Antiretroviral Drug Transporters and Metabolic Enzymes in Circulating Monocytes and Monocyte-Derived Macrophages of ART-Treated People Living With HIV and HIV-Uninfected Individuals

Abstract

Membrane-associated drug transport proteins and drug metabolic enzymes could regulate intracellular antiretroviral (ARV) drug concentrations in HIV-1 target cells such as myeloid cells. We investigated the expression of these transporters and enzymes in monocyte subsets and monocyte-derived macrophages (MDMs) isolated from peripheral blood mononuclear cells (PBMCs) of HIV-uninfected individuals (HIV-negative) and people living with HIV receiving viral suppressive antiretroviral therapy (ART; HIV+ART) and examined plasma and intracellular ARV concentrations. Monocytes were isolated from PBMCs of 12 HIV-negative and 12 HIV+ART donors and differentiated into MDMs. The mRNA and protein expression of drug transporters and metabolic enzymes were analyzed by quantitative real-time polymerase chain reaction and flow cytometry, respectively. ARV drug concentrations were quantified in plasma, PBMCs, monocytes, and MDMs by LC-MS/MS. The mRNA expression of relevant ARV transporters or metabolic enzymes, ABCB1/P-gp, ABCG2/BCRP, ABCC1/MRP1, ABCC4/MRP4, SLC22A1/OCT1, SLC29A2/ENT2, CYP2B6, CYP2D6, and UGT1A1, was demonstrated in monocytes and MDMs of 2 to 4 HIV-negative donors. P-gp, BCRP, and MRP1 proteins were differentially expressed in classical, intermediate, and nonclassical monocytes and MDMs of both HIV+ART and HIV-negative donors. Intracellular concentrations of ARVs known to be substrates of these transporters and metabolic enzymes were detected in monocytes of HIV+ART donors but were undetectable in MDMs. In this study, we demonstrated the expression of drug transporters and metabolic enzymes in monocytes and MDMs of HIV-negative and HIV+ART individuals, which could potentially limit intracellular concentrations of ARVs and contribute to residual HIV replication. Further work is needed to assess the role of these transporters in the penetration of ARVs in tissue macrophages.

INTRODUCTION

Since the implementation of antiretroviral therapy (ART) for the treatment of HIV type 1 (HIV-1) infection, the associated morbidity and mortality has significantly declined.^1,2 ART targets different stages of the HIV-1 life cycle and has been effective in suppressing the plasma viral load. However, ART does not lead to HIV eradication, and viral rebound occurs after treatment cessation.^1,3 Noteworthy, subtherapeutic antiretroviral (ARV) drug concentrations in both cellular and anatomic HIV reservoirs could contribute, partly, to inadequate viral suppression and facilitate the evolution of drug-resistant virus and persistent infection.⁴

ARV permeability into cellular and anatomic HIV reservoirs is highly regulated by the physicochemical and plasma protein–binding properties of the drugs and a dynamic interplay among drug uptake, metabolism, and efflux processes.^5-9 The disposition of ARVs across the cell membrane involves influx or efflux by the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies of membrane-associated drug transport proteins, respectively. In addition, interactions of ARVs with cytochrome P450 (CYP450) and uridine diphosphate–dependent glucuronosyltransferase (UGT) metabolic enzymes could result in enhanced drug metabolism and clearance.^10,11 These drug transporters and metabolic enzymes have been shown to alter the intracellular accumulation of ARVs and other substrates in HIV-1 cellular reservoirs such as CD4⁺ T-cells.^9,12,13

