Interaction between dolutegravir and folate transporters and receptor in human and rodent placenta.

BACKGROUND: Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta. METHODS: Human placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays. FINDINGS: In placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions. INTERPRETATION: These findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy. FUNDING: Funded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.

Impact of in-utero antiretroviral drug exposure on expression of membrane-associated transporters in mouse placenta and fetal brain.

OBJECTIVE: Although antiretroviral therapy (ART) during pregnancy is effective in limiting vertical HIV transmission, adverse outcomes persist amongst uninfected children exposed to antiretroviral drugs in utero. Membrane-associated drug transporters, metabolic enzymes, and tight junction proteins play important roles in adult antiretroviral drug disposition and toxicity; however, the fetal expression of these proteins in the context of ART, and their impact on in-utero antiretroviral drug distribution remain poorly understood. This study aimed to characterize the role of these proteins in modulating in-utero antiretroviral drug exposure. METHODS: Pregnant mice were exposed to an ART regimen consisting of lamivudine, abacavir, atazanavir, and ritonavir, at clinically relevant doses. Fetal brain, liver, placenta amniotic fluid, and maternal plasma were collected on gestational day 18.5 and concentration of antiretroviral drugs in fetal tissues was measured by LC/MS/MS, whereas transporter expression was assessed by qPCR. RESULTS: Abacavir and lamivudine were detected in fetal brain and amniotic fluid, whereas atazanavir and ritonavir were detected in amniotic fluid only. Robust mRNA expression of key transporters was observed in adult and fetal tissues, and sex differences were identified in the expression of Abcc1 and Slc29a1 in the placenta. Antiretroviral drug exposure was associated with a reduction in relative placental Abcg2, Abcc1, and Slc29a1 expression. CONCLUSION: These findings identify a novel effect of fetal sex and antiretroviral drug treatment on the expression of placental transporters in a mouse model, and characterize the penetration of lamivudine and abacavir into fetal brain, uncovering a potential role of transporters in modulating fetal exposure to antiretroviral drugs.

Differential effects of antiretroviral drug toxicity in male versus female children who are HIV-exposed but uninfected.

: In recent years, widespread use of antiretroviral therapy (ART) during pregnancy has been increasingly effective in reducing risk of vertical transmission of HIV, with over 80% of pregnant women living with HIV now accessing ART, and a 41% reduction in new infections in children between 2010 and 2018. Despite these strides, the developmental toxicity of widely administered antiretroviral drugs (ARVs) remains poorly described and existing literature often fails to account for fetal and infant sex as a variable. Recent reports have identified associations between in-utero exposure to commonly used antiretroviral regimens and alteration in neurodevelopment, growth, and metabolism amongst children who are HIV-exposed but uninfected, with findings of sex differences in the prevalence and severity of ARV toxicity. These differences are potentially explained by variable exposure to ARV drugs in utero or exacerbation of existing sex-linked risk factors. Fetal ARV exposure is mediated by placental and fetal drug transporters and metabolic enzymes, which may contribute to the manifestation of sex differences. Existing evidence of sex differences in ARV toxicity in fetal development is concerning, and demands further research to guide optimal treatment options for maternal health and prevention of vertical HIV transmission.

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.

A licensing step links AID to transcription elongation for mutagenesis in B cells.

Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.

Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor.

Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread in macrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In line with these findings, HIV-1-resistant intestinal myeloid cells express higher levels of miR-221 than peripheral blood monocytes. Thus, miR-221/miR-222 act as effectors of the antiviral host response activated during macrophage infection that restrict HIV-1 entry.

Regulation of CD4 Receptor and HIV-1 Entry by MicroRNAs-221 and -222 during Differentiation of THP-1 Cells.

Human immunodeficiency virus type-1 (HIV-1) infection of monocyte/macrophages is modulated by the levels of entry receptors cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5), as well as by host antiviral restriction factors, which mediate several post-entry blocks. We recently identified two microRNAs, miR-221 and miR-222, which limit HIV-1 entry during infection of monocyte-derived macrophages (MDMs) by down-regulating CD4 expression. Interestingly, CD4 is also down-regulated during the differentiation of monocytes into macrophages. In this study, we compared microRNA expression profiles in primary monocytes and macrophages by RNAseq and found that miR-221/miR-222 are enhanced in macrophages. We took advantage of the monocytic THP-1 cell line that, once differentiated, is poorly susceptible to HIV-1. Accordingly, we found that CD4 levels are very low in THP-1 differentiated cells and that this down-regulation of the virus receptor is the result of miR-221/miR-222 up-regulation during differentiation. We thus established a THP-1 cell line stably expressing a modified CD4 (THP-1-CD4R) that is not modulated by miR-221/miR-222. We show that in contrast to parental THP-1, this line is productively infected by HIV-1 following differentiation, sustaining efficient HIV-1 CD4-dependent replication and spread. This new THP-1-CD4R cell line represents a useful tool for the study of HIV-1-macrophage interactions particularly in contexts where spreading of viral infection is necessary.