-MDMs compared to controls, as well as in control-MDMs treated with cART (tenofovir, emtricitabine, and raltegravir). Our metabolic data further confirmed a direct link between reduced expression of IKAROS, increased phosphorylation of p70S6K, and high levels of mitochondrial respiration in PLWH-MDMs, while downregulation of IKAROS in control-MDMs resulted in increased mitochondrial respiration and increased levels of phosphorylated p70S6K. Importantly, pharmacological restoration of IKAROS expression and function significantly reduced mitochondrial respiration of PLWH-MDMs and cART-treated MDMs from controls further indicating that IKAROS could be a new therapeutic target for the improvement of monocyte fitness in PLWH.

2. Results

2.1. PLWH-MDMs show elevated mitochondrial respiration and glycolysis.

To test the metabolic requirement of MDMs from PLWH on cART and from age-, sex-, and race-matched control subjects we performed real-time bioenergetic assays 5 days after cell isolation. Results in Figure 1A show that both basal respiration and maximal respiration were significantly higher in PLWH-MDMs compared to controls, which also translated to the significantly higher spare respiratory capacity (Figure 1B). In the glycolysis stress test (Figure 1C), we also observed a statistically significant increase of ECAR in PLWH-MDMs compared to controls. Results in Figure 1D further show that glycolysis and glycolytic capacity were significantly higher in PLWH-MDMs, while the glycolytic reserve was comparable to controls. In terms of ATP production, both controls- and PLWH-MDMs had statistically significant more ATP produced by mitochondria compared to the portion of ATP produced by glycolysis (Figure 1E). However, PLWH-MDMs displayed higher production of ATP from both mitochondria and glycolysis than controls (Figure 1E). These data strongly indicate that PLWH-MDMs have unusual metabolic adaptation characterized by highly active mitochondrial respiration and glycolysis. Furthermore, these metabolic differences between PLWH- and control-MDMs were maintained after culturing the cells for 1 day, 3 days or 7 days (Figure 1F). To better understand the potential genes involved in the abnormal metabolic profile of PLWH-MDMs, we evaluated the expression of genes involved in glycolysis and mitochondrial respiration (Table 2). Expression levels of differentially regulated genes were determined in freshly isolated monocytes (T0) and 7-day cultured MDMs obtained from Ctrls (T0 n=9, 7d n=13) and PLWH (T0 n=19, 7d n=18). Figure 2 shows a list of differentially regulated genes in PLWH- and Ctrls-MDMs compared to their own T0 (Figure 2A), and scatter plots of the normalized cycle threshold values (Figure 2B). GLUT1 was the most upregulated and IRF4 and TXNIP were the most down-regulated genes. LDHA was upregulated in both PLWH- and control-MDMs, although it was significantly lower in PLWH. Interestingly, the stress sensor SOD2 was upregulated in controls but downregulated in PLWH, perhaps indicating an impaired response to stress in these cells. Another stress sensor and inhibitor of glycolysis, TXNIP [21], was about 4 times less in PLWH compared to controls. IRF4 was downregulated in both groups of MDMs (PLWH and Ctrls) compared to their own T0, but to a significantly greater extent in PLWH-MDMs. AMPK is a key regulator of cellular homeostasis, and its expression was significantly lower in PLWH- compared to Ctrls-MDMs. Expression levels of a key metabolic enzyme, ACLY, were significantly lower in PLWH-MDMs compared to Ctrls. Other genes were upregulated (G6PD, IDH1, HK2, LDHB, HK3, TXTP, LDLR, PPARGC1B, and SDHA) or downregulated (SIRT1 and SIRT3) to a comparable extent in both Ctrls and PLWH cells cultured for 7d compared to their T0. Altogether, these data show significant changes in the expression of selected genes involved in metabolic pathways in freshly isolated monocytes as well as 7-day cultured MDMs from PLWH compared to controls.

Figure 1. Increased mitochondrial respiration and glycolysis of MDMs from PLWH compared to controls.

A) Metabolic profile of MDMs from PLWH (n=12) and Ctrls (n=10). Real-time Oxygen Consumption Rate (OCR) was measured using Seahorse metabolic bioanalyzer following execution of the mitochondrial stress test, in which OCR values are recorded after sequential injection of Oligomycin (OM), FCCP and Rotenone/Antimycin (Rot/AA). B) Scatter plot showing calculated basal, maximal, and spare respiratory capacity (maximal – baseline). Two-tailed Mann-Whitney analysis; **** p<0.0001. C) The same control and PLWH MDMs were used in the glycolytic stress test. In this experiment, the real-time extracellular acidification rate (ECAR) was measured in glucose starved cells that were exposed to sequential injection of glucose (Gluc), Oligomycin (OM), and 2-deoxyglucose (2-DG). D) Scatter plot showing calculated glycolysis, glycolytic capacity, and glycolytic reserve. *** p<0.001, **** p<0.0001. In all graphs, OCR and ECAR values were normalized for the number of cells (see also Materials and Methods). Data represent average values with SEM obtained from independent experiments, each in quadruplicate. E) Rate of ATP production calculated from mitochondrial and glycolytic systems of MDMs from controls and PLWH. Two-tailed Mann-Whitney tests were used for statistical analysis; *mitoATP or glycoATP in PLWH vs Ctrls, *** p<0.001, **** p<0.0001; ^# p<0.05, ^#### p<0.0001. F) Mitochondrial stress test was performed on monocytes/MDMs from PLWH and from HIV (−) controls after 1 day, 3 days, and 7 days of culture. Cells were maintained in complete medium containing GM-CSF (25ng/ml). Data represent average values with SEM obtained from 3 PLWH and 3 HIV (−) controls, each in quadruplicate. Statistical significance was calculated using a two-tailed Mann-Whitney test: baseline (1–3) p≤0.05; measurements 7 and 8 at day 1 had p=0.002, while measurement 9 had p=0.003. On day 3, measurements 7, 8, and 9 had all p<0.0001. On day 7, p=0.007 (7), and p=0.02 (8 and 9).

Table 2.

List of genes analyzed by RT-qPCR.

Gene
ACTB	Actin B
ACLY	ATP citrate lyase
AMPK	AMP-activated protein kinase
G6PC3	Glucose-6-phosphatase catalytic subunit 3
G6PD	Glucose-6-phosphate dehydrogenase
SLC2A1/GLUT1	Solute carrier family 2 member 1
H6PD	Hexose-6-phosphate dehydrogenase
HK2	Hexokinase 2
HK3	Hexokinase 3
IDH1	Isocitrate dehydrogenase (NADP (+)) 1
IRF4	Interferon regulatory factor 4
LDHA	Lactate dehydrogenase B
LDHB	Lactate dehydrogenase A
LDLR	LDL receptor
OPA1	OPA1 mitochondrial dynamin like GTPase
PKM	Pyruvate kinase isozymes M1/M2
PPARGC1B	PPARG coactivator 1 alpha
SDHA	Succinate dehydrogenase A
SIRT1	Sirtuin 1
SIRT3	Sirtuin 3
SOD2	Superoxide dysmutase 2
TXNIP	Thioredoxin interacting protein
SLC25A1/TXTP	Solute carrier family 25 member 1

Figure 2. Expression of genes regulating cell metabolism.

A) List of differentially regulated genes analyzed in 7-day cultured MDMs from PLWH (n=18) and controls (n=13), each relative to their T0 (PLWH, n=19; Ctrls, n=9). P- values are indicated. FC: fold change (2^−ΔΔCt). B) Scatter plots of selected genes showing the greatest difference in the expression between PLWH and controls. Data represent normalized Cts (ΔCt). Note that higher Cts indicate lower expression. Mann-Whitney two-tailed test with Bonferroni correction analysis; * p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; **** p≤ 0.0001.

2.2. cART-treated control-MDMs have a metabolic profile comparable to PLWH-MDMs.

Since all PLWH are on cART and have low or undetectable viral load (Table I), we asked if cART contributes to these metabolic changes. We selected the most common combination of ART drugs in our cohort at the concentrations previously reported [18]. MDMs from HIV (−) controls were cultured for 5 days in the absence or presence of cART (FTC 40 nM, RAL 4 nM, TDF 500 nM). As a positive control for trained immunity, we treated cells with BGP [22–24]. Results in Figure 3A show a significant increase of mitochondrial respiration in cART-treated or BGP-treated MDMs compared to untreated cells. Significant differences in OCR values can be further appreciated in the bar graphs showing basal and maximal respiration, as well as spare respiratory capacity (Figure 3B). In addition, both BGP-treated and cART-treated MDMs demonstrated a significant increase in glycolysis and glycolytic capacity (Figure 3C–D). To summarize, cART treated MDMs show increased glycolysis suggestive of a trained phenotype, as well as increased oxidative phosphorylation, which is typical of activated cells.

Table 1.

General demographics of study cohort subjects. Subsets of age (46–66)-, sex - and race- matched PLWH with low or undetectable viral load and controls from this larger cohort were utilized in this study.

	PLWH	Controls
Sample size	n=121	n=94
Men	58%	48%
Women	42%	52%
Avg Age (range)	54 (39–71)	47 (20–69)
Black	79%	31%
White	18%#	62%#
High Viral Load (>400 copies/ml)	2%
Undetectable/Low Viral Load (≤20 copies/ml)	78%
CD4 T cell count	586 cells/mm3 (range 98 to 1614)
cART	100%
Smoking	42%
Hypertension	43%
Diabetes	23%
HCV	3%

^#χ² indicates statistically significant difference in race distribution within the group.

Figure 3. cART-treated MDMs from HIV (−) controls display a trained metabolic profile.

Primary monocytes were treated with cART: FTC (40 nM), TDF (500 nM), and RAL (4 nM). BGP (10 ng/ml) was added overnight to cART-untreated cells. A) Metabolic profile after mitochondrial stress tests. B) Bar graph showing calculated basal respiration, maximal respiration and spare respiratory capacity. C) Metabolic profile following glycolysis stress tests. D) Bar graph showing calculated glycolysis, glycolytic capacity, and glycolytic reserve. In all panels, data represent average values with SEM obtained from 3 HIV (−) controls, each in quadruplicate. P-values were calculated using two-tailed Mann-Whitney tests. * p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; **** p≤ 0.0001.

Stimulation of human macrophages with LPS is known to increase glycolysis and oxidative phosphorylation [25, 26]. We then evaluated the metabolic profile of MDMs from PLWH and controls upon 2 hrs treatment with LPS (1 μg/ml). Control cells responded to LPS with a significant increase in mitochondrial respiration, while the response from PLWH-MDMs did not increase in a statistically significant manner (Figure 4A). The differences between MDMs from PLWH and controls are additionally illustrated in the bar graphs relative to OCR values at baseline, maximal respiration, and spare respiratory capacity (Figure 4B). Furthermore, MDMs from both PLWH and controls showed an increase in glycolysis, although PLWH-MDMs had a significantly higher increase than control cells (Figure 4C–D). Unlike control cells, LPS treatment of PLWH-MDMs did not increase the glycolytic reserve (Figure 4D). We additionally assessed the effect of LPS on control-MDMs treated with cART. Results in Figure 4E–H show mitochondrial and glycolysis profiles of cART-treated cells with and without LPS stimulation surprisingly similar to the profiles of PLWH-MDMs (Figure 4A–D). Like PLWH-MDMs, cART-treated control-MDMs had increased mitochondrial respiration and the addition of LPS did not further enhance OCR (Figure 4E), confirming the presence of limited spare respiratory capacity of these cells.

Figure 4. Bioenergetic profiles after stimulation with LPS reveal a reduced ability of MDMs from PLWH or cART-treated control cells to respond to energy demands.

Metabolic profiles of MDMs from PLWH (n=4) and Ctrls (n=4) unexposed or exposed to LPS (1 μg/ml) for 2 hrs and assayed in the mitochondrial (A) and glycolytic (C) stress tests. B) Bar graph showing calculated basal respiration, maximal respiration, and spare respiratory capacity. D) Bar graph showing calculated glycolysis, glycolytic capacity, and glycolytic reserve. E, F, G, H) Bioenergetic profiles of MDMs from HIV (−) controls (n=4) untreated (UNT) or cART-treated (cART) exposed or unexposed to LPS for 2 hrs. In all graphs, P-values were calculated using two-tailed Mann-Whitney tests; ** p≤ 0.05, ** p≤0.01, *** p≤ 0.001, **** p≤ 0.0001.

2.3. The abnormal increase in oxidative phosphorylation and glycolysis of PLWH-MDMs and cART-treated control-MDMs is mTOR-dependent.

The beta-glucan-trained metabolic profile of macrophages can be reverted by inhibition of mTOR [24, 27]. Our data indicated an abnormally trained phenotype of PLWH-MDMs (Figures 1–3); as such, we asked whether the mTOR inhibitor rapamycin could revert the high mitochondrial respiration of these cells. Rapamycin (10nM) efficiently inhibited phosphorylation of p-70S6K in both control- and PLWH-MDMs (Figure 5E). Data in Figure 5A–B further demonstrates a rapamycin-dependent decrease of mitochondrial respiration in PLWH-MDMs. Baseline (B) and maximal (stressed, S) values of OCR and ECAR from the mitochondrial stress test indicate the glycolytic phenotype of both groups of MDMs and the transition to an aerobic phenotype under stress conditions; however, PLWH-MDMs are more aerobic than controls at both baseline and in stressed conditions, and rapamycin effectively reverted this phenotype. In the glycolytic stress test rapamycin treatment resulted in a reduction of glycolysis and glycolytic capacity in control cells, and those differences were significantly higher in PLWH-MDMs (Figure 6C–D)

Figure 5. The increased mitochondrial respiration and glycolysis of PLWH-derived MDMs is mTOR-dependent.