Cells of the monocyte/macrophage lineage have been demonstrated to contain HIV-1 DNA in individuals on ART.^14-16 Peripheral blood monocytes are composed of 3 major subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺, and nonclassical CD14⁺CD16⁺⁺, with different trafficking potential and effector functions at homeostasis and during viral infection, including HIV.^17-19 Although monocytes are naturally resistant to productive HIV infection, they are precursors for macrophages that represent important HIV infection targets in peripheral tissues.^20,21 Furthermore, Ancuta et al¹⁹ demonstrated that CD16⁺ monocytes promote high levels of HIV replication on differentiation into macrophages and interaction with T-cells. Therefore, it is important to understand the mechanisms of HIV-1 persistence in these cell types. The expression or function of several ABC drug efflux transporters such as P-glycoprotein (P-gp), breast cancer–resistant protein (BCRP), multidrug-resistant associated proteins (MRPs), and metabolic enzymes, including CYP3A4, CYP2B6, CYP2D6, and UGT1A1, was previously demonstrated in in vitro monocytic cell lines and peripheral blood monocytes and monocyte-derived macrophages (MDMs) obtained from uninfected individuals (HIV-negative).^22-26 In addition, mRNA expression of SLC transporters such as OATP2B1, CNT3, ENT3, and OCT1 has been reported in peripheral monocytes or MDMs of HIV-negative individuals.²⁷ However, the expression of these drug transporters and metabolic enzymes and their potential roles in the disposition of ARVs in macrophages and monocytes obtained from individuals living with HIV (HIV-positive) are not well characterized. Moreover, to the best of our knowledge, data on the intracellular concentration of different classes of ARVs in monocytes and macrophages of individuals living with HIV on ART (HIV+ART) are lacking.

In this study, we investigated the mRNA and protein expression of several ABC (ie, P-gp, BCRP, MRP1, and MRP4) and SLC [ie, organic anion transporting polypeptide (OATP2B1) and organic cation transporter (OCT1)] transporters known to be involved in the disposition of commonly used ARVs and metabolic enzymes (ie, CYP3A4, CYP2B6, CYP2D6, and UGT1A1), in both monocytes and MDMs obtained from HIV-negative and HIV+ART donors. Furthermore, we quantified the concentrations of ARVs in plasma, peripheral blood mononuclear cells (PBMCs), monocytes, and MDMs obtained from HIV+ART donors to determine the extent of drug penetration into these cell types.

MATERIALS AND METHODS

Sample Population

HIV-negative and HIV+ART study donors were recruited at the McGill University Health Centre (see Tables 1a and 1b, Supplemental Digital Content, http://links.lww.com/QAI/B641). Leukaphereses were performed and PBMCs were isolated by Ficoll gradient centrifugation and frozen in liquid N₂ for future use, as previously described.^28,29 The plasma viral load was measured using the Amplicor HIV-1 monitor ultrasensitive method (Roche) (detection limit 40 HIV-RNA copies/mL). The estimated infection date was determined using clinical and laboratory data.

Ethics Statement

The study samples collected from the HIV+ART and HIV-negative donors were in accordance with the guidelines included in the Declaration of Helsinki and with approval from the McGill University Health Centre and CHUM-Research Centre Institutional Review Boards. All donors signed a written informed consent form for their participation in the study.

Magnetic-Activated Cell Sorting of Monocytes

Monocytes were isolated from PBMCs by negative selection using the Monocyte Isolation Kit following manufacturer’s protocol (MACS, Pan Monocyte Isolation kit, human, Miltenyi Biotech, Auburn, CA). The purity of monocytes (95%–98%) was determined by flow cytometry after staining the cells with anti-CD3, anti-CD4, anti-HLA-DR, anti-CD14, and anti-CD16 antibodies.

Generation of Monocyte-Derived Macrophages

To obtain MDMs, highly pure monocytes were cultured in 48-well plates (2 × 10⁶ monocytes/well) in the presence of 10 ng/mL macrophage colony-stimulating factor (M-CSF) (R&D Systems). Media containing M-CSF were refreshed every 2 days. Cells were harvested at day 6 for quantitative real-time polymerase chain reaction (qPCR), flow cytometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses.

