kclpure.kcl.ac.uk · Web viewPurified total CD4+ T cells were obtained from colorectal tissue and...

Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7

Integrin CD4+ T cells: Implications for HIV Microbicides

Indrani Mukhopadhya†, §, Graeme I Murrayǁ, Linda Duncan§, Raif Yuecel§, Robin Shattockǂ,

Charles Kelly#, Francesco Iannelli┴, Gianni Pozzi┴, Emad M El-Omar§, Georgina L Hold§, ‡

and Karolin HijaziI†, §, ‡, *

† University of Aberdeen Dental School and Hospital, Aberdeen, UK.

§ Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK.

ǁ Department of Pathology, School of Medicine & Dentistry, University of Aberdeen,

Aberdeen, UK.

ǂ Mucosal Infection & Immunity Group, Section of Infectious Diseases, Imperial College,

London, UK.

# King’s College London, Dental Institute, Mucosal & Salivary Biology, London, UK.

┴ Laboratory of Molecular Microbiology and Biotechnology, Department of Medical

Biotechnologies, University of Siena, Siena, Italy.

Corresponding Author:

* University of Aberdeen Dental School and Hospital, Cornhill Road, Foresterhill, Aberdeen,

AB25 2ZR. UK. Tel: +44 (0) 1224-555153. E-mail: [email protected]

‡ denotes authors contributed equally.

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mailto:[email protected]

ABSTRACT

CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and

dissemination. As such they are also the primary target for anti-retroviral (ARV)-based rectal

microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T

cells determine ARV distribution across the cell membrane and, most likely, efficacy of

microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T

lymphocytes and compare gene expression with circulating α4β7+CD4+ T cells which traffic

to the intestine and have been shown to be preferentially infected by HIV-1. Purified total

CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic

separation. CD4+ T cells expressing α4β7 integrin were isolated by fluorescence-activated

cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15

efflux and uptake drug transporter genes were quantified using Taqman qPCR assays.

Expression of efflux transporters MRP3, MRP5, BCRP and uptake transporter CNT2 was

significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells

(p=0.01-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated significantly higher

expression of OATPD compared to colorectal CD4+ T cells (p=0.001). To the best of our

knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and

peripheral α4β7+CD4+ T cells. The qualitative and quantitative differences in drug

transporter gene expression profiles between α4β7+CD4+ T cells and total mucosal CD4+ T

cells may have significant implications for the efficacy of rectally delivered ARV-

microbicides. Most notably, we have identified efflux drug transporters that could be targeted

by selective inhibitors or beneficial drug-drug interactions to enhance intracellular

accumulation of antiretroviral drugs.

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KEY WORDS: efflux drug transporters; uptake drug transporters; gene expression; CD4+ T

cells; α4β7 integrin; colorectal mucosa

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TABLE OF CONTENTS GRAPHIC

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INTRODUCTION

The intestinal immune system plays a key role in HIV-1 mucosal transmission,

dissemination, CD4+ T cell depletion and persistence.1, 2 Naïve lymphocytes residing in

resting inductive sites of gastrointestinal associated lymphoid tissue (GALT) are activated by

HIV-1 antigens and travel to the systemic circulation via mesenteric lymph nodes. Activated

lymphocytes then ‘home’ back to the diffuse effector compartment in the lamina propria

mediated by the homing receptor α4β7 integrin (referred to as α4β7 hereafter).3 This system

is exploited by HIV-1, as its envelope glycoprotein gp120 binds to α4β7+CD4+ T cells

ensuring successful infection of GALT and subsequent widespread dissemination. 4

Antiretroviral (ARV)-containing rectal microbicides aimed at pre-exposure prophylaxis

should prevent infection of resident colorectal CD4+ T cells including the preferentially

infected α4β7+CD4+ T cells.5 Absorption of ARV drugs through the mucosa and

concentration within target CD4+ T cells are determined by expression of drug transporters.

