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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 LCMS/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 HIVnegative 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. [ABSTRACT FROM AUTHOR] more...

HIV reservoirs persist in gut-homing CD4+ T cells of people living with HIV and receiving antiretroviral therapy, but the antigenic specificity of such reservoirs remains poorly documented. The imprinting for gut homing is mediated by retinoic acid (RA), a vitamin A-derived metabolite produced by dendritic cells (DCs) exhibiting RA-synthesizing (RALDH) activity. RALDH activity in DCs can be induced by TLR2 ligands, such as bacterial peptidoglycans and fungal zymosan. Thus, we hypothesized that bacterial/fungal pathogens triggering RALDH activity in DCs fuel HIV reservoir establishment/outgrowth in pathogenreactive CD4+ T cells. Our results demonstrate that DCs derived from intermediate/nonclassical CD16+ compared with classical CD162 monocytes exhibited superior RALDH activity and higher capacity to transmit HIV infection to autologous Staphylococcus aureus-reactive T cells. Exposure of total monocyte-derived DCs (MDDCs) to S. aureus lysates as well as TLR2 (zymosan and heat-killed preparation of Listeria monocytogenes) and TLR4 (LPS) agonists but not CMV lysates resulted in a robust upregulation of RALDH activity. MDDCs loaded with S. aureus or zymosan induced the proliferation of T cells with a CCR5+integrin b7+CCR6+ phenotype and efficiently transmitted HIV infection to these T cells via RALDH/RA-dependent mechanisms. Finally, S. aureus- and zymosan-reactive CD4+ T cells of antiretroviral therapy-treated people living with HIV carried replication-competent integrated HIV-DNA, as demonstrated by an MDDC-based viral outgrowth assay. Together, these results support a model in which bacterial/fungal pathogens in the gut promote RALDH activity in MDDCs, especially in CD16+ MDDCs, and subsequently imprint CD4+ T cells with gut-homing potential and HIV permissiveness. Thus, nonviral pathogens play key roles in fueling HIV reservoir establishment/outgrowth via RALDH/RA-dependent mechanisms that may be therapeutically targeted. [ABSTRACT FROM AUTHOR] more...

Background: Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. Methods: As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and had a comparable amount of sleep, physical exercise and light exposure. Plasma levels of bacterial lipopolysaccharide (LPS) and fungal (1→3)-β-D-Glucan (BDG) translocation markers, along with markers of intestinal damage fatty acid binding protein (I-FABP) and regenerating islet-derived protein-3α (REG3α) were assessed by ELISA or the fungitell assay. Results: Participants had a median age of 57 years old (range 50 to 63). Plasma levels of BDG and REG3α did not vary significantly over the course of the study. In contrast, a significant increase of LPS was detected between 12:00 and 16:00 (Z-score: − 1.15 ± 0.18 vs 0.16 ± 0.15, p = 0.02), and between 12:00 and 24:00 (− 1.15 ± 0.18 vs 0.89 ± 0.26, p < 0.001). The plasma levels of I-FABP at 16:00 (− 0.92 ± 0.09) were also significantly lower, compared to 8:00 the first day (0.48 ± 0.26, p = 0.002), 4:00 (0.73 ± 0.27, p < 0.001) or 8:00 on secondary day (0.88 ± 0.27, p < 0.001). Conclusions: Conversely to the fungal translocation marker BDG and the gut damage marker REG3α, time of blood collection matters for the proper evaluation for LPS and I-FABP as markers for the risk of inflammatory non-AIDS co-morbidities. These insights are instrumental for orienting clinical investigations in PLWH. [ABSTRACT FROM AUTHOR] more...