Combination antiretroviral therapy (ART) effectively reduces the blood HIV viral load and dramatically decreases HIV-1-related mortality and morbidity. However, one of the major obstacles preventing the eradication of HIV-1 infection is the existence of cellular and anatomic reservoirs in which the virus can persist despite treatment, such as memory CD4+ T cells, myeloid cells, the brain, the male and female genital tracts and lymphoid tissues [1–3]. It has been proposed that ineffective viral suppression, the development of viral drug resistance and persistent infection in HIV-1 reservoirs could, in part, be attributed to low antiretroviral drug concentrations reached in tissues, such as lymph nodes, brain, and testes [4–8]. Antiretroviral distribution into tissues is primarily dependent on drug physicochemical properties (i.e. molecular weight, particle size, pKa, lipophilicity), drug serum protein binding, as well as a dynamic interplay between membrane drug uptake/efflux and cellular metabolic processes. Antiretroviral uptake into, or efflux out of, cells involves the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies of biological membrane-associated drug transport proteins, respectively [9,10]. Furthermore, many antiretrovirals are metabolized by cytochrome P450 (CYP450) phase I metabolic enzymes, while a few undergo phase II conjugation by uridine diphosphate-dependent glucuronosyltransferases (UGTs) [11–13]. Multiple drug interactions of antiretrovirals with drug transporters and metabolic enzymes during ART could contribute to subtherapeutic concentrations in cells and tissues known to be HIV-1 reservoirs.
Several HIV-1 cellular reservoirs have been documented, but CD4+ T cells are the main targets of HIV-1 infection and are the best characterized reservoirs to date . Moreover, the central, transitional and effector memory CD4+ T-cell subsets are proposed as the major cellular reservoirs for HIV-1 in virally suppressed individuals on ART [14–16]. It is anticipated that drug efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and the multidrug resistance-associated proteins (MRPs), as well as metabolic enzymes including CYP3A4, known to play an important role in antiretroviral distribution and metabolism, are functionally expressed in CD4+ T-cell subsets . Previous reports have identified ABC transporters P-gp, BCRP and MRP1-2 in human CD4+ and CD8+ T cells isolated from uninfected blood [17–20], and associated the expression of P-gp and MRP1-2 with low accumulation of HIV protease inhibitors lopinavir and saquinavir in these cells [17,19]. However, the expression of these transporters and metabolic enzymes in naive and various memory T-cell subsets is not well characterized. Moreover, their functional expression has not been examined in the T cells infiltrating the testis, an anatomic compartment where we previously demonstrated low concentrations of antiretrovirals known to interact with drug efflux transporters and drug metabolic enzymes .
Immune-privileged organ systems that are protected by blood--tissue barriers are ideal anatomical reservoirs for HIV-1 [21–23]. Such systems include the brain and the testis, which are relatively shielded from leukocyte trafficking . Studies by Jenabian et al. in 2016 demonstrated that the testis is a distinctive anatomical reservoir for HIV-1 persistence. Specifically, they demonstrated a higher number of effector-memory T cells in HIV-1 infected and uninfected testicular tissue, compared with matched peripheral blood mononuclear cells (PBMCs), as well as substantially higher CCR5 expression on testicular T cells. Higher CCR5 expression and T-cell immune activation (co-expression of CD38 and HLA-DR) in the testis of HIV-1-infected individuals suggest higher permissiveness for HIV-1 infection in testicular tissue . Furthermore, we previously demonstrated a potential role of drug efflux transporters and metabolic enzymes in limiting the tissue concentrations of several antiretrovirals in the testis of HIV-1-infected men on therapy when compared with their plasma concentrations . Therefore, we propose that the functional expression of these efflux transporters and metabolic enzymes could limit the permeability of antiretroviral substrates in T cells infiltrating the testis. Low antiretroviral penetration in these HIV target T cells could contribute to the establishment of viral reservoirs or residual viral replication in the testis. For this study, we obtained testicular tissue from uninfected men undergoing bilateral orchiectomy for gender reaffirmation. We assessed the expression of several ABC drug efflux transporters and metabolic enzymes in testicular T cells and matched PBMCs, as well as examined the function of efflux transporters known to interact with many antiretrovirals. In addition, we investigated the expression of ABC transporters and the metabolic enzyme CYP3A4 in naive CD4+ T cells, as well as central, transitional and effector memory CD4+ T cells isolated from PBMCs.