The ATP-binding cassette (ABC) and solute carrier (SLC) transporter families are the

predominant efflux and uptake drug transporters, respectively. Their expression in

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submucosal CD4+ T cells determines intracellular concentration, and ultimately efficacy, of

ARV drugs.6

To date there are no data on the expression of drug transporters in resident colorectal CD4+ T

cells and most of our knowledge is extrapolated from data relating to peripheral blood

mononuclear cells (PBMC).6-9 This study investigates drug transporter gene expression in

colorectal CD4+ T cells and peripheral gut-homing α4β7+CD4+ T cells. Analyses included

the efflux and uptake transporters which were most highly expressed in circulating CD4+ T

cells, those involved in transport of ARV drugs and those which expression is induced or

suppressed by ARV drugs.10, 20 Understanding of the balance of ARV drugs efflux and uptake

across the cell membrane of colorectal CD4+ T cells is critical for designing rectal

microbicides with maximal efficacy as well as predicting drug resistance and drug-drug

interactions.

EXPERIMENTAL SECTION

Tissue and blood samples

Fresh, morphologically normal human colonic mucosal samples were obtained from four

subjects (age range 54-73 years, 3 male and 1 female) undergoing surgery for left-sided

colorectal cancer at Aberdeen Royal Infirmary through NHS Grampian Biorepository (tissue

bank request number TR000037). Tissue for drug transporter analyses was sampled at a

10cm-margin from the tumour. Absence of tumour at the resective margin was documented

morphologically and histologically to ensure that assessment was done on normal colorectal

tissue. Patients did not receive cytotoxic drugs, immunosuppressive drugs or antibiotics in the

four weeks prior to surgery. Blood samples were collected from eight healthy donors (age

range 22-45 years; 3 male and 5 female) under ethical approval from the University of

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Aberdeen Ethics Review Board (CERB/2014/1/987). Subjects were recruited after written

informed consent and the study was conducted according to local Research Ethics Committee

policies and the Declaration of Helsinki.

Isolation of CD4+ T cells from whole blood and colorectal mucosal samples

Whole blood samples were collected in BD Vacutainer™ EDTA tubes (Fisher Scientific,

UK) and peripheral blood mononuclear cells (PBMC) were isolated from freshly collected

blood by density gradient centrifugation using LymphoprepTM (Stemcell Technologies)

following manufacturer’s instructions. The dissected colonic mucosal samples were placed in

15 ml FalconTM tubes containing DMEM (Sigma, UK) supplemented with 10 % foetal calf

serum (FCS, Sigma, UK), 2 mM L-glutamine (Sigma, UK) and antibiotics (100 U penicillin,

100 µg of streptomycin, and 50 µg of gentamicin/ml, Sigma UK). Samples were immediately

placed on ice for transfer to the laboratory and the isolation procedure was initiated within an

hour of collection. Tissue samples were washed and dissected into ~2 mm pieces in a sterile

Petri dish using a disposable scalpel. Samples were next transferred to a gentleMACS™ C

tube (Miltenyi Biotec Ltd) containing 4 ml digestion buffer (DMEM/F12 supplemented with

1% penicillin/streptomycin, 12 mM HEPES and 2 mg/mL Collagenase) and dissociated using

the 37C_h_TDK_2 setting of a gentleMACS™ Octo Dissociator with heaters (Miltenyi

Biotec Ltd) for 1 hour 1 minute. The process was repeated twice and the cell suspension was

filtered through a 70 µm cell strainer (BD Biosciences). The resulting single-cell suspension

was washed with phosphate buffered saline (PBS) and counted using a haemocytometer with

trypan blue exclusion to analyse cell yield. Purified total CD4+ T cells were obtained from

both PBMC and colorectal tissue cell suspensions by negative selection (~ 90 % purity) using

magnetic activated cell sorting (MACS) LS columns (Miltenyi Biotec Ltd) and human CD4+

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T Cell Isolation Kit for the isolation of untouched CD4+ T cells (Miltenyi Biotec Ltd)

following manufacturer’s instructions.