Total RNA Extraction, cDNA Synthesis, and Quantitative PCR

Total RNA was isolated from monocytes or MDMs of HIV-negative donors using TRIzol reagent (Invitrogen, Carlsbad, CA). Two microgram of DNase-treated total RNA was reverse transcribed with a high-capacity cDNA reverse transcriptase kit (Applied Biosystems, Waltham, MA) using a Mastercycler ep Realplex 2S thermal cycler (Eppendorf, Mississauga, ON, Canada). The mRNA expression levels of drug transporters and metabolic enzymes were then analyzed by qPCR using TaqMan primers designed and validated by Life Technologies (see Table 2, Supplemental Digital Content, http://links.lww.com/QAI/B641). All reactions were performed in triplicates with each 20 μL reaction containing 200 ng of cDNA, 1 μL of 20 × primer mix, and 10 μL of TaqMan qPCR mastermix. The expression of each gene of interest is presented as normalized RNA levels (arbitrary units) relative to the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene using the ΔC_t method, where ΔC_t is equal to the C_t value of the gene of interest minus the C_t value of GAPDH. GAPDH was confirmed as an appropriate housekeeping gene in our analyses with consistent levels of expression in both monocytes and MDMs obtained from HIV-negative and HIV+ART donors.

Flow Cytometry

The antibodies used in the flow cytometry analyses are listed in the Supplemental Digital Content (see Table 3, http://links.lww.com/QAI/B641). The cell viability marker LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Burlington, ON, Canada) was used to exclude dead cells. The data acquisition was performed using BD-LSRII cytometer and analyzed using FlowJo (Tree Star, Inc., Ashland, OR) software. Fluorescence minus one (FMO) strategy was used in setting the gates for positive events, as previously described (see Figure 1, Supplemental Digital Content, http://links.lww.com/QAI/B641).^30,31

ARV Quantification in Plasma, PBMCs, Monocytes, and MDMs

ARV quantification in plasma and cell lysates from PBMCs, monocytes, and MDMs of HIV+ART participants was performed in the Antiviral Pharmacology Laboratory, University of Nebraska Medical Center, applying LC-MS/MS, according to previously validated methods.³² In brief, abacavir, atazanavir, efavirenz, rilpivirine, tenofovir, lamivudine, emtricitabine, elvitegravir, dolutegravir, and cobicistat were extracted from cell lysates or plasma using 70:30 methanol/water, mixed with ¹³C internal standards (ISs), and proteins were precipitated using an acetonitrile step. To analyze tenofovir diphosphate, carbovir triphosphate, emtricitabine triphosphate, and lamivudine triphosphate in cell lysates, the phosphorylated drug was isolated from interferences, metabolites, and unphosphorylated drug using an ion exchange solid-phase extraction. Phosphorylated drug was then desalted with a reversed-phase extraction, dried, and reconstituted before analysis. Final sample extracts were separated and quantified using a Shimadzu Nexera ultrahigh-performance liquid chromatograph attached to an AB Sciex 5500 qTrap mass spectrometer (plasma, PL) or an AB Sciex 6500 triple quadrupole mass spectrometer (intra-cellular, IC). Ion pairs (tenofovir/emtricitabine/abacavir/lamivudine/atazanavir/rilpivirine/dolutegravir/elvitegravir/ cobicistat, positive; efavirenz, negative) were monitored in multiple reaction-monitoring mode. Analyte peaks were normalized to the corresponding IS peaks, except for lamivudine, for which no IS was available and was instead normalized to the emtricitabine-IS. The linear ranges were as follows: tenofovir/emtricitabine 10–1500 ng/mL (PL), 2.5–1000 fmol/sample (IC); abacavir/lamivudine 20–5000 ng/mL (PL), carbovir 50–10,000 fmol/sample (IC), lamivudine 1000–200,000 fmol/sample (IC); atazanavir 20–20,000 ng/mL (PL), 0.4–200 fmol/sample (IC); rilpivirine 5–2500 ng/mL (PL), 2.73–546 fmol/sample (IC); dolutegravir 20–10,000 ng/mL (PL), 0.477–238 fmol/sample (IC); elvitegravir 30–6000 ng/mL (PL), 0.893–447 fmol/sample (IC); and cobicistat 30–6000 ng/mL (PL), 0.525–258 fmol/sample (IC). The interbatch %CV for the quality control samples in the phosphorylated NRTI methods (tenofovir diphosphate, emtricitabine triphosphate, carbovir triphosphate, and lamivudine triphosphate) and the PI/NNRTI/INSTI methods (atazanavir, efavirenz, rilpivirine, dolutegravir, elvitegravir, and cobicistat) were 3.8%–7.7% and 1.1%−7.4%, respectively. Absolute mean relative errors to the theoretical target quality control samples were <5.6% for phosphorylated NRTI methods and <8.1% for the PI/NNRTI methods.