Materials and methods
Study donors and ethics statement
Testicular tissue and matched blood samples were obtained from 15 uninfected individuals undergoing elective orchiectomy in collaboration with Dr Jean-Pierre Routy at the McGill University Health Center, and Drs Pierre Brassard and Maud Bélanger from the Metropolitan Centre of Plastic Surgery in Montréal, Quebec, Canada. Donors were eligible for the study if they were males 18 years or older and willing to give informed consent undergoing bilateral orchiectomy for sex reassignment after having obtained legal agreement (Table 1). Six weeks prior to surgery, hormonal therapy was withheld to decrease risks of postoperative thrombotic complications. Donors were excluded if they had recent acute illness (less than 3 months); recent sexually transmitted infection; active cancer or uncontrolled coagulation disorder. This study obtained scientific and ethical approval from the research institute of McGill University Health Center, and participant written consent was obtained at the Centre Métropolitain de Chirurgie (Montreal, Quebec, Canada).
Peripheral blood mononuclear cells and testicular interstitial cell isolation
Testicular tissue and matched blood samples from the study donors were packaged with ice and shipped to our laboratory overnight. Following the protocol of Ponte et al., the tunica albuginea and major blood vessels were removed from the testis before mincing and incubation in 0.1 mg/ml liberase buffer (Liberase-DL; Sigma Aldrich, Oakville, Ontario, Canada). The testis tissue was rinsed in digestion buffer (25 ml RPMI media containing 500 μl Liberase-DL), minced and equally divided into 50 ml tubes. The tissue was incubated at 37 °C for 1.5 h while mixing by inversion every 5 min for the first 20 min. Enzymatic activity was stopped by adding 5 ml heat inactivated-FBS. FACS buffer (PBS + 2% FBS) was then added to each tube up to 50 ml followed by centrifugation for 1 min at 300 rpm to allow sedimentation of seminiferous tubules without pelleting immune cells. The supernatant containing interstitial cells was recuperated and filtered through a 70 μm cell strainer. The interstitial cells were washed twice by centrifugation at 1800 rpm for 15 min, and red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Cells were washed again prior to counting. PBMCs were isolated from matched blood samples by Ficoll-paque density gradient centrifugation. Additional PBMCs from HIV-uninfected donors were used for phenotypic analyses. Testicular interstitial cells or PBMCs were counted using trypan blue to evaluate cell viability before proceeding to transport assays or staining for flow cytometric analysis. We confirmed that tissue digestion did not affect the expression of the drug transporters and metabolic enzymes by evaluating their expression in PBMC samples treated with or without Liberase-DL.
PBMCs or testicular interstitial cells (2 × 106) were distributed into 96-well plates for staining with appropriate antibodies. After washing with FACs buffer, cells were stained extracellularly with fluorochrome-conjugated monoclonal antibodies to detect surface receptors (CD45, CD3, CD4, CD8, CD45RA, CCR7, CD27), in the presence of a viability dye. Cells were then fixed and permeabilized prior to intracellular staining of ABC transporters (P-gp, BCRP and MRP1) and drug metabolic enzymes (CYP3A4 and UGT1A1) using the True Nuclear Transcription Factor permeabilization kit (BioLegend, San Diego, California, USA) and manufacturer's protocol. Fluorescence minus one color (FMO) controls were used to discriminate autofluorescence from positive signals (Fig. S1–4, http://links.lww.com/QAD/B722, http://links.lww.com/QAD/B723, http://links.lww.com/QAD/B724, http://links.lww.com/QAD/B725, http://links.lww.com/QAD/B726). The binding specificity of the antibodies used to detect the ABC transporters was evaluated in wild-type cells compared with cells stably transfected to overexpress each transporter. For metabolic enzymes, antibodies were compared in cells that display high or very low expression of these proteins. Cells were acquired on a Beckman Coulter Cytoflex-S benchtop cytometer. Data analysis was performed using FlowJo software v10 (FlowJo LLC, Ashland, Oregon, USA) and GraphPad prism 7 (GrapdPad Software Inc., San Diego, California, USA). See Table S-1, http://links.lww.com/QAD/B720 for a list of viability dyes, antibodies and fluorochrome conjugates used in flow cytometric assessment.
Peripheral blood mononuclear cell phenotyping
For phenotypic analysis, the associated expression of CD3, CD4, CD45RA, CCR7, and CD27 were used to measure the frequency of naive, central memory, transitional memory and effector memory T cells as previously reported . Central memory T cells were distinguished by co-expression of CCR7 and CD27 (CD45RA-CCR7+CD27+), whereas transitional memory and effector memory T cells were negative for CCR7 receptor. CD27 further distinguished between the transitional memory (CD45RA-CCR7-CD27+) and effector memory (CD45RA-CCR7-CD27-) phenotypes. Naive T cells were identified as CD45RA+CCR7+CD27+. The expression of P-gp, BCRP, MRP1 and CYP3A4 was evaluated in these T-cell phenotypes.