Isolation of α4β7+CD4+ T cells by Fluorescent antibody-based cell sorting

Fresh PBMCs were stained with fluorochrome conjugated specific antibodies to human

CD45-V450, CD3-PE-CF594, CD4-APC-Cy7, Integrin β7-APC and CD49d (Integrin α4

chain)-PerCP-Cy™5.5 obtained from BD Biosciences at 4°C for 35 minutes. Targeted cells

were then sorted on a BD Influx™ cell sorter according to the sorting strategy shown in Fig.

1. Sort purity was ~ 98% for each sample. Cells were initially gated according to size and

density by Forward scatter (FSC) vs Side scatter (SSC) plot. Once the lymphocyte population

was identified and gated, a process of sequential gating identified α4β7+CD4+ T cell

populations by their extracellular phenotypic markers. Data were analysed using the FlowJo

Analysis Software (Version 10.1).

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Figure 1: Representative flow cytometry analysis of CD45+CD3+CD4+α4β7+ T-cell subgroups. A hierarchical gating strategy identified

single lymphocytes in peripheral blood mononuclear cells (PBMC) using scatter plot (FSC vs SSC). Subsequent gating analysis on fluorescence

allowed the identification of CD45+ lymphocytes, CD3+CD4+ helper T-cells and the target subset of α4β7+CD4+ T lymphocytes (CD49d vs

Integrin β7).

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CD45+ CD3+ CD4+ α4β7+CD45+ CD3+ CD4+

CD45+Lymphocytes Singlets

Quantitative RT-PCR

RNA, cDNA preparation and quantitative RT-PCR were done according to methods

described previously.10 Fifteen drug transporter genes and 2 housekeeper genes (PPIA and

PGK1) were analysed using Taqman® gene expression assays (Life Technologies, UK). Out

of the 15 drug transporter genes selected for the study, twelve transporters (Pg-p, BCRP,

MRP1-5, MRP10, CNT2, ENT2, OATPE and OATPD) were chosen as they were the most

relevant in ARV transport. The other three transporters (LAT2, TAP1 and TAP2) were

included as they were found to be amongst the most highly expressed genes in a preliminary

study where we analysed eighty-four drug transporter genes in total CD4+ T cells isolated

from PBMC using TaqMan® Array 96-well Fast plates for Human Drug Transporters (Life

Technologies) (data not shown). Relative gene expression was determined using the

comparative CT method11 where gene expression of total CD4+ T cells or a Human universal

reference RNA (Clontech, CA, USA) were used as reference.

Statistical methods

Expression differences between mucosal and circulating CD4+ T cells were assessed by two-

tailed t-test with significance at P<0.05. Pearson’s product moment correlation (r) was used

for individual sample comparisons and hierarchical clustering of ∆CT values. Principal

component analysis (PCA) was also based on ∆CT values. SPSS 23 (IBM Corp; Armonk,

NY) was used for all analyses.

RESULTS

Drug transporter mRNA expression in colorectal CD4+ T cells

In colorectal CD4+T cells, MRP3 and CNT2 were the highest expressed efflux and uptake

transporters, respectively. Expression levels of all 15 drug transporters in total colorectal

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CD4+ T cells is shown in Fig. 2. The overall expression profile was variable between

subjects with 3 out of 4 individuals showing no significant differences of expression (r=0.19-

0.89, p=0.502-0.0001) as confirmed by hierarchical clustering (Fig. 2B) and PCA analyses

(Fig. 2C).

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Figure 2: Comparison of drug transporter mRNA expression between colorectal CD4+

T cells and circulating CD4+ T cells isolated from PBMC. Panel A shows mean (± SD)

relative quantitation values of mRNA expression for the 15 drug transporter genes (*P<0.05;

**P<0.01 and ***P<0.001). Panel B shows hierarchical clustering of mRNA expression of

15 drug transporter genes in the 3 groups: CD4+ T cells from colorectal tissue (n=4),

α4β7+CD4+ T cells from PBMC (n=5) and CD4+ T cells isolated from PBMC (n=3).