RESULTS

mRNA Expression of Drug Transporters and Metabolic Enzymes in Monocytes and MDMs

We investigated the mRNA expression of the following: (1) drug efflux transporters, ABCB1/P-gp, ABCG2/BCRP, ABCC1/MRP1, and ABCC4/MRP4; (2) uptake transporters, SLC22A1/OCT1 and SLC29A2/ENT2, and (3) metabolic enzymes, CYP3A4, CYP2B6, CYP2D6, and UGT1A1, in monocytes and MDMs isolated from HIV-negative donors (donor ID HIV-negative Nos. 1–4). Many ARVs are known to interact with these transporters and metabolic enzymes and can serve as substrates, inhibitors, or inducers. We detected mRNA expression of these transporters and metabolic enzymes in both monocytes and MDMs with ABCC1, demonstrating relatively high expression in both cell types (Figs. 1A-F).

FIGURE 1.

Relative mRNA expression of drug efflux/uptake transporters and metabolic enzymes in human monocytes and MDMs. The mRNA expression of drug efflux transporters, ABCB1 (P-gp), ABCG2 (BCRP), ABCC1 (MRP1), and ABCC4 (MRP4) (A and B), uptake transporters SLC22A1 (OCT1) and SLC29A2 (ENT2) (C and D), and metabolic enzymes CYP2D6, CYP2B6, and UGT1A1 genes (E and F) were analyzed in monocytes or macrophages isolated from HIV-negative donors (HIV-negative Nos. 1–4) by qPCR using TaqMan Gene Expression Assay (see Table 2, Supplemental Digital Content, http://links.lww.com/QAI/B641 for the list of primers). The results are expressed as mean relative mRNA expression ± SD normalized to the housekeeping gene GAPDH.

P-gp, BCRP, and MRP1 Protein Expression in HIV+ART and HIV-negative Monocytes and MDMs

We then investigated the protein expression of the drug transporters and metabolic enzymes by performing immunoblotting assays; however, we were unable to detect robust expression of these proteins in the monocytes and MDMs by immunoblotting. Therefore, we performed flow cytometry analyses and detected the expression of several efflux transporters in monocyte subsets and MDMs of HIV-negative and HIV+ART donors. Unfortunately, we were not able to evaluate the expression of the SLC transporters and metabolic enzymes by flow cytometry because of unavailability of appropriate antibodies validated for this technique.