Fluorescent substrate accumulation assays
To examine the functional activity of P-gp in PBMCs or testicular interstitial cells, 2 × 106 cells were incubated with 1 μmol/l rhodamine-123 (rho-123; Sigma Aldrich, Oakville, Ontario, Canada), known substrate of P-gp, or 1 μmol/l rho-123 in the presence of 5 μmol/l of the selective P-gp inhibitor valspodar (PSC833; Sigma Aldrich) for 30 min at 37 °C. Cells were washed with cold PBS then subsequently incubated with substrate-free medium in the presence or absence of PSC833 for 1 h at 37 °C. As rho-123 could potentially interact with MRP1 , 10 μmol/l of the MRP inhibitor MK571 (Sigma Aldrich) was added to the cells. To examine the function of BCRP, experiments were performed in similar conditions as described for P-gp, using 1 μmol/l of the BCRP fluorescent substrate mitoxantrone (Sigma Aldrich) and 5 μmol/l of the inhibitor fumitremorgin C (FTC; Sigma Aldrich). Mitoxantrone could also interact with P-gp  and as such 5 μmol/l PSC833 was added to the incubation media for the BCRP transport assays. After the last incubation, cells were washed with cold PBS and stained with antibodies in FACS buffer prior to flow cytometric analysis of T-cell subsets. The intracellular fluorescence of these substrates was determined using a 488 nm laser and 525/40 nm bandpass filter to detect rho-123 or a 638 nm laser and 660/20 bandpass filter to detect mitoxantrone. Drug efflux was determined by measuring the relative increase in median fluorescent intensity (MFI) of the substrates in the presence or absence of the inhibitor in CD4+ and CD8+ T-cell subsets. The percentage-change in intracellular fluorescence was then calculated for cells incubated with substrate only (control) compared with cells incubated with substrate plus inhibitor.
All experiments were performed in cells isolated from the blood or testes of at least three donors. The expression of drug efflux transporters and metabolic enzymes was compared between blood and testicular compartments using the nonparametric Wilcoxon ranked t-test for paired analyses. For the functional assays, comparison between groups was performed as applying the unpaired t-test. For PBMC phenotyping, comparisons between naive and memory phenotypes were performed using the repeated-measures one-way analysis of variance with the Bonferroni's correction for multiple comparison. Statistical analyses were performed using GraphPad Prism 7.03 software (GraphPad Software Inc.), P less than 0.05 was considered statistically significant.
ATP-binding cassette transporters and metabolic enzymes are expressed in human testicular and peripheral blood mononuclear cells T-cell subsets
The testicular interstitial cell suspension consisted of a variety of cell types that exhibited different levels of autofluorescence . To distinguish autofluorescence from antibody-specific signals, and to discriminate leucocytes from nonhematopoietic cells, interstitial cells were first gated on cells positive for the pan-leucocyte CD45 marker and then dead cells were excluded. To identify T-cell subsets, we next gated on cells that were positive for the T-cell marker CD3 and subsequently on CD4 or CD8. The same gating strategy was applied to the matched PBMCs (see gating strategy in Fig. S-5, http://links.lww.com/QAD/B727). It is important to note that the expression of CD4 on the T cells in the testis is reduced following treatment with Liberase-DL enzymes [27–29]. Therefore, testicular ‘CD4+’ T cells were gated as ‘CD3+CD8-’ T cells. Although CD8+ T cells are not primarily infected with HIV in vivo, they have been shown to harbor HIV proviral DNA and further spread the virus to CD4+ T cells following infection in vitro. Furthermore, the CD8+ subset is the most abundant T-cell subset in the testis . Therefore, for completeness, we included CD8+ T cells in our analyses. Among CD4+ T cells in the testes of six donors, the frequency of cells (expressed as mean ± SD), which expressed P-gp, BCRP and MRP1 was 96.1 ± 3.4, 87.9 ± 10.1 and 14.1 ± 17.3%, respectively. Their frequencies were not significantly different in CD4+ T cells gated from matched PBMCs (Fig. 1a--c). Among testicular CD8+ T cells, P-gp demonstrated 93.4 ± 6% frequency, whereas BCRP and MRP1 were expressed in 88.3 ± 8.9 and 11.4 ± 15% of this cell subset. MRP1 expression was significantly lower in testicular CD8+ T cells compared with circulating CD8+ T cells (Fig. 1d--f).
