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.

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.

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.