Total monocytes were enriched from PBMCs of 12 HIV+ART (donor ID HIV+ART Nos. 1–12) and 12 HIV-negative (donor ID HIV-negative Nos. 1–12) donors by negative selection using MACS. To identify total monocyte population, we gated on live cells that were negative for the T-cell markers CD3 and CD4 and positive for CD14/CD16. The cells were further gated into classical (CD14⁺⁺ CD16⁻), intermediate (CD14⁺⁺ CD16⁺⁺), and nonclassical (CD14+/CD16⁺⁺) monocytes (Fig. 2A), and the expression of P-gp, BCRP, and MRP1 was investigated in these subsets (Figs. 2B-D). To obtain MDMs, monocytes were cultured in the presence of M-CSF for 6 days. MDMs acquired the expression of CD4, and most cells were positive for CD14 and CD16 (Fig. 3A). The expression of P-gp, BCRP, and MRP1 was analyzed in the CD14⁺CD16⁺ MDM cell population (Fig. 3B). The results are reported as the median percentage of cells expressing these transporters. Overall, we detected P-gp, BCRP, and MRP1 protein expression in both HIV+ART and HIV-negative monocytes and MDMs.

FIGURE 2.

Expression of BCRP, MRP1, and P-gp proteins in monocyte subsets of HIV-negative and HIV+ART donors. A, Flow cytometry gating strategy for monocytes isolation from PBMCs of a representative donor. Panels from left to right, monocytes were selected based on high forward scatter and intermediate side scatter. Viable (positive for vivid) single HLA-DR+ cells were positively selected and CD3⁺CD4⁺ cells were excluded. Monocyte subsets, that is, classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺), and nonclassical (CD14⁺CD16⁺⁺) were sorted based on the expression of CD14 and CD16. B, BCRP, C, MRP1, and D, P-gp expression in monocyte subsets were compared in classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺), and nonclassical (CD14⁺CD16⁺⁺) monocytes isolated from HIV-negative and HIV+ART donors using the appropriate antibodies, as listed in the Supplemental Digital Content (see Table 3,http://links.lww.com/QAI/B641). Data are expressed as median percentage of monocyte subsets expressing BCRP, MRP1, and P-gp proteins isolated from HIV-negative and HIV+ART participants, n = 12/group. Statistical significance between groups was determined by Friedman or 2-way RM ANOVA tests using Dunn or Sidak multiple comparisons tests, respectively. *P < 0.05; **P < 0.01; and ***P < 0.001.

FIGURE 3.

Expression of BCRP, MRP1, and P-gp proteins in MDMs of HIV-negative and HIV+ART participants. A, Gating strategy for MDMs isolated from a representative donor. Panels from left to right, MDMs were selected based on high forward scatter and intermediate side scatter. Viable (positive for vivid) single cells were positively selected, and CD3⁺CD4⁺ cells were excluded. MDMs (CD14⁺CD16⁺⁺) were sorted based on the expression of CD14 and CD16. B, BCRP, MRP1, and P-gp expression were compared in MDMs isolated from HIV-negative or HIV+ART participants for BCRP (left panels), MRP1 (middle panels), and P-gp (right panels) using the appropriate antibodies listed in the Supplemental Digital Content (see Table 3, http://links.lww.com/QAI/B641). Data are expressed as median percentage of MDMs expressing BCRP, MRP1, and P-gp proteins isolated from HIV-negative or HIV+ART participants, n = 12/group. Comparisons between groups were performed using the Mann–Whitney U test.

Specifically, among the monocyte subsets, BCRP demonstrated a significantly higher median frequency in intermediate monocytes (10.8%) compared with that in classical monocytes (4.8%) of HIV+ART donors (Fig. 2B). The expression of MRP1 was also detected, with significantly higher frequencies in the intermediate (13.2%) compared with those in classical (1.5%) monocytes of HIV+ART donors and in both intermediate (11%) and nonclassical (8.1%) compared with those in classical (2.3%) monocytes of HIV-negative donors (Fig. 2C). On the other hand, the expression of P-gp was detected at a significantly lower frequency in nonclassical (41.4%) monocytes compared with that in the classical (61.2%) subset of HIV+ART donors (Fig. 2D). The expression of BCRP, MRP1, or P-gp were compared between HIV-negative and HIV+ART monocyte subsets, and a trend of lower frequencies of BCRP and P-gp could be observed in cells of the HIV+ART donors; however, this was not statistically significant (Figs. 2B-D). Similarly, in the MDMs, we did not observe significant differences in the frequency of cells expressing BCRP, MRP1, or P-gp in the HIV-negative compared with that in HIV+ART groups (Fig. 3B).