The frequency of CD4+ and CD8+ T cells expressing CYP3A4 and UGT1A1 demonstrated high inter-individual variability in both testis and PBMCs (Fig. 2). In the testis, CYP3A4 was expressed in 65.4 ± 43.5% of CD4+ and 67.1 ± 42.5% of CD8+ T cells, whereas the frequency of this metabolic enzyme in PBMCs was 51.4 ± 32.6 and 56.3 ± 33.8% in CD4+ and CD8+ T-cell subsets, respectively. UGT1A1 demonstrated 17 ± 13 and 16 ± 13.7% frequencies in testicular CD4+ and CD8+ T cells, respectively. In PBMCs, UGT1A1 demonstrated 27.8 ± 15.2 and 25 ± 13.2% frequencies in CD4+ and CD8+ T-cell subsets, respectively. Overall, a trend demonstrating lower frequencies of MRP1 and UGT1A1 or higher frequencies of CYP3A4 in testis compared with PBMCs could be observed; however, these differences were not statistically significant because of the high interindividual variability and low sample size. On the other hand, P-gp and BCRP consistently demonstrated high frequencies in CD4+ and CD8+ T cells isolated from both compartments. The MFI of each transporter and metabolic enzyme was also analyzed in the matched testicular and circulating T cells and the results demonstrated a trend toward higher CYP3A4 expression in testicular T cells (Fig. S-6, http://links.lww.com/QAD/B728).
P-glycoprotein and breast cancer resistance protein display functional activity in T-cell subsets isolated from testis and peripheral blood mononuclear cells
Transport assays were performed using testicular interstitial cells or PBMCs isolated from at least three separate donors. The CD4+ and CD8+ T-cell subsets were gated and identified as described in the previous section, then the fluorescence intensity of rho-123 or mitoxantrone was evaluated in these subsets. Inhibiting P-gp function with PSC833 resulted in modest but significant increases in the fluorescence intensity of rho-123 in testicular T cells (37% in CD4+ and 35% in CD8+) and PBMCs (20% in CD4+ and 25% in CD8+), indicating higher retention of the substrate in these cells (Fig. 3). Similarly, the BCRP substrate mitoxantrone was significantly increased in T-cell subsets isolated from PBMCs (50% in CD4+ and 46% in CD8+) and testes (31% in CD4+ and 27% in CD8+) in the presence of the BCRP inhibitor FTC. Overall, these results demonstrate the potential for P-gp and BCRP to alter the intracellular accumulation of their substrates in T-cell subsets, specifically in the human testis. We did not measure the functional activity of MRP1 as its expression in the testis was very low and we had a limited number of samples.
ATP-binding cassette transporters and CYP3A4 are expressed in CD4+-naive and memory T-cell phenotypes and demonstrate a trend towards lower frequencies in naive T cells
The proportion of CD4+-naive or central, transitional and effector memory CD4+ T-cell phenotypes were evaluated in PBMCs obtained from four individuals (Table S-2, http://links.lww.com/QAD/B721). The expression of P-gp, BCRP, MRP1 or CYP3A4 was then determined in these T-cell phenotypes (Fig. 4). Gating strategies are shown in Fig. S-7, http://links.lww.com/QAD/B729. P-gp displayed high and consistent expression with average frequencies above 85% in each T-cell phenotype. Although the expression of BCRP, MRP1 and CYP3A4 was more variable across individuals, these proteins demonstrated significantly lower frequencies in the naive T cells when compared with the memory subsets, a trend that was also observed when the expression levels (measured by MFI) of these proteins were analyzed (Fig. S-8, http://links.lww.com/QAD/B730).
The persistence of long-lived HIV-1-containing cell reservoirs despite ART is a major barrier to HIV-1 eradication. These cells harbor integrated copies of HIV-1 proviral DNA within their genome, remain transcriptionally silent and can evade recognition and clearance by the immune system and ART. Moreover, a low concentration of antiretrovirals within gut-derived mononuclear cells has been shown to correlate with ongoing low-level virus replication, sufficient to maintain a state of immune activation . In addition to drug physicochemical and plasma/tissue protein binding properties, drug transporters and metabolic enzymes present in T-cell subsets could contribute to low drug concentrations intracellularly. We previously characterized the functional expression and/or regulation of drug efflux transporters at the human and mouse blood--testis barrier [30–32], but to the best of our knowledge their expression and functional activity in testicular immune cells have not been examined.
In this study, we demonstrated protein expression of P-gp, BCRP, MRP1, CYP3A4 and UGT1A1 in CD4+ and CD8+ T cells isolated from PBMCs and testis using multicolor flow cytometry analyses. Furthermore, we demonstrated functional activity of P-gp and BCRP, two highly expressed ABC efflux transporters, in the T cells isolated from blood and testis compartments. We detected lower expression levels of CD4 in testis compared with PBMCs (Fig. S-5, http://links.lww.com/QAD/B727). This is most likely because of collagenase treatment, which has been shown to reduce CD4 expression in several tissues including the testis [27–29,33]. Although the study of Jenabian et al. confirmed the presence of CD4+ T-cell subsets in testicular interstitial T cells that were obtained by mechanically disrupting the tissue , we cannot rule out the possibility of CD3+CD4−CD8− (double negative) T cells, a previously identified HIV reservoir , in our testis analyses.
