Genetic haute couture to block HIV-1 at front doors.

The chemokine receptors CCR5 and CXCR4 are "front doors" for HIV-1 infection in host cells, and their targeting represents a potential solution for a cure. Dudek et al.1 now propose a new gene editing strategy to simultaneously block CCR5- and CXCR4-mediated HIV-1 entry in autologous hematopoietic stem and progenitor cells (HSPCs).

Metformin Enhances Antibody-Mediated Recognition of HIV-Infected CD4+ T-Cells by Decreasing Viral Release.

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T-cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T-cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T-cells by HIV-1 Envelope antibodies. Thus, metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Alterations in Th17 Cells and Non-Classical Monocytes as a Signature of Subclinical Coronary Artery Atherosclerosis during ART-Treated HIV-1 Infection.

Cardiovascular disease (CVD) remains an important comorbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed in the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) > 5%) revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by computed tomography angiography scan (CTAScan) as the total (TPV) and low attenuated plaque volume (LAPV), in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4+ T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1, and triglyceride levels; lower Th17/Treg ratios; and classical monocyte expansion. Among HIV+, TPV+ versus TPV- exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9lowHLADRhigh monocytes, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9lowHLADRhigh monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.

IL-32 Drives the Differentiation of Cardiotropic CD4+ T Cells Carrying HIV DNA in People With HIV.

Interleukin 32 (IL-32) is a potent multi-isoform proinflammatory cytokine, which is upregulated in people with HIV (PWH) and is associated with cardiovascular disease (CVD) risk. However, the impact of IL-32 isoforms on CD4 T-cell cardiotropism, a mechanism potentially contributing to heart inflammation, remains unknown. Here we show that IL-32 isoforms ß and γ induce the generation of CCR4+CXCR3+ double positive (DP) memory CD4 T-cell subpopulation expressing the tyrosine kinase receptor c-Met, a phenotype associated with heart-homing of T cells. Our ex vivo studies on PWH show that the frequency of DP CD4 T cells is significantly higher in individuals with, compared to individuals without, subclinical atherosclerosis and that DP cells from antiretroviral-naive and treated individuals are highly enriched with HIV DNA. Together, these data demonstrate that IL-32 isoforms have the potential to induce heart-homing of HIV-infected CD4 T cells, which may further aggravate heart inflammation and CVD in PWH.

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people.

IMPORTANCE: Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

Near full-length HIV sequencing in multiple tissues collected postmortem reveals shared clonal expansions across distinct reservoirs during ART.

HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

Early ART reduces viral seeding and innate immunity in liver and lungs of SIV-infected macaques.

Identifying immune cells and anatomical tissues that contribute to the establishment of viral reservoirs is of central importance in HIV-1 cure research. Herein, we used rhesus macaques (RMs) infected with SIVmac251 to analyze viral seeding in the liver and lungs of either untreated or early antiretroviral therapy-treated (ART-treated) RMs. Consistent with viral replication and sensing, transcriptomic analyses showed higher levels of inflammation, pyroptosis, and chemokine genes as well as of interferon-stimulating gene (ISG) transcripts, in the absence of ART. Our results highlighted the infiltration of monocyte-derived macrophages (HLA-DR+CD11b+CD14+CD16+) in inflamed liver and lung tissues associated with the expression of CD183 and CX3CR1 but also with markers of tissue-resident macrophages (CD206+ and LYVE+). Sorting of myeloid cell subsets demonstrated that CD14+CD206-, CD14+CD206+, and CD14-CD206+ cell populations were infected, in the liver and lungs, in SIVmac251-infected RMs. Of importance, early ART drastically reduced viral seeding consistent with the absence of ISG detection but also of genes related to inflammation and tissue damage. Viral DNA was only detected in CD206+HLA-DR+CD11b+ cells in ART-treated RMs. The observation of pulmonary and hepatic viral rebound after ART interruption reinforces the importance of early ART implementation to limit viral seeding and inflammatory reactions.

Identification of aryl hydrocarbon receptor as a barrier to HIV-1 infection and outgrowth in CD4+ T cells.

The aryl hydrocarbon receptor (AhR) regulates Th17-polarized CD4+ T cell functions, but its role in HIV-1 replication/outgrowth remains unknown. Genetic (CRISPR-Cas9) and pharmacological inhibition reveal AhR as a barrier to HIV-1 replication in T cell receptor (TCR)-activated CD4+ T cells in vitro. In single-round vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection, AhR blockade increases the efficacy of early/late reverse transcription and subsequently facilitated integration/translation. Moreover, AhR blockade boosts viral outgrowth in CD4+ T cells of people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Finally, RNA sequencing reveals genes/pathways downregulated by AhR blockade in CD4+ T cells of ART-treated PLWH, including HIV-1 interactors and gut-homing molecules with AhR-responsive elements in their promoters. Among them, HIC1, a repressor of Tat-mediated HIV-1 transcription and a tissue-residency master regulator, is identified by chromatin immunoprecipitation as a direct AhR target. Thus, AhR governs a T cell transcriptional program controlling viral replication/outgrowth and tissue residency/recirculation, supporting the use of AhR inhibitors in "shock and kill" HIV-1 remission/cure strategies.