ARV Quantification in HIV+ART Plasma, PBMCs, Monocytes, and MDMs

We further quantified the concentration of ARVs in plasma, PBMCs, monocytes, and MDMs obtained from 10 HIV+ART donors using LC-MS/MS analysis. We did not obtain plasma samples for 2 donors, HIV+ART1 and HIV+ART2; as such, they were excluded from the analysis. We detected plasma concentrations of dolutegravir (1310–3640 ng/mL), elvitegravir (745.4 ng/mL), tenofovir (33.5–167 ng/mL), emtricitabine (36.3–916.9 ng/mL), lamivudine (217–1169 ng/mL), efavirenz (923.1 ng/mL), rilpivirine (8.5–18.4 ng/mL), abacavir (84.1–1563 ng/mL), and cobicistat (172.1 ng/mL) (Table 1). Overall, the plasma concentrations of these ARVs were similar to previously reported therapeutic concentration ranges.^32-36 It must be noted that a number of factors related to analytical or donor variability could explain the differences in intracellular concentrations observed between our group and others. In PBMCs, the concentrations of ARVs were lower than those reported in the literature,^33,37,38 except for one donor (HIV+ART7) who demonstrated emtricitabine levels comparable with previously reported values,³⁹ (Table 1). Overall, monocytes demonstrated higher concentrations of these drugs compared with PBMCs, a trend that was previously reported for tenofovir diphosphate and emtricitabine triphosphate in monocytes and further attributed to the larger volume of these cells compared with B and T lymphocytes primarily found in PBMCs.^33,40 The ARV concentrations were below the limit of quantification in the MDMs; as such, we did not present these results in Table 1. We suspect that differentiation of monocytes to MDMs for 6 days in vitro could have potentially contributed to degradation of intracellular ARVs in these cells.

TABLE 1.

ARV Concentrations in Plasma, PBMCs, and Monocytes of HIV+ART Donors

Patient ID	Drug	Plasma Concentration, ng/mL*	Intracellular Concentration, fmol/106 Cells†
		Plasma	PBMC	Monocyte
HIV+ART3	Tenofovir-DF	167	8.5	22.8
	Emtricitabine	916.9	457.7	1111
HIV+ART4	Tenofovir-DF	73.13	26.87	83.45
	Emtricitabine	120.6	1256.5	2927.5
	Nelfinavir	NQ	NQ	NQ
HIV+ART#5	Abacavir	BLQ	BLQ	BLQ
	Lamivudine	311	496.9	826.5
	Delavirdine	NQ	NQ	NQ
HIV+ART6	Tenofovir-DF	68.85	24.655	49.085
	Emtricitabine	163.4	1919.5	3472.5
	Cobicistat	172.1	8.955	12.635
	Elvitegravir	745.4	4.734	BLQ
HIV+ART7	Tenofovir-DF	57.32	27.975	49.81
	Emtricitabine	73.46	2787	3588
	Efavirenz	923.1	BLQ	BLQ
HIV+ART8	Tenofovir-DF	41.94	30.88	60.1
	Rilpivirine	18.4	BLQ	BLQ
	Dolutegravir	1310	BLQ	BLQ
HIV+ART9	Tenofovir-AF	BLQ	BLQ	BLQ
	Emtricitabine	BLQ	BLQ	BLQ
HIV+ART10	Tenofovir-DF	33.53	28.67	BLQ
	Emtricitabine	36.34	787	BLQ
	Rilpivirine	8.54	BLQ	BLQ
HIV+ART11	Abacavir	1563	25.11	31.89
	Lamivudine	1169	4487	8315
	Dolutegravir	3640	BLQ	BLQ
HIV+ART12	Abacavir	BLQ	BLQ	BLQ
	Lamivudine	217	BLQ	BLQ
	Dolutegravir	BLQ	BLQ	BLQ

^*NRTIs were measured as parent compounds in plasma.