Distances between samples and assays were calculated for hierarchical clustering based on

the ΔCT values using Pearson’s correlation. Two endogenous control genes (PGK1 and

PPIA) were used for normalization and the ΔCT values were calculated by subtracting the

mean of the CT values of the endogenous controls from the CT value of the gene for each

sample. The ΔCT value of the neutral/middle expression level (mean) is set such that red

indicates an increase with a ΔCT value below the middle level, and green indicates a

decrease, with a ΔCT value above the middle level. The relationship between colours and

normalized values of gene expression is illustrated in the key at the top of the figure. The

rows of the heat map represent 15 drug transporter genes and the columns represent samples.

Panel C shows the principal component analysis of gene expression profiles generated from

the three different groups. Relative contribution of the ∆Ct variance is shown by two major

principal components (PC1 and PC2) plotted in two dimensions. Pgp = P-glycoprotein, MRP

= Multidrug Resistance-associated Protein, BCRP = Breast Cancer Resistance Protein, CNT

= Concentrative Nucleoside Transporter, ENT = Equilibrative Nucleoside Transporter, LAT

= Large-neutral Amino Acid Transporter, OATP = Organic Anion-Transporting

Polypeptides, TAP = Transporter associated with Antigen Processing.

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A B C

MRP3BCRPOATPDMRP4ENT2MRP5CNT2OATPEP-gpMRP10LAT2TAP1TAP2MRP1MRP2

BL_5 BL_4BL_2BL_8BL_13BL_6BL_7BL_12CRS3CRS2CRS4CRS7

CD4+ T cells from colonic tissue

α4β7+ CD4+ T

cells from PBMC

CD4+ T cells from PBMC

Drug transporter mRNA expression in total and α4β7+CD4+ T cells circulating cells

The efflux and uptake drug transporters highest expressed in total circulating CD4+ T cells were

MRP1 and LAT2, respectively (Fig. 2B). Expression of α4β7 integrin on circulating CD4+ T

cells ranged from 5-15% (representative sample shown in Fig. 1) of the total CD4+ T cells

between the individuals and was consistent with previous findings.12, 13 In circulating

α4β7+CD4+ T cells, the most expressed efflux and uptake transporters, were P-gp and OATPD,

respectively. Expression levels of all 15 drug transporters in α4β7+CD4+ T cells relative to the

total CD4+ T cells is shown in Fig. 2. There was no expression of CNT2. No significant

difference was noted between individual subjects for either α4β7+CD4+ T cells (r=0.72-0.97,

p=0.004-0.0001) or total CD4+ T cells (r=0.91-0.97, p<0.0001) as confirmed by hierarchical

clustering (Fig. 2B) and PCA analyses (Fig. 2C).

Comparison between expression in colorectal CD4+ T cells and circulating α4β7+CD4+ T

cells

Comparison of expression of drug transporters between colorectal CD4+ T cells and circulating

α4β7+CD4+ T cells showed high expression of P-gp for both cell types with no statistical

difference noted between each other (Fig. 2A). Expression of efflux transporters MRP3, MRP5

and BCRP was significantly higher in colorectal CD4+ T cells (p=0.01-0.03 respectively).

Among uptake transporters, CNT2 and OATPE were predominantly expressed in colorectal

CD4+ T cells (p=0.005-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated

significantly higher expression of OATPD, LAT2 and TAP2, respectively (p=0.001-0.04).