ABC transporters and metabolic enzymes are well known to be involved in the distribution of many antiretrovirals . Moreover, HIV or ART could alter the expression of these transporters. Specifically, our group demonstrated higher expression of these efflux transporters in intestinal and rectosigmoid colon biopsies obtained from HIV-infected, ART-treated individuals compared with therapy-naive patients [35,36]. The expression and/or function of P-gp, BCRP and MRP1 was previously demonstrated in peripheral blood CD4+ and CD8+ T cells isolated from uninfected donors [17,19,37]. Additionally, the expression of CYP3A4 was also reported in total peripheral blood lymphocytes of uninfected volunteers by flow cytometry, but not in the T-cell subsets . UGT1A1 expression was previously reported at the mRNA level in rat peripheral blood lymphocytes , and at the mRNA and protein levels in rat peritoneal macrophages ; however, the expression of this metabolic enzyme in human T-cell subsets has not been previously demonstrated. We were able to detect expression of UGT1A1 in both peripheral and testicular T-cell subsets by flow cytometry but its functional activity in these cells remains to be determined. To the best of our knowledge, the expression of these transporters and metabolic enzymes in T-cell subsets isolated from the testis has not been documented, these proteins could play an important role in limiting antiretroviral permeability in HIV-1 target cells present in this tissue.
Due to practical limitations in obtaining human testicular tissue, previous studies examining the testicular compartment in the context of ART penetration have utilized the simian immunodeficiency virus (SIV)-infected cynomolgus macaque model, which displays similar immunological disease progression characteristics to HIV-1 infection in humans. Matusali et al. measured concentrations of several antiretrovirals including raltegravir, lopinavir, tenofovir-diphosphate and emtricitabine-triphosphate in genital tract tissues of SIV-infected macaques, and showed that the concentrations of these drugs were consistently lower in the testis compared with the prostate and seminal vesicles of animals despite therapeutic drug levels in blood and seminal fluid. Furthermore, ART did not decrease SIV DNA levels in testis and other tissues, such as epididymis, seminal vesicles and urethra, suggesting the presence of nonproductively infected cells in these tissues . We previously reported that several antiretrovirals demonstrated lower concentrations in the testicular tissue of HIV-1-infected men compared with their plasma levels, with drugs such as darunavir displaying subtherapeutic levels in the testis . Suboptimal antiretroviral concentrations in testicular T cells could contribute to the formation of viral reservoirs in the testes [7,23]. Although Coombs et al. reported that genital tract tissues, such as the seminal vesicles and urethral glands are important sources of seminal HIV-1, an earlier study by Parjanpe et al., reported that HIV-1 in seminal cells originated from the rete testis and epididymis, after performing phylogenetic analyses on autoptic tissue. The study also demonstrated that T lymphocytes were the predominant cells displaying HIV-1 infection in the testes . Overall, the testis presents as a complex anatomic and pharmacologic tissue compartment, which could contribute to HIV-1 reservoirs and persistent HIV-1 infection.
To our knowledge, the expression of efflux transporters and metabolic enzymes in central, transitional and effector memory T-cell subsets has not previously been demonstrated. We also characterized the expression of the efflux transporters and CYP3A4 in CD4+-naive and memory T-cell phenotypes sorted from PBMCs. Phenotypic analysis of PBMCs revealed that P-gp, BCRP, MRP1 and CYP3A4 were expressed in the naive, central memory, transitional memory and effector memory CD4+ T cells. However, these proteins were significantly lower in the naive T cells compared with the memory T-cell phenotypes, except for P-gp, which was highly expressed in all T-cell subsets. This finding corroborates the work of Zhang et al., which demonstrated the expression of MRP1 in total CD45RO+ memory T cells but barely detectable levels in CD45RO-naive T cells . Efflux transporters including P-gp and MRP1 have been demonstrated to play a physiological role in the immune function of lymphocytes by participating in the secretion of cytokines and other inflammatory molecules [44–47]. Furthermore, their expression has also been shown to increase and correlate with T-cell activation [38,48,49], potentially resulting in higher expression in the memory T-cell phenotypes. The expression of these transporters and metabolic enzymes in these cell types, particularly in the central, transitional and effector memory T-cell phenotypes, suggest their potential roles in limiting antiretroviral penetration in memory T-cell reservoirs present in blood and at tissue sites.
Testicular tissues were obtained from individuals undergoing orchiectomies for sex reassignment and receiving hormonal therapy. It is well documented that nuclear receptors, such as the estrogen receptor and the progesterone receptor can be activated by hormones including estradiol and progesterone, respectively , which could potentially regulate the expression and activity of drug transporters and metabolic enzymes in different cell systems [51–54]. The direct effect of these hormones on drug transporters and metabolic enzymes in testicular T cells is unknown, however, as hormonal therapy was ceased at least 6 weeks prior to surgery, it is unlikely that the trends we observed in our study were influenced by these hormones. Furthermore, in a previous related study, Ponte et al. demonstrated that hormonal therapy did not affect the histology of the testicular interstitium between participants who were on hormonal therapy compared with controls .