^†The phosphorylated metabolites of the NRTIs were quantified in PBMCs and monocytes.

BLQ, below limit of quantification; NQ, not quantified; Tenofovir-DF, tenofovir disoproxil fumarate; Tenofovir-AF, tenofovir alafenamide.

DISCUSSION

It is anticipated that several drug transporters and metabolic enzymes involved in ARV disposition and tissue distribution could regulate the intracellular concentrations of ARVs in viral reservoirs such as myeloid cells of HIV+ART individuals. Using several human and rodent models, our group and others have demonstrated a role of ABC transporters in limiting the permeability of ARVs in several cell types including T-lymphocytes, brain microglia, and astrocytes^41-46 and at blood–tissue barriers such as the blood–intestinal barrier, blood–testis barrier, and the blood–brain barrier.^8,47-50 Furthermore, using testicular tissue from HIV-negative and HIV-positive donors undergoing sex reassignment surgery, we previously demonstrated the expression of drug transporters and metabolic enzymes relevant to ART and their potential involvement in limiting ARV concentrations in the testis when compared with the drug plasma concentrations.⁷ In addition, we recently demonstrated expression or function of drug efflux transporters and metabolic enzymes in T-cells isolated from the testis and blood of HIV-negative donors.⁹ Limited data are available on the expression of drug transporters and metabolic enzymes relevant to ART in human monocytes and MDMs, and to the best of our knowledge, their expression in these cells obtained from HIV+ART individuals has not been previously documented.

In this work, qPCR analyses revealed mRNA expression of several ABC transporters known to participate in the efflux of ARVs in human monocytes and MDMs. We further examined the protein expression of P-gp, BCRP, and MRP1 in classical, intermediate, and nonclassical monocyte subsets and in MDMs obtained from HIV-negative and HIV+ART+ individuals. Overall, the flow cytometry analyses revealed that BCRP and MRP1 were least frequent in the classical monocyte subset, whereas P-gp showed higher frequency in this subset, compared with that in intermediate or nonclassical monocytes. In the context of HIV infection, P-gp and BCRP demonstrated consistent trends toward higher frequencies in the monocyte subsets of HIV− individuals compared with those in HIV+ART individuals; however, this did not reach statistical significance, most likely because of the large degree of interindividual variability. Our group previously demonstrated lower mRNA or protein expression of transporters such as P-gp, BCRP, and MRP2 in upper gastrointestinal and rectosigmoid colon tissue biopsies of ART-naïve HIV-positive individuals compared with tissue biopsies of HIV-negative individuals,^51,52 suggesting that HIV-1 could alter the expression of these transporters in HIV reservoirs. Furthermore, studies by our group have previously demonstrated in vitro in rat and human astrocytes that exposure to the proinflammatory cytokine IL-6 or HIV envelope glycoprotein, gp120, significantly decreased P-gp expression and function.^53,54 By contrast, other studies demonstrated an increase in P-gp and MRP1 mRNA expression in mouse astrocytes and mouse blood–brain barrier after exposure to HIV viral protein, Tat.^55,56 Overall, these studies suggest that HIV or isolated viral proteins could have differential effects on drug transporter expression.