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Heat maps were generated to visualize hierarchical clustering between the three different groups

of samples and gene expression levels (Fig 2B). The mRNA expression profile of all the 15

target genes in the samples were tested by hierarchical cluster analysis of ∆Ct values. Analyses

indicated that samples fell into two distinct clusters as indicated by the branching shown above

Figure 2B. The circulating total CD4+ and α4β7+CD4+ T cells fell into one cluster and the

colorectal CD4+ T cells clustered separately from them. The distinct patterns of gene expression

between the three different sample groups studied were elucidated by PCA (Figure 2C). The

relative contribution of the ∆Ct variance was shown by two major principal components PC1 and

PC2, plotted in two dimensions of the scatterplot. The PC1 and PC2 used in the graphical

representation account for 62.9 % and 24.1 % respectively of the total variation in the mRNA

expression. The circulating total CD4+ cells, α4β7+CD4+ T cells and the colorectal CD4+ T

cells clustered separately and were distinguishable indicating that they varied from one another.

DISCUSSION

This is the first study to investigate transporters involved in efflux/uptake of ARV drugs in

colorectal CD4+ T cells as well as the homing population of circulating α4β7+CD4+ T cells.

Resident colorectal CD4+ T cells showed significant differences in drug transporter gene

expression when compared with peripheral total CD4+ and α4β7+CD4+ T cells. Anatomically,

colorectal resected samples contain intraepithelial and lamina propria CD4+ T cells. The latter

include those from inductive sites (organized lymphoid nodules) and effector sites (diffuse

lamina propria). Previous estimates of α4β7 expression in CD4+ T cells ranged from 30% in

intraepithelial cells to around 70% in lamina propria cells.14 Therefore, colorectal CD4+ drug

transporter gene expression represents the amalgamation of all subtypes, of which α4β7+CD4+ T

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cells are a variable component. Our findings have important clinical implications as all CD4+ T

cells subtypes in the colorectal mucosa are potentially susceptible to HIV-1 infection and

therefore contribute to virus spread from the anorectal route.

Highly expressed drug transporters in colorectal CD4+ T cells were the efflux transporters

MRP3, P-gp, MRP5 and BCRP (in decreasing order of expression) and uptake transporters

CNT2 and OATPE. A summary of the differential expression of drug transporters in colorectal

CD4+ T cells and peripheral α4β7+CD4+ T cells is summarized in Table 1 with its implications

on potential ARV substrates and inhibitors.

Protease inhibitors (PIs) including darunavir, in the pipeline for development of vaginal

microbicides, are substrates of P-gp15, 16, highly expressed in both colorectal CD4+ T cells and

peripheral α4β7+CD4+ T cells. P-gp as well as BCRP, expressed at significantly higher levels in

colorectal CD4+ T cells, also mediate efflux of nucleoside/nucleotide reverse transcriptase

inhibitors (NRTIs) such as tenofovir disoproxil fumarate and abacavir.17, 18 This could adversely

impact concentration of these drugs within colorectal CD4+ T cells. Equally, expression of

MRP5 may reduce intracellular concentrations of phosphorylated nucleoside analogues such as

stavudine which are MRP5 substrates.19 This could also be true for NRTIs like tenofovir, which

may be impacted by the high expression of MRP4 in both colorectal CD4+ T cells and peripheral

α4β7+CD4+ T cells. However, other efflux transporters which reduce intracellular accumulation

of antiretroviral drugs (MRP1 and MRP2)20 were not highly expressed in these cells. In addition,

the highly expressed efflux transporter MRP3 is inhibited by NRTIs as well as non-nucleoside

reverse transcriptase inhibitors (NNRTIs) like efavirenz, emtricitabine and delaviridine in vitro21

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which may therefore preferentially accumulate in colorectal CD4+ T cells. Among uptake

transporters, CNT2 was exclusively expressed in colorectal CD4+ T cells. This may result in

enhanced intracellular concentration of certain nucleoside analogue drugs like didanosine.22

Drug transporter gene expression in peripheral CD4+ T cells has been well characterized

previously8, 9, 20, 23 but has been documented in α4β7+CD4+ T cells for the first time in this study.