Overall, our results demonstrated for the first time, the expression and function of key drug efflux transporters and metabolic enzymes in T cells isolated from the testis. The expression and function of these efflux transporters and metabolic enzymes was found similar in circulating and testicular T cells, however, our sample size was very small. Future experiments performed in a larger sample size, as well as in HIV-infected cells will be necessary to compare the functional activity of the transporters in T cells isolated from PBMCs and testes and examine their roles in modulating intracellular antiretroviral concentrations. In addition, other pharmacokinetic properties, such as the tissue protein binding and unbound tissue concentrations of these drugs in the testis should be investigated as they are also known to regulate drug tissue and intracellular concentrations .
In conclusion, ABC drug efflux transporters and metabolic enzymes, which have known interactions with many antiretrovirals are highly expressed in T cells. Further studies are needed to elucidate their regulation and function during T-cell activation and differentiation to memory phenotypes, particularly in the context of HIV infection. The functional expression of efflux transporters and metabolic enzymes could alter the intracellular accumulation and subsequently reduce the therapeutic efficacy of many antiretroviral substrates in T cells infiltrating the testis and other tissue compartments. Moreover, HIV RNA and DNA has been detected in the testis of individuals on ART [23,43,56]. It is clear that the testes have the potential to harbor HIV reservoirs, and are capable of restricting drug permeability; however, more studies are needed to fully elucidate the contribution of this tissue to HIV persistence. Understanding drug distribution in the testis, both at the blood--testis barrier and in the interstitial leukocytes, will be important when developing strategies to eradicate HIV.
We would like to thank Dr Pierre Brassard and Dr Maud Bélanger from the Metropolitan Centre of Plastic Surgery in Montréal, Canada, as well as the individuals from the Orchid study group who donated their tissues. We are grateful to Angie Massicotte for coordinating all the tissue collection and shipment, and to Dr Rosalie Ponte and Dr Franck Dupuy for providing expertise and guidance with the methods on testicular tissue processing and T-cell isolation and analysis by flow cytometry. We also thank Mr. Olanrewaju Kayode for his assistance with cell isolation from tissues. Finally, we acknowledge the Centre of Pharmaceutical Oncology at the Leslie Dan Faculty of Pharmacy for the use of the flow cytometry equipment.
Funding: This work was supported by the University of Toronto, Leslie Dan Faculty of Pharmacy Internal Grant 923465 (to R.B.) and by the Canadian Institutes of Health Research Catalyst Operating Grant 296635 and Canadian HIV Cure Enterprise Team Grant HIG-133050 (to J.-P.R. and R.B.) in partnership with the Canadian Foundation for AIDS Research and the International AIDS Society. R.B. is a career scientist of the Ontario HIV Treatment Network.
Authors’ contributions: S.-K.W.-A. and R.B. conceived and designed the experiments; S.-K.W.-A., M.T.H. and J.G. performed the experiments; S.-K.W.-A. and R.B. analyzed the data; R.K., J.-P.R. and R.B. contributed reagents/materials/analysis tools; S.-K.W.-A. and R.B. wrote the manuscript. All the authors have edited and approved the contents of the manuscript.
Conflicts of interest
There are no conflicts of interest.
1. Else LJ, Taylor S, Back DJ, Khoo SH. Pharmacokinetics of antiretroviral drugs in anatomical sanctuary sites: the male and female genital tract
. Antivir Ther
2. Palmer S, Josefsson L, Coffin JM. HIV reservoirs and the possibility of a cure for HIV infection
. J Intern Med
3. Smith MZ, Wightman F, Lewin SR. HIV reservoirs and strategies for eradication
. Curr HIV/AIDS Rep
4. Choo EF, Leake B, Wandel C, Imamura H, Wood aJJ, Wilkinson GR, et al. Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes
. Drug Metab Dispos
5. Le Tortorec A, Dejucq-Rainsford N. HIV infection of the male genital tract - consequences for sexual transmission and reproduction
. Int J Androl
6. Cory TJ, Schacker TW, Stevenson M, Fletcher CV. Overcoming pharmacologic sanctuaries
. Curr Opin HIV AIDS
7. Fletcher CV, Staskus K, Wietgrefe SW, Rothenberger M, Reilly C, Chipman JG, et al. Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues
. Proc Natl Acad Sci U S A
8. Huang Y, Hoque M, Jenabian M, Vyboh K, Whyte S, Sheehan N, et al. Antiretroviral drug transporters and metabolic enzymes in human testicular tissue - potential contribution to HIV-1 sanctuary site
. J Antimicrob Chemother
9. Kis O, Robillard K, Chan GNY, Bendayan R. The complexities of antiretroviral drug-drug interactions: role of ABC and SLC transporters