Differential Impact of IL-32 Isoforms on the Functions of Coronary Artery Endothelial Cells: A Potential Link with Arterial Stiffness and Atherosclerosis.

Chronic inflammation is associated with higher risk of cardiovascular disease (CVD) in people living with HIV (PLWH). We have previously shown that interleukin-32 (IL-32), a multi-isoform proinflammatory cytokine, is chronically upregulated in PLWH and is linked with CVD. However, the mechanistic roles of the different IL-32 isoforms in CVD are yet to be identified. In this study, we aimed to investigate the potential impact of IL-32 isoforms on coronary artery endothelial cells (CAEC), whose dysfunction represents a major factor for atherosclerosis. Our results demonstrated that the predominantly expressed IL-32 isoforms (IL-32ß and IL-32γ) have a selective impact on the production of the proinflammatory cytokine IL-6 by CAEC. Furthermore, these two isoforms induced endothelial cell dysfunction by upregulating the expression of the adhesion molecules ICAM-I and VCAM-I and the chemoattractants CCL-2, CXCL-8 and CXCL-1. IL-32-mediated expression of these chemokines was sufficient to drive monocyte transmigration in vitro. Finally, we demonstrate that IL-32 expression in both PLWH and controls correlates with the carotid artery stiffness, measured by the cumulated lateral translation. These results suggest a role for IL-32-mediated endothelial cell dysfunction in dysregulation of the blood vessel wall and that IL-32 may represent a therapeutic target to prevent CVD in PLWH.

Immuno-metabolic control of the balance between Th17-polarized and regulatory T-cells during HIV infection.

Th17-polarized CD4+ effector T-cells together with their immunosuppressive regulatory T-cell (Treg) counterparts, with transcriptional profiles governed by the lineage transcription factors RORγt/RORC2 and FOXP3, respectively, are important gatekeepers at mucosal interfaces. Alterations in the Th17/Treg ratios, due to the rapid depletion of Th17 cells and increased Treg frequencies, are a hallmark of both HIV and SIV infections and a marker of disease progression. The shift in Th17/Treg balance, in favor of increased Treg frequencies, contributes to gut mucosal permeability, immune dysfunction, and microbial translocation, subsequently leading to chronic immune activation/inflammation and disease progression. Of particular interest, Th17 cells and Tregs share developmental routes, with changes in the Th17 versus Treg fate decision influencing the pro-inflammatory versus anti-inflammatory responses. The differentiation and function of Th17 cells and Tregs rely on independent yet complementary metabolic pathways. Several pathways have been described in the literature to be involved in Th17 versus Treg polarization, including 1) the activity of ectonucleotidases CD39/CD73; 2) the increase in TGF-ß1 production; 3) a hypoxic environment, and subsequent upregulation in hypoxia-inducible factor-1α (HIF-1α); 4) the increased mTOR activity and glycolysis induction; 5) the lipid metabolism, including fatty acid synthesis, fatty acids oxidation, cholesterol synthesis, and lipid storage, which are regulated by the AMPK, mevalonate and PPARγ pathways; and 6) the tryptophan catabolism. These metabolic pathways are understudied in the context of HIV-1 infection. The purpose of this review is to summarize the current knowledge on metabolic pathways that are dysregulated during HIV-1 infection and their impact on Th17/Treg balance.

A Blood Immunological Signature of Subclinical Coronary Artery Atherosclerosis in People Living with HIV-1 Receiving Antiretroviral Therapy.

Cardiovascular disease (CVD) remains an important co-morbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed on the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) >5%), revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by Computed tomography angiography scan (CTAScan) as total (TPV) and low attenuated plaque volume (LAPV) in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4 + T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells, were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1 and triglyceride levels, lower Th17/Treg ratios, and classical monocyte expansion. Among HIV + , TPV + versus TPV - exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9 low HLADR high monocyte, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9 low HLADR high monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.

Targeting Th17 cells in HIV-1 remission/cure interventions.