SLC transport proteins primarily serve to mediate the uptake of several ARVs intracellularly. However, we were not able to confirm their protein expression in the monocytes and MDMs because of unavailability of suitable antibodies validated for flow cytometry. Drug metabolic enzymes including CYP3A4, CYP2B6, CYP2D6, and UGT1A1 are known to interact with many ARVs from all classes. Although we did not detect CYP3A4 mRNA expression in our samples, we detected mRNA expression of CYP2B6, CYP2D6, and UGT1A1 in both monocytes and MDMs. The mRNA and the corresponding protein expression of CYP3A4, CYP2D6, and CYP2B6 was previously demonstrated in the U937 macrophage cell line.²⁵ In addition, Baron et al⁵⁷ detected CYP2B6 and CYP3A4 mRNA expression in human blood monocytes and MDMs and demonstrated induction in the expression of CYP3A4 in cells exposed to phenobarbital. A previous study by Tochigi et al²⁶ reported mRNA expression and immunohistochemical staining of UGT1A1 in rat peritoneal MDMs. However, to our knowledge, this is the first report of UGT1A1 mRNA expression in human circulating monocytes and MDMs.

In our LC/MS/MS analyses, the plasma concentrations of ARVs in most donors were within the therapeutic ranges reported elsewhere. For example, dolutegravir (1310–3640 ng/mL) and elvitegravir (745 ng/mL) were readily detectable in the plasma and were within the range of therapeutic concentrations of 137–5091 ng/mL and 496–2660 ng/mL, respectively.^34,35 Interestingly, ARV concentrations were below the quantification limit in the MDMs, whereas we could detect these compounds in the matched plasma, PBMCs, and monocytes of HIV+ART individuals. Lower intracellular concentrations of NRTIs in MDMs exposed to HIV-1 and ARVs in vitro, and compared with other cells such as lymphocytes, have been previously reported.³⁹ In particular, the intracellular concentrations of the active phosphorylated metabolites of the NRTIs were 5- to 140-fold lower in MDMs compared with those in lymphocytes, independent of the cellular activation states.³⁹ Primary human MDMs were shown to have diminished deoxynucleoside kinase activities and reduced ability to phosphorylate NRTIs such as zidovudine when compared with those in T-lymphoblasts.⁵⁸ Furthermore, Jorajuria et al⁵⁹ demonstrated a significant increase in zidovudine and indinavir anti-HIV activity in the presence of P-gp or MRP inhibitors in vitro, suggesting the involvement of these transporters in regulating antiviral penetration in MDMs. Therefore, in addition to drug physicochemical and protein-binding properties, active transport by drug efflux transporters and impaired NRTI phosphorylation could contribute, partly, to the low or undetectable ARV concentrations in the HIV+ART MDMs of our study.

It is important to note that isolation of monocytes and differentiation processes of MDMs could most likely impact ARV concentrations in these cell types. In particular, MDMs were differentiated over 6 days, and the stability or degradation of the ARVs during this period was unknown. We anticipate that this process likely contributed to the undetectable drug concentrations in the MDMs of our donors. Macrophages are primarily found in tissues, requiring access to tissue biopsies to isolate them. However, it is very challenging to obtain human tissue biopsies, and most often, research groups obtain these cells through in vitro differentiation of cultured monocytes.^{23,27,39,58,59} To fully understand ARV penetration in macrophages in vivo, future studies are needed to investigate ARV concentrations in macrophages isolated directly from the tissues of individuals living with HIV on ART.

In summary, we present novel data demonstrating the expression of several SLC and ABC transporters and drug metabolic enzymes in human circulating monocyte subsets and MDMs directly isolated from ART-treated individuals with HIV and respective noninfected controls. Furthermore, we were able to quantify ARV concentrations in matching plasma, PBMCs, and monocytes of these individuals. To the best of our knowledge, we are the first to report intracellular concentrations of drugs belonging to the integrase inhibitor and nonnucleotide reverse transcriptase inhibitor class of ARVs in monocytes obtained directly from individuals living with HIV. Drug transport proteins and metabolic enzymes could potentially limit intracellular concentrations of several ARVs in monocytes and macrophages and contribute to residual HIV-1 replication or maintenance of the HIV reservoir in these cell types. Our findings should prompt future investigations on ARV penetration and efficacy in myeloid cells, particularly in tissue-resident myeloid cells present in sanctuary sites such as the brain and testes, which are known to display low ARV penetration.^7,60,61