. Trends Pharmacol Sci
10. Alam C, Whyte-Allman S-K, Omeragic A, Bendayan R. Role and modulation of drug transporters in HIV-1 therapy
. Adv Drug Deliv Rev
11. Andrade CH, de Freitas LM, de Oliveira V. Twenty-six years of HIV science: an overview of anti-HIV drugs metabolism
. Brazilian J Pharm Sci
12. Li X, Chan WK. Transport, metabolism and elimination mechanisms of anti-HIV agents
. Adv Drug Deliv Rev
13. Pal D, Mitra AK. MDR- and CYP3A4-mediated drug-drug interactions
. J Neuroimmune Pharmacol
14. Kulpa DA, Chomont N. HIV persistence in the setting of antiretroviral therapy: when, where and how does HIV hide?
. J virus Erad
15. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation
. Nat Med
16. Saksena NK, Wang B, Zhou L, Soedjono M, Shwen Ho Y, Conceicao V. HIV reservoirs in vivo and new strategies for possible eradication of HIV from the reservoir sites
. HIV/AIDS (Auckl)
17. Janneh O, Owen A, Chandler B, Hartkoorn RC, Hart CA, Bray PG, et al. Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP
18. Ford J, Hoggard PG, Owen A, Khoo SH, Back DJ. A simplified approach to determining P-glycoprotein expression in peripheral blood mononuclear cell subsets
. J Immunol Methods
19. Janneh O, Jones E, Chandler B, Owen A, Khoo SH. Inhibition of P-glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes
. J Antimicrob Chemother
20. Oselin K, Mrozikiewicz P, Pahkla R, Roots I. Quantitative determination of the human MRP1 and MRP2 mRNA expression in FACS-sorted peripheral blood CD4+, CD8+, CD19, and CD56+ cells
. Eur J Haematol
21. Shechter R, London A, Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates
. Nat Rev Immunol
22. Eisele E, Siliciano RF. Perspective redefining the viral reservoirs that prevent HIV-1 eradication
23. Jenabian M, Costiniuk CT, Mehraj V, Ancuta P, Bendayan R, Brassard P, et al. Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults
24. Ponte R, Dupuy FP, Brimo F, Mehraj V, Brassard P, Belanger M, et al. ORCHID study group. Characterization of myeloid cell populations in human testes collected after sex reassignment surgery
. J Reprod Immunol
25. Twentyman PR, Rhodes T, Rayner S. A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes
. Eur J Cancer
26. Consoli U, Van NT, Neamati N, Mahadevia R, Beran M, Zhao S, et al. Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines
27. Van Damme N, Baeten D, De Vos M, Demetter P, Elewaut D, Mielants H, et al. Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers
. J Immunol Methods
28. Schreurs RRCE, Drewniak A, Bakx R, Corpeleijn WE, Geijtenbeek THB, van Goudoever JB, et al. Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses
. J Immunol Methods
29. Mulder WMC, Koenen H, van de Muysenberg AJC, Bloemena E, Wagsfaff J, Scheper RJ. Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment
. Cancer Immunol Immunother
30. Robillard KR, Chan GNY, Zhang G, La Porte C, Cameron W, Bendayan R. Role of P-glycoprotein in the distribution of the HIV protease inhibitor atazanavir in the brain and male genital tract
. Antimicrob Agents Chemother
31. Robillard KR, Hoque T, Bendayan R. Expression of ATP-binding cassette membrane transporters in rodent and human sertoli cells: relevance to the permeability of antiretroviral therapy at the blood-testis barrier
. J Pharmacol Exp Ther
32. Whyte-Allman S-K, Hoque MT, Jenabian M-A, Routy J-P, Bendayan R. Xenobiotic nuclear receptors PXR and CAR regulate antiretroviral drug efflux transporters at the blood-testis barrier
. J Pharmacol Exp Ther
33. De Rose R, Fernandez CS, Hedger MP, Kent SJ, Winnall WR. Characterisation of macaque testicular leucocyte populations and T-lymphocyte immunity
. J Reprod Immunol
34. Cheney K, Kumar R, Purins A, Mundy L, Ferguson W, SHaw D, et al. HIV type 1 persistence in CD4-/CD8- double negative T cells from patients on antiretroviral therapy
. AIDS Res Hum Retroviruses
35. Kis O, Sankaran-walters S, Walmsley SL, Dandekar S, Bendayan R. HIV-1 alters intestinal expression of drug transporters and metabolic enzymes: implications in antiretroviral drug disposition
36. De Rosa MF, Robillard KR, Kim CJ, Hoque MT, Kandel G, Kovacs C, et al. Expression of membrane drug efflux transporters in the sigmoid colon of HIV-infected and uninfected men
. J Clin Pharmacol
37. Zhang J-C, Deng Z-Y, Wang Y, Xie F, Sun L, Zhang F-X. Expression of breast cancer resistance protein in peripheral T cell subsets from HIV-1-infected patients with antiretroviral therapy
. Mol Med Rep
38. Liptrott NJ, Khoo SH, Back DJ, Owen A. Detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells using flow cytometry
. Phys Lett
39. Wu T-Y, Huang Y, Zhang C, Su Z-Y, Boyanapalli S, Khor TO, et al. Pharmacokinetics and pharmacodynamics of 3,3′-diindolylmethane (DIM) in regulating gene expression of phase II drug metabolizing enzymes
. J Pharmacokinet Pharmacodyn
40. Tochigi Y, Yamashiki N, Ohgiya S, Ganaha S, Yokota H. Isoform-specific expression and induction of UDP-glucuronosyltransferase in immunoactivated peritoneal macrophages of the rat
. Drug Metab Dispos
41. Matusali G, Dereuddre-Bosquet N, Le Tortorec A, Moreau M, Satie A-P, Mahé D, et al. Detection of Simian Immunodeficiency Virus in Semen, Urethra, and Male Reproductive Organs during Efficient Highly Active Antiretroviral Therapy
. J Virol
42. Coombs RW, Lockhart D, Ross SO, Deutsch L, Dragavon J, Diem K, et al. Lower genitourinary tract sources of seminal HIV
. J Acquir Immune Defic Syndr
43. Paranjpe S, Craigo J, Patterson B, Ding M, Barroso P, Harrison LEE, et al. Subcompartmentalization of HIV-1 quasispecies between seminal cells and seminal plasma indicates their origin in distinct genital tissues
. AIDS Res Hum Retroviruses
44. Zhang J, Alston MA, Huang H, Rabin RL. Human T cell cytokine responses are dependent on multidrug resistance protein-1
. Interntional Immunol
45. Pawlik A, Baskiewicz-Masiuk M, Machalinski B, Safranow K, Gawronska-Szklarz B. Involvement of P-glycoprotein in the release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone
. J Pharm Pharmacol
46. Drach BJ, Gsur A, Hamilton G, Zhao S, Angerler J, Fiegl M, et al. Involvement of P-glycoprotein in the transmembrane transport of interleukin-2 (IL-2), IL-4, and interferon-y in normal human T lymphocytes
47. Giraud C, Manceau S, Declèves X. Influence of development, HIV infection, and antiretroviral therapies on the gene expression profiles of ABC transporters in human lymphocytes
. J Clin Pharmacol
48. Ebert C, Perner F, Wolleschak D, Schnoder T, Fischer T, Heidel H. Expression and function of ABC-transporter protein ABCB1 correlates with inhibitory capacity of Ruxolitinib in vitro and in vivo
49. Gupta S, Kim CH, Tsuruo T, Gollapudi S. Preferential expression and activity of multidrug resistance gene 1 product (P-glycoprotein), a functionally active efflux pump, in human CD8+ T cells: a role in cytotoxic effector function
. J Clin Immunol
50. Sever R, Glass CK. Signaling by nuclear receptors
. Cold Spring Harb Perspect Biol
51. Hartz AMS, Mahringer A, Miller DS, Bauer B. 17-B-Estradiol: A powerful modulator of blood-brain barrier BCRP activity
. J Cereb Blood Flow Metab
52. Chang FW, Fan HC, Liu JM, Fan TP, Jing J, Yang CL, et al. Estrogen enhances the expression of the multidrug transporter gene ABCG2—increasing drug resistance of breast cancer cells through estrogen receptors
. Int J Mol Sci
53. Coles LD, Lee IJ, Voulalas PJ, Eddington ND. Estradiol and progesterone-mediated regulation of P-gp in P-gp overexpressing cells (NCI-ADR-RES) and placental cells (JAR)
. Mol Pharm
54. Sarlis NJ, Gourgiotis L. Hormonal effects on drug metabolism through the CYP system: perspectives on their potential significance in the era of pharmacogenomics
. Curr Drug Targets Immune Endocr Metab Disord
55. Faed EM. Protein binding of drugs in plasma, interstitial fluid and tissues: effect on pharmacokinetics
. Eur J Clin Pharmacol
56. Lamers SL, Rose R, Maidji E, Agsalda-Garcia M, Nolan DJ, Fogel GB, et al. HIV DNA is frequently present within pathologic tissues evaluated at autopsy from cART-treated patients with undetectable viral load
. J Virol