Since the discovery of HIV-1, progress has been made in deciphering the viral replication cycle and mechanisms of host-pathogen interactions that has facilitated the implementation of effective antiretroviral therapies (ARTs). Major barriers to HIV-1 remission/cure include the persistence of viral reservoirs (VRs) in long-lived CD4+ T cells, residual viral transcription, and lack of mucosal immunity restoration during ART, which together fuel systemic inflammation. Recently, T helper (Th)17-polarized cells were identified as major contributors to the pool of transcriptionally/translationally competent VRs. In this review, we discuss the functional features of Th17 cells that were elucidated by fundamental immunology studies in the context of autoimmunity. We also highlight recent discoveries supporting the possibility of extrapolating this knowledge toward the identification of new putative Th17-targeted HIV-1 remission/cure strategies.

Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification.

Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4+ T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4+ T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6+CD4+ T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6+CD4+ T cells from peripheral tissues, such as the colon.

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

IL-17A reprograms intestinal epithelial cells to facilitate HIV-1 replication and outgrowth in CD4+ T cells.

The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4+ T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4+ T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH.

A Tale of Two Viruses: Immunological Insights Into HCV/HIV Coinfection.

Nearly 2.3 million individuals worldwide are coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Odds of HCV infection are six times higher in people living with HIV (PLWH) compared to their HIV-negative counterparts, with the highest prevalence among people who inject drugs (PWID) and men who have sex with men (MSM). HIV coinfection has a detrimental impact on the natural history of HCV, including higher rates of HCV persistence following acute infection, higher viral loads, and accelerated progression of liver fibrosis and development of end-stage liver disease compared to HCV monoinfection. Similarly, it has been reported that HCV coinfection impacts HIV disease progression in PLWH receiving anti-retroviral therapies (ART) where HCV coinfection negatively affects the homeostasis of CD4+ T cell counts and facilitates HIV replication and viral reservoir persistence. While ART does not cure HIV, direct acting antivirals (DAA) can now achieve HCV cure in nearly 95% of coinfected individuals. However, little is known about how HCV cure and the subsequent resolution of liver inflammation influence systemic immune activation, immune reconstitution and the latent HIV reservoir. In this review, we will summarize the current knowledge regarding the pathogenesis of HIV/HCV coinfection, the effects of HCV coinfection on HIV disease progression in the context of ART, the impact of HIV on HCV-associated liver morbidity, and the consequences of DAA-mediated HCV cure on immune reconstitution and HIV reservoir persistence in coinfected patients.

Overt IL-32 isoform expression at intestinal level during HIV-1 infection is negatively regulated by IL-17A.

OBJECTIVES: Untreated HIV infection was previously associated with IL-32 overexpression in gut/intestinal epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people with HIV (PWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN: Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PWH (HIV + ART; n = 17; mean age: 56 years; CD4+ cell counts: 679 cells/µl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. METHODS: Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A and HIV. IL-32α/ß/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS: IL-32ß/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32ß mRNA levels being predominantly expressed in both compartments and at significantly higher levels in HIV + ART compared to HIVneg. IL-17A transcripts were only detectable in SCB, with increased IL-17A levels in HIVneg compared with HIV + ART and negatively correlated with IL-32ß mRNA levels. IL-32ß/γ/ε isoform mRNA were detected in HT-29 cells upon exposure to TNF-α, Poly I:C (TLR3 agonist), Flagellin (TLR-5 agonist) and HIV. IL-17A significantly decreased both IL-32 ß/γ/ε mRNA and cell-associated IL-32 protein levels induced upon TNF-α and Poly I:C triggering. CONCLUSION: We document IL-32 isoforms abundant in the colon of ART-treated PWH and reveal the capacity of the Th17 hallmark cytokine IL-17A to attenuate IL-32 overexpression in a model of inflamed IEC.

Loss of CD96 Expression as a Marker of HIV-Specific CD8+ T-Cell Differentiation and Dysfunction.

Persistent immune activation and inflammation in people living with HIV (PLWH) are associated with immunosenescence, premature aging and increased risk of non-AIDS comorbidities, with the underlying mechanisms not fully understood. In this study, we show that downregulation of the T-cell immunoglobulin receptor CD96 on CD8+ T cells from PLWH is associated with decreased expression of the co-stimulatory receptors CD27 and CD28, higher expression of the senescence marker CD57 and accumulation of a terminally differentiated T-cell memory phenotype. In addition, we show that CD96-low CD8+ T-cells display lower proliferative potential compared to their CD96-high counterparts and that loss of CD96 expression by HIV-specific CD8+ T-cells is associated with a suboptimal response to HIV antigens. In conclusion, our results suggest that CD96 marks CD8+ T-cells with competent responses to HIV and the loss of its expression might be used as a biomarker for CD8+ T-cell senescence and dysfunction in PLWH.

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.