PCA analysis shows that α4β7+CD4+ T cells are distinct from total circulating as well as

colorectal CD4+ T cells. Previous studies have demonstrated enhanced ENT2 expression in

peripheral CD4+ T cells upon activation.7 In contrast, our study showed modest expression of

ENT2 which could be explained by the quiescent state of cells here analysed. We observed

differential expression of OATP uptake transporters between colorectal CD4+ and α4β7+CD4+

circulating T cells with OATPD predominantly expressed in α4β7+CD4+ T cells and OATPE

expressed primarily in colorectal CD4+ T cells. PIs are both substrates and inhibitors of OATP

transporters20 and their uptake by the two CD4+ T cell types may be differentially reduced. Two

other transporters, LAT2 and TAP2 were expressed more in α4β7+CD4+ T cells. The former is

primarily involved in transport of amines whilst the latter is involved in the transport of antigens

from the cytoplasm to the endoplasmic reticulum for association with MHC class I molecules

and could point to other functions of α4β7+CD4+ T cells. While their role has not been

documented in the transport of any class of ARV drugs, expression of LAT2 in a colorectal

epithelial cell line was significantly upregulated by stimulation with tenofovir. 10

CONCLUSIONS

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In conclusion, this study provides novel gene expression data of key drug transporters in

colorectal CD4+ T cells and confirms that these cells are distinct from their α4β7+CD4+

circulating counterparts. High expression of P-gp and MRP3 in colorectal CD4+ T cells may

significantly affect drug concentration in these cells after administration of rectal microbicidal

preparations. While targeted proteomic measurements would be desirable to corroborate our

findings, upregulation of MRP mRNA levels correlates with increased expression in

corresponding protein. 24, 25 We acknowledge that the age range of the participants is only partly

reflective of the HIV PrEP target population. Future larger scale studies may investigate drug

transporter expression in CD4+ T cells in relation to age. Nonetheless, our data along with our

previous findings of drug transporter expression in colorectal epithelium 10 will inform

optimisation of rectal microbicides through beneficial combinations of ARV drugs or addition of

selective inhibitors of efflux transporters. Functional assessment of drug transporters expressed

in colorectal CD4+ T cells is needed to elucidate their role in distribution of topically-applied

ARV drugs. In vivo pharmacokinetic studies on patients with HIV infection may be the ideal

model to analyse drug transporters in colorectal CD4+ T cells and document their response to

ARV microbicides. This is, however, hampered by the very low yield of CD4+ T cells from

endoscopic colorectal biopsies. This study is a useful surrogate to assess the potential effect of

rectal ARV microbicides on colorectal CD4+ T cells which are the target cells for anorectal

acquisition of HIV-1 infection.

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Table1: Relative mRNA expression of drug transporters in colorectal CD4+ T cells and peripheral α4β7+CD4+ T cells and implications on potential ARV substrates and inhibitors. An arbitrary classification system was assigned to the data, designating relative expression levels >2 as high mRNA expression, levels between 2 and 1 as moderate mRNA expression, levels between 1 and 0.1 as low mRNA expression and levels below 0.1 as unexpressed.

GeneTotal CD4+ T

cells from colonic tissue

(n=4)

α4β7+CD4+ T cells from

PBMC (n=5)

ARV substrateAdvantage/

disadvantage of ARV substrate

ARV inhibitorAdvantage/

disadvantage of ARV inhibitor

Pgp +++ +++

NRTI's (tenofovir, abacavir), PIs (ritonavir, lopinavir, atazanavir, saquinavir, and indinavir), maraviroc and raltegravir15-18, 20, 26

Decreased intracellular drug concentration

PI's (ritonavir, lopinavir, tipranavir, and nelfinavir), NNRTI (efavirenz)20, 27

Increased intracellular drug concentration

MRP1 + +++PIs (ritonavir, lopinavir, atazanavir, saquinavir, and indinavir),emtricitabine20


PIs ( ritonavir, lopinavir, atazanavir, saquinavir, and indinavir)20, 21


MRP2 + ++PIs (ritonavir, lopinavir, atazanavir, saquinavir, and indinavir), NRTI's (tenofovir)20


