Regulation of ABC Drug Efflux Transporters in Human T-Cells Exposed to an HIV Pseudotype.

ATP-binding cassette (ABC) drug efflux transporters could contribute to low intracellular concentrations of antiretroviral drugs in HIV-1 cell reservoirs and sanctuary sites. Furthermore, the functional expression of these transporters could be induced in activated T-cells. Therefore, we investigated the expression of ABC drug efflux transporters in human T-cells exposed to an HIV pseudotype virus (pHIVNL4-3), and further examined the potential involvement of the mammalian target of rapamycin (mTOR) signaling pathway in regulating their expression following exposure to pHIVNL4-3. Additionally, we investigated the contribution of the drug efflux transporters to the inflammatory response following pHIVNL4-3-induced T-cell activation. Human peripheral blood mononuclear cells (PBMCs) were exposed to HIV-1 envelope glycoprotein gp120IIIB, pHIVNL4-3 and/or mTOR inhibitors. The expression of ABC transporters, T-cell activation marker CD69, mTOR and pHIVNL4-3 was assessed in CD4+ T-cells by Flow cytometry. mRNA and protein levels of proinflammatory cytokines (IL6, TNFα and INFγ) were examined in PBMCs by qPCR and ELISA analyses, respectively, following exposure to pHIVNL4-3 with or without inhibitors of mTOR or ABC transporters. The expression of ABC transporters (P-glycoprotein, breast cancer resistance protein and multi-drug resistance associated protein-1) was significantly increased in CD4+ T-cells exposed to pHIVNL4-3. Treatment with mTOR inhibitors attenuated pHIVNL4-3-induced transporter expression, as well as mRNA and protein levels of IL6, TNFα and INFγ. Additionally, inhibition of P-gp or MRP1 activity resulted in lower concentrations of proinflammatory cytokines in supernatants of PBMC exposed to pHIVNL4-3. Herein we present novel data demonstrating that upregulation of ABC drug efflux transporters could involve the mTOR signaling pathway in CD4+ T-cells exposed to an HIV pseudotype. These transporters could limit antiretroviral drug penetration in HIV target T-cells. Furthermore, ABC transporters could potentially contribute to HIV-associated proinflammatory cytokine secretion.

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.

HIV-1 Sanctuary Sites-the Role of Membrane-Associated Drug Transporters and Drug Metabolic Enzymes.

Despite significant advances in the treatment of human immunodeficiency virus-1 (HIV) infection with highly active antiretroviral drug therapy, the persistence of the virus in cellular and anatomic reservoirs is a major obstacle preventing total HIV eradication. Viral persistence could result from a variety of contributing factors including, but not limited to, non-adherence to treatment and adverse drug reactions, latently infected cells carrying replication-competent virus, drug-drug interactions, and inadequate antiretroviral drug (ARV) concentrations reached in several anatomic sites such as the brain, testis, and gut-associated lymphoid tissues. The distribution of ARVs at specific sites of infection is primarily dependent on drug physicochemical properties and drug plasma protein binding, as well as drug efflux, influx, and metabolic processes. A thorough understanding of the functional roles of drug transporters and metabolic enzymes in the disposition of ARVs in immune cell types and tissues that are characterized as HIV reservoirs and sanctuaries is critical to overcome the challenge of suboptimal drug distribution at sites of persistent HIV infection. This review summarizes the current knowledge related to the expression and function of drug transporters and metabolic enzymes in HIV cellular and anatomic reservoirs, and their potential contribution to drug-drug interactions and insufficient drug concentration at these sites.

Drug efflux transporters and metabolic enzymes in human circulating and testicular T-cell subsets: relevance to HIV pharmacotherapy.

OBJECTIVES: ATP-binding cassette (ABC) drug efflux transporters and drug metabolic enzymes could reduce antiretroviral concentrations in HIV target cells. The testis has been demonstrated to be a sanctuary site, displaying suboptimal antiretroviral concentrations and persistent HIV infection. Therefore, we compared the expression and function of ABC transporters and metabolic enzymes in CD4 and CD8 T cells isolated from human testis and peripheral blood mononuclear cells (PBMCs), and assessed their expression in circulating naive and memory CD4 T-cell phenotypes. DESIGN: Testicular tissue and blood were collected from 15 uninfected donors undergoing gender affirmation surgery. Testicular interstitial cells were isolated by enzymatic digestion, whereas PBMCs were isolated from blood by density gradient centrifugation. The expression and/or function of ABC transporters and metabolic enzymes were examined in blood and testicular T-cell subsets by flow cytometry. RESULTS: ABC transporters (P-gp, BCRP, MRP1) and metabolic enzymes (CYP3A4, UGT1A1) were expressed in testicular and circulating CD4 and CD8 T cells, as well as in circulating naive, central, transitional, and effector memory T-cell phenotypes. MRP1 demonstrated lower frequencies in T cells from testis compared with PBMCs, as well as in circulating naive T cells compared with the memory T-cell phenotypes. Functional activity of P-gp and BCRP was detected in T-cell subsets from testis and PBMCs. CONCLUSION: Our findings demonstrate for the first time that antiretroviral drug efflux transporters and metabolic enzymes are functionally expressed in T-cell subsets infiltrating the human testis. These transporters and enzymes can reduce antiretroviral intracellular concentrations, potentially contributing to residual HIV replication in the testis, and negatively impact HIV cure strategies.

Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier.

Poor antiretroviral drug (ARV) penetration in the testes could be due, in part, to the presence of ATP-binding cassette (ABC) membrane-associated drug efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) expressed at the blood-testis barrier (BTB). The functional expression of these transporters is known to be regulated by ligand-activated nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in various tissues. This study aimed to investigate in vitro and ex vivo the role of PXR and CAR in the regulation of ABC transporters at the BTB. Both PXR and CAR proteins were expressed in human testicular tissue and in mouse TM4 Sertoli cells (an in vitro cell line model of the BTB). In addition, we demonstrated an upregulation of P-gp, Bcrp, and Mrp4 mRNA and protein expression, after exposure to PXR or CAR ligands in TM4 cells. Small interfering RNA downregulation of PXR or CAR attenuated the expression of these transporters, suggesting the direct involvement of these nuclear receptors in regulating P-gp, Bcrp, and Mrp4 in this system. In an ex vivo study using freshly isolated mouse seminiferous tubules, we found that exposure to PXR or CAR ligands, including ARVs, significantly increased P-gp expression and function. Together, our data suggest that ABC transporters could be regulated at the BTB during chronic treatment with ARVs that can serve as ligands for PXR and CAR, which could in turn further limit testicular ARV concentrations.

Role and modulation of drug transporters in HIV-1 therapy.

Current treatment of human immunodeficiency virus type-1 (HIV-1) infection involves a combination of antiretroviral drugs (ARVs) that target different stages of the HIV-1 life cycle. This strategy is commonly referred to as highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART). Membrane-associated drug transporters expressed ubiquitously in mammalian systems play a crucial role in modulating ARV disposition during HIV-1 infection. Members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter superfamilies have been shown to interact with ARVs, including those that are used as part of first-line treatment regimens. As a result, the functional expression of drug transporters can influence the distribution of ARVs at specific sites of infection. In addition, pathological factors related to HIV-1 infection and/or ARV therapy itself can alter transporter expression and activity, thus further contributing to changes in ARV disposition and the effectiveness of HAART. This review summarizes current knowledge on the role of drug transporters in regulating ARV transport in the context of HIV-1 infection.