A, B) Mitochondrial respiration profiles of Ctrls- and PLWH -derived MDMs untreated or treated with rapamycin (10nM) overnight. Energy map of OCR and ECAR normalized values at baseline (B) and after FCCP (stressed, S) addition in the indicated samples. C, D) Metabolic profiles following glycolytic stress test. In all graphs, two-tailed Mann-Whitney tests were applied; * p≤ 0.05, ** p≤0.01, *** p≤ 0.001. E) Western blot showing rapamycin-induced inhibition of p-70S6K phosphorylation.

Figure 6. The increased mitochondrial respiration and glycolysis of cART-treated Ctrls-MDMs is mTOR-dependent.

A, B) Mitochondrial and glycolytic stress test profiles of HIV (−) MDMs exposed to cART for 5 and untreated or treated with rapamycin for 24 hrs. Data represent average values with SEM obtained from 4 HIV (−) controls, each in quadruplicate. In all graphs, two-tailed Mann-Whitney tests were applied; * p≤ 0.05, ** p≤0.01, *** p≤ 0.001. C) Western blot showing rapamycin-induced inhibition of p-70S6K phosphorylation.

Interestingly, rapamycin treatment reverted the increased mitochondrial respiration and glycolysis of cART-treated control MDMs (Figure 6A–C). These data also indicate a potential therapeutic value of rapamycin, which could be used to reverse, at least partially, MDMs metabolic hyperactivity in PLWH.

2.4. Role of IKAROS in the enhanced respiration of PLWH-MDMs

We have previously demonstrated a role of IKAROS in the hyper-responsiveness of monocytes/MDMs from PLWH to inflammatory stimuli [11]. Here, we sought to determine whether this important transcription factor could also play a role in the altered metabolic profile of PLWH-MDMs. To mimic downregulation of IKAROS, which we observed in PLWH-MDMs [Figure 9A and [11]], we used a siRNA to treat control-MDMs, which express IKAROS at significantly higher levels [Figure 9A and [11]].

Figure 9. Repression of mTOR activity by IKAROS.

A) Western blots showing IKAROS and P-p70S6K expression levels in 5 HIV (−) control and 5 PLWH MDMs. Unpaired two-tailed t-test; * p≤ 0.05. B) Pearson’s correlation between maximal respiration and IKAROS expression for the indicated samples as in A). C) IKAROS and P-p70S6K expression levels in HIV (−) controls (n=4) 72 hrs after nucleofection (left panel), 24 hrs after lenalidomide treatment (middle panel), or 5 days after cART treatment (right panel).

Nucleofection of freshly isolated monocytes from controls with a siRNA targeting IKAROS (50 nM) efficiently downregulated the expression of this protein by nearly 50 % compared to the non-targeting siRNA control (si-NC) (Figure 7A). Decreased expression of IKAROS in these cells was sufficient in producing a significant increase of mitochondrial respiration (Figure 7B). In an alternative approach, we targeted IKAROS expression in control-MDMs using the selective inhibitor lenalidomide [28] at the concentrations of 0.1 and 1 μM (Figure 7B). In a dose-dependent manner, overnight exposure of control-MDMs to lenalidomide resulted in a decreased expression of IKAROS, and a significant increase of mitochondrial respiration (Figure 7B).

Figure 7. Downregulation of IKAROS expression in MDMs from HIV (−) controls enhances mitochondrial respiration, while upregulation of IKAROS expression reduces mitochondrial respiration of MDMs from PLWH.

A) Western blot showing downregulation of IKAROS expression in MDMs from HIV (−) control subjects 3 days after nucleofection with siRNA. Quantification (Image J) of IKAROS bands after normalization with GAPDH expression is also indicated (n=7). ** p≤0.01. Mitochondrial stress test profiles of Ctrls-MDMs transfected with siNC or siIK. Measurements 7–9 in the mitochondrial stress test had p<0.005. B) Western blot showing IKAROS expression in MDMs from HIV (−) control subjects untreated or treated with lenalidomide (LEN) at the indicated concentrations. Quantification after normalization with GAPDH obtained from 3 HIV (−) subjects is also indicated. * Statistical significance (p<0.05) of LEN 0.1 μM/1μM vs UNT. The Mitochondrial stress test was performed on MDMs untreated or treated with lenalidomide, each in quadruplicate. p=0.01 at all baseline points, p=0.04, 0.02, and 0.005 at measurements 7, 8, and 9 respectively. C) Western blot showing the effect of tucidinostat (TUC) on IKAROS expression in MDMs obtained from PLWH, and relative quantification normalized by GAPDH (n=3). Lysates from HIV (−) subjects were used as controls for IKAROS levels. * p<0.05 between PLWH-MDMs untreated or treated with tucidinostat vs controls; # p<0.05 between tucidinostat-treated vs untreated PLWH-MDMs. Mitochondrial stress test performed on MDMs from PLWH untreated (UNT) or treated with different concentrations of tucidinostat (TUC). Statistical significance and p values at baseline vs UNT were p<0.05 (Tuc 2 μM), p=0.01 (Tuc 4 μM), and p≤0.004 (Tuc 8 μM). P values after FCCP injection were p≤0.04 (Tuc 2 μM), p≤0.005 (Tuc 4 μM), and ≤p=0.001 (Tuc 8 μM). SEM is indicated. In all blots, vertical dark lines indicate non-contiguous lanes. P-values for OCR data were calculated using two-tailed Mann-Whitney tests. P-values for the quantification of Western blots were done using unpaired, two-tailed t-test in Excel.

Next, we attempted to up-regulate IKAROS expression in PLWH-MDMs using a variety of methods that resulted either in high toxicity or very low transfection efficiency. Among the pharmacological approaches, the selective histone deacetyl-transferase 1,2,3 (HDACi) inhibitor tucidinostat was previously reported to up-regulate the expression of IKAROS [29]. We therefore evaluated the effect of tucidinostat on IKAROS expression and mitochondrial respiration in PLWH-MDMs. Figure 7C again shows that IKAROS expression is reduced in PLWH-MDMs compared to controls, as we previously observed [11], and that tucidinostat can increase IKAROS levels in these cells. Importantly, increased levels of IKAROS paralleled with a reduction of mitochondrial respiration (Figure 7C), further indicating a possible pharmaceutical intervention to reduce mitochondrial respiration of PLWH-MDMs to levels comparable to control cells.

While IKAROS appears to be a strong candidate for the abnormal metabolic profile of PLWH-MDMs, we considered another key regulator of metabolism. Thioredoxin interacting protein (TXNIP) is a negative regulator of glycolysis [30], supports mitochondrial respiration [31], it is a target of IKAROS [32], and we found lower expression of TXNIP mRNA in PLWH-MDMs compared to controls (Figure 2A). To mimic the reduced levels of TXNIP observed in PLWH-MDMs and determine the role of this factor in the increased glycolysis and mitochondrial respiration, we downregulated TXNIP in control-MDMs and compared the metabolic profiles of these cells to cells nucleofected with IKAROS siRNA(siIK) or with the negative control (siNC) (Figure 8A). While downregulation of IKAROS affected both glycolysis and mitochondrial respiration, downregulation of TXNIP resulted in increased glycolysis (Figure 8B), but not mitochondrial respiration (Figure 8C), further confirming the broader function of IKAROS in regulating cellular metabolism.

Figure 8. Downregulation of IKAROS, but not TXNIP, recapitulates the abnormal metabolic profile of PLWH-MDMs.

A) Western blot showing downregulation of TXNIP and IKAROS after nucleofection of control-MDMs with siTXNIP (siTX) and siIK compared to the non-targeting control siRNA (siNC). Metabolic profiles after glycolytic (B) and mitochondrial (C) stress tests in the indicated experimental conditions. Bar graphs with ECAR and OCR data with statistical significance are also shown. Data represent average values with SEM obtained from 3 HIV (−) controls, each in quadruplicate. * p<0.05, ** p≤ 0.01.

As both IKAROS expression (Figures 7 and 8) and mTOR activity (Figures 5 and 6) seem to critically regulate the metabolic reprogramming of PLWH-MDMs, we evaluated a possible connection between IKAROS and Phospho-p70S6K, a downstream effector of mTOR. First, we determined how the expression of IKAROS could parallel the expression of P-p70S6K in PLWH and control-MDMs. On average, MDMs from PLWH (n=5) had about 50% less IKAROS protein and 50% more P-p70S6K than controls (n=5) (Figure 9A). One control-sample (#2) and one PLWH-sample (#4) had low and high IKAROS levels that correlated with high and low P-p70S6K, respectively; confirming the inverse relationship between these two proteins. Interestingly, there is a strong correlation between IKAROS expression and maximal respiration (R²=9.6, Figure 9B), as determined by Pearson’s correlation using OCR data and GAPDH-normalized IKAROS expression levels from the samples indicated in Figure 9A. Furthermore, downregulation of IKAROS by either siRNA, lenalidomide, or cART in control-MDMs resulted in increased levels of Phospho-p70S6K (Figure 9C), further supporting our hypothesis of a role of IKAROS/mTOR axis in PLWH-MDMs metabolic dysfunction.

3. Discussion

The goal of our study was to determine differences in the energy metabolism, and the molecular mechanisms underlying those differences, between PLWH and HIV (−) controls. All PLWH in our cohort were on cART and had low or undetectable viral load; they were prevalently Blacks, and more than 40% had other conditions, such as hypertension and diabetes, while most subjects in the control group were Whites (Table I). To minimize demographic differences between PLWH and controls, we utilized a subset of PLWH that was matched for age (46–66 years old), sex, and race with the control group.

Monocytes and macrophages are essential components of our innate immune system; they are present both in tissues as resident cells and can be recruited from the circulating pool of monocytes in the blood [33]. The function of monocytes/macrophages to mount a balanced immune response and their ability to switch from pro-inflammatory (M1) to anti-inflammatory (M2) phenotypes in response to environmental cues requires epigenetic and metabolic reprogramming [34]. In our previous study, we found that PLWH-MDMs are defective in establishing endotoxin tolerance compared to MDMs from HIV (−) individuals [11]. Evidence suggested that these monocytes were “trained” toward a pro-inflammatory phenotype characterized by increased production of inflammatory cytokines [11, 35] and attenuation of negative feedback mechanisms [11]. Furthermore, we have identified IKAROS as a key player in the negative regulation of inflammatory responses [11]. Since trained immunity is characterized by metabolic and epigenetic reprogramming, in the present study we evaluated the metabolic profile of PLWH-MDMs and the role of IKAROS in the metabolism of these cells. We unexpectedly found increased mitochondrial respiration and glycolysis of PLWH-MDMs compared to control-MDMs (Figure 1). Of note, the addition of oligomycin after glucose to human primary macrophages does not induces a dramatic increase in ECAR (Figures 1C, 3C, 4C, and 4G), which is observed in mouse macrophages [26, 36, 37].

M1-polarized human macrophages utilize mostly mitochondria for ATP production [26], and we also found higher mitochondria-derived ATP compared to glycolysis-derived ATP in both control- and PLWH-MDMs, although the latter had significantly higher amounts of ATP produced by mitochondria and glycolysis (Figure 1E). The pro-inflammatory nature of MDMs from PLWH on cART has been described by others [38] and us [11]. As glycolysis and oxidative phosphorylation are associated with production of pro-inflammatory cytokines in human MDMs [26, 34, 39], perhaps was not surprising that MDMs from PLWH, which are highly inflammatory [11, 38], showed increased glycolysis and mitochondrial respiration compared to controls. Importantly, the increased mitochondrial respiration was observed in early monocyte cultures (1 day), as well as in MDMs cultured for 3 and 7 days (Figure 1F), suggesting a long-lasting metabolic reprogramming of these cells. This finding is in line with a recent study showing that PLWH on cART have sustained immunometabolic reprogramming evaluated through the analysis of serum metabolites [40].

To corroborate OCR and ECAR data, we evaluated the expression profile of key genes involved in glycolysis and mitochondrial respiration in freshly isolated monocytes (T0) as well as 7 days cultured MDMs obtained from PLWH and HIV (−) controls. Higher expression of free radical sensors SOD2 and TXNIP in PLWH-derived monocytes at T0 may correlate with increased oxidative stress in these cells, while a reduction in AMPK expression may correlate with impaired metabolic homeostasis. Oxidative damage has been observed in PLWH, attributed to cART treatments, and potentially associated with insulin-resistance [41]. In addition, AMPK has been shown to repress mTOR pathway [42, 43], and therefore, reduced expression of AMPK in PLWH-derived MDMs may indicate increased mTOR activity, supporting the higher phosphorylation of p70S6K in these cells compared to controls (Figure 9). MDMs from PLWH show statistically significant differences in IRF4, and TXNIP compared to controls (Figure 2). Bae et al. demonstrated the importance of the MYC/LDH/IRF4 axis in regulating early glycolysis in the pro-inflammatory polarization of macrophages [44]. We did not investigate expression levels of MYC in our experimental setting, but the reduced expression levels of IRF4 in PLWH-derived MDMs compared to controls (Figure 2), may indicate a dysfunctional IRF4-mediated signaling pathways. RNA and protein levels of IKAROS are both significantly lower in PLWH MDMs compared to controls [11] (Figure 9A), paralleling with the lower expression of genes previously identified as downstream targets of IKAROS, such as TXNIP [45] and IRF4 [46, 47].

Among the factors potentially responsible for the abnormal metabolic behavior of PLWH-MDMs, we hypothesized a role for cART in modifying monocyte metabolism, since all PLWH had low or undetectable viral load (Table 1) and are on cART on average for longer than 5 years. While the general toxicity of cART is well-documented [48–52], its effect on monocyte/MDMs metabolism remain elusive. Van der Heijden et al. have evaluated transcriptional and functional reprogramming of circulating monocytes and MDMs from PLWH on cART and, similar to us [11], they found a sustained pro-inflammatory phenotype of monocytes from PLWH [35]. Our previous work further confirmed the pro-inflammatory phenotype of monocytes/MDMs from PLWH on cART and identified at least one mechanism involved in the aberrantly trained phenotype of these cells [11]. We found that lower expression of IKAROS results in attenuated pro-inflammatory negative feedback [11]. To the best of our knowledge, the present study is the first to investigate bioenergetic profiles of MDMs from PLWH and controls and to identify at least one molecular mechanism underlying the abnormal metabolic reprogramming of PLWH-derived cells. In addition, we investigated the effect of a three-drug cART regimen (FTC, RAL, TDF) on the metabolic profile of MDMs. We selected this combination of antiretrovirals because it includes the most common drugs, particularly tenofovir, administered to PLWH in our cohort. As a positive control, we used beta-glucan peptide (BGP), which is known to train immune cells through increased glycolysis (Figure 3A–B) [2, 53]. Unexpectedly, cART-treated cells showed an increase in glycolysis comparable to the BGP-treated cells, further indicative of a cART-induced trained phenotype (Figure 3C–D). Furthermore, like PLWH-MDMs, cART-treated MDMs showed a significant increase in mitochondrial respiration (Figures 1 and 3), typical of activated cells [34, 54].

Various regimens of cART have been shown to exert a negative impact on T-cell mitochondrial respiration [18, 19]. Similar data were obtained for mitochondrial respiration evaluated in PBMCs and T cell subtypes from healthy controls treated acutely with TDF [16, 55] or FTC [16]. One study found that M2-MDMs treated for 7 days with cART had a reduction in mitochondrial respiration compared to cART-untreated cells [20], and our data show that cART increases mitochondrial respiration of M1-MDMs. Since M2-macrophages display higher respiration than M1-macrophages [34, 56], the effect of cART on decreasing or increasing mitochondrial respiration could therefore depend on the M2 or M1 activation status, respectively.

In line with previous studies [26], we found an increase in both mitochondrial respiration and glycolysis of control MDMs upon LPS stimulation (Figure 4). Our results indicate that PLWH-MDMs while being able to respond to high energy demands, have limited ability to metabolically adapt to other stimuli such as LPS. Notably, treatment of control-MDMs with cART and further exposure of the cells to LPS (Figure 4E–H), resulted in metabolic profiles overlapping those obtained from PLWH-MDMs. Overall, metabolic profiles upon LPS challenge confirm the aberrantly trained phenotype of MDMs obtained from PLWH or from controls after treatment with cART. As trained immunity induced by beta-glucan is dependent on mTOR pathway [24], and MDMs from PLWH show metabolic profiles similar to BGP treated cells (Figure 3), we found that the mTOR inhibitor rapamycin significantly reduced mitochondrial respiration and glycolysis of MDMs from PLWH (Figure 5) suggesting a potential treatment to reprogram the abnormally trained phenotype of MDMs in PLWH. Of note, rapamycin effectively reduced the abnormal mitochondrial respiration and glycolysis of cART treated MDMs from controls (Figure 6), at least at the tested concentration and combination of antiretrovirals.

Downregulation of IKAROS expression by siRNA or the IKAROS-specific inhibitor lenalidomide [28] upregulated mitochondrial respiration of control-MDMs (Figure 7A–B) imitating the metabolic profile of MDMs from PLWH (Figure 1), while upregulating IKAROS expression through the HDAC inhibitor tucidinostat [29] decreased mitochondrial respiration of MDMs from PLWH (Figure 7C). Given the role of TXNIP in regulating cell metabolism, we evaluated the effect of this protein on MDMs metabolism. The knockdown of TXNIP in control-MDMs recapitulated the increased glycolysis observed in PLWH-MDMs without having a significant impact on mitochondrial respiration. Therefore, compared to IKAROS downregulation, siTXNIP recapitulated only one aspect of the altered phenotype of PLWH-MDMs, confirming the role of TXNIP in the negative regulation of glycolysis and strengthening the prominent role of IKAROS expression in the abnormal metabolic phenotype of PLWH-MDMs.

Higher IKAROS expression paralleled with lower expression of Phospho-p70S6K in MDMs from controls, while this relationship is lost in PLWH-MDMs (Figure 9A). Downregulation of IKAROS expression in control cells by siRNA, lenalidomide, and cART treatment, resulted in increased levels of P-p70S6K (Figure 9C). This finding is in line with a previous report describing IKAROS as a repressor of mTOR activity in B-cell acute lymphoblastic leukemia, a tumor characterized by a single-copy deletion of IKZF1 and increased activation of AKT/mTOR pathway [57]. Altogether, those data strongly support the presence of an IKAROS/mTOR defective axis in PLWH-MDMs. Besides an association between IKAROS expression and P-p70S6K, we found a strong correlation between IKAROS expression and maximal respiration (Figure 9B), suggesting that IKAROS expression could be used to stratify HIV patients with altered metabolic profile, although more samples are needed to support this finding.

In summary, these data demonstrate a very specific metabolic adaptation of monocytes isolated from PLWH, which involves an mTOR-dependent highly increased mitochondrial respiration and glycolysis. This unexpected finding prompted us to hypothesize that PLWH-MDMs are in a hyperactive metabolic mode in which both glycolysis and mitochondrial respiration contribute to the high-energy demands of these aberrantly activated cells. It also implies that PLWH-MDMs can utilize different energy sources, and therefore, could be more resistant to adapt their metabolic profile to environmental cues. This is important, since dependency on a specific energy source is a critical regulatory mechanism that may control, at least in part, the M1/M2 switch of monocytes. Therefore, the enhanced metabolism of PLWH-MDMs, or cART-treated controls, while broadening their energy sources, can decrease the regulatory ability of these cells to quickly respond to environmental cues, compromising their function by “locking” PLWH-MDMs in the M1 pro-inflammatory activated phenotype.

4. Materials and Methods

4.1. Clinical Samples, Study Approval

Blood samples were obtained from PLWH on cART and race-, sex-, and age-matched HIV (−) volunteers currently recruited in the HIV-Clinical Tumor Biorepository (HCTB) core facility (Table I), following previously described protocols [11, 58–60] and approved by the LSUHSC-NO Institutional Review Board (IRB). Written informed consent was received prior to participation in this study. Freshly isolated monocytes and cultured MDMs from PLWH tested negative for viral RNA and DNA [11].

4.2. Primary cells.

CD14⁺/CD16⁻ cells were isolated [EasySep^™ Direct Human Monocyte Isolation Kit (STEMCELL Technologies)] from 50 ml of whole blood. Cells were plated in RPMI 1640 (Gibco) supplemented with 1 mM Sodium Pyruvate, 2 mM Glutamax, 10 % FBS and human recombinant GM-CSF (25 ng/ml, R&D Systems). The medium was replaced every 3 days. For transfection experiments, monocytes were nucleofected with Human Monocyte Nucleofector^™ Kit (Lonza). Human IKZF1 and human TXNIP Smart Pool siRNA (siIK, siTX) and non-targeting control siRNA (siNC) were from Horizon and used at the concentration of 50 nM in all experiments.

4.3. Reagents and Antibodies.

Emtricitabine (FTC), Tenofovir Disoproxil Fumarate (TDF) and Raltegravir (RAL) were from Selleckchem. Beta-glucan peptide (BGP) was from InvivoGen. LPS was from Sigma-Aldrich. Rapamycin was from Millipore Sigma. TXNIP, IKAROS and P-p70S6K antibodies were from Cell Signaling Technology and GAPDH antibody was from Santa Cruz Biotechnology. Lenalidomide and tucidinostat were from Selleckchem.

4.4. Cell metabolism assays.

The metabolism of MDMs was evaluated using the Seahorse XFe96 Flux Analyzer (Agilent). Mitochondrial function was analyzed using the Cell Mito Stress Test kit. This test measures the Oxygen Consumption Rate (OCR) at baseline and after the sequential injections of oligomycin (2 μM), FCCP (4 μM), and rotenone/antimycin A (1 μM). The last injection included Hoechst dye for cell counting and normalization (4 μM).

Glycolysis was assessed with the Glycolysis Stress Test kit. The test measures ECAR at baseline and after three consecutive injections of glucose (10 mM), oligomycin (2 μM), and 2-deoxy-D-glucose together with Hoechst dye (2-DG, 50 mM). In all experiments, OCR and ECAR values were normalized by the number of cells. The total number of cells was determined by extrapolation using a cell-type specific calibration curve.

4.5. Quantitative RT-PCR.

RNA was isolated using the miRNeasy Mini Kit (Qiagen) and 320 ng of total RNA were reverse transcribed to cDNA using the RT² First Strand Kit (Qiagen). SYBRGreen mix was from Qiagen. All primers were purchased from Origene. Real-time PCR was performed using a Roche Light Cycler 480 Real-Time PCR System. Each sample was assessed in duplicate and actin beta was used as reference gene. The relative quantification of gene expression was calculated using the comparative Ct (2^−ΔΔCt) method as we previously described [11, 58, 61]. Data were then converted to log- normalized fold change, and the standard error was calculated following formulas described previously [62].

4.6. Statistical analysis.

Real-time PCR data were analyzed using GenEx 7 software. All data are presented as mean ± SEM and graphed using GraphPad Prism 9 and Excel software packages. Two-tailed non-parametric tests were used and p ≤ 0.05 was considered statistically significant.

Highlights.

• Monocyte-derived macrophages from PLWH display enhanced mitochondrial respiration and glycolysis.

• Dysfunctional IKAROS/mTOR axis drives the PLWH-macrophages metabolic phenotype.

• HIV antiretrovirals alter both expression of IKAROS and metabolism of macrophages.