NNRTI (efavirenz, emtricitabine, delaviridine), NRTI's (tenofovir, emtricitabine and lamivudine), PI's (ritonavir, saquinavir)20, 21


MRP3 +++ + None known Not applicable

NNRTI (efavirenz, emtricitabine, delaviridine), NRTI's (tenofovir, emtricitabine and lamivudine)20, 21


MRP4 +++ +++ NRTI's (tenofovir, abacavir, lamivudine and zidovudine)19


None known Not applicable

MRP5 +++ +++ NRTI's (tenofovir, stavudine and zidovudine)19



MRP10 +++ +++ None known Not applicable None known Not applicable

19

269270271272

273

BCRP +++ ++NRTI's (tenofovir, didanosine, abacavir, lamivudine and zidovudine)28


PI's (ritonavir, lopinavir, tipranavir, and nelfinavir), NNRTI (efavirenz, emtricitabine, delaviridine)29


CNT2 +++ - NRTI's (Didanosine and zidovudine)22



ENT2 +++ +++ NRTI's (Didanosine and zidovudine)30



LAT2 ++ +++ None known Not applicable None known Not applicable

OATPD +++ +++ PIs (lopinavir,saquinavir, and

darunavir)20


PIs (lopinavir,saquinavir, and darunavir)20


OATPE +++ + PIs (lopinavir,saquinavir, and darunavir)20


PIs (lopinavir,saquinavir, and darunavir)20


TAP1 ++ +++ None known Not applicable None known Not applicable

TAP2 + +++ None known Not applicable None known Not applicable

RQ = Relative Quantification

+++High

expression, Mean RQ

>2

++Moderate

expression, Mean RQ

1-2

+Low

expression, Mean RQ

<1

-Unexpressed, Mean RQ <

0.1

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ACKNOWLEDGEMENT

This work was supported by the European Union's Seventh Framework Programme for research,

technological development and demonstration under grant agreement No 305316 as part of the

MOTIF (Microbicides Optimisation Through Innovative Formulation for Vaginal and Rectal

Delivery) project. We would like to extend our thanks to all the study participants for their

invaluable contribution and to Grampian Biorepository staff for help with collection of fresh

colorectal resection tissue.

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REFERENCES

1. Xu, H.; Wang, X.; Veazey, R. S. Mucosal immunology of HIV infection. Immunol

Rev 2013, 254, (1), 10-33.

2. Lackner, A. A.; Lederman, M. M.; Rodriguez, B. HIV pathogenesis: the host. Cold

Spring Harb Perspect Med 2012, 2, (9), a007005.

3. Kempster, S. L.; Kaser, A. alpha4beta7 integrin: beyond T cell trafficking. Gut 2014,

63, (9), 1377-9.

4. Arthos, J.; Cicala, C.; Martinelli, E.; Macleod, K.; Van Ryk, D.; Wei, D.; Xiao, Z.;

Veenstra, T. D.; Conrad, T. P.; Lempicki, R. A.; McLaughlin, S.; Pascuccio, M.; Gopaul, R.;

McNally, J.; Cruz, C. C.; Censoplano, N.; Chung, E.; Reitano, K. N.; Kottilil, S.; Goode, D.

J.; Fauci, A. S. HIV-1 envelope protein binds to and signals through integrin alpha4beta7,

the gut mucosal homing receptor for peripheral T cells. Nat Immunol 2008, 9, (3), 301-9.

5. McGowan, I.; Dezzutti, C. Rectal microbicide development. Curr Top Microbiol

Immunol 2014, 383, 117-36.

6. Hu, M.; Patel, S. K.; Zhou, T.; Rohan, L. C. Drug transporters in tissues and cells

relevant to sexual transmission of HIV: Implications for drug delivery. J Control Release

2015.

7. Minuesa, G.; Purcet, S.; Erkizia, I.; Molina-Arcas, M.; Bofill, M.; Izquierdo-Useros,

N.; Casado, F. J.; Clotet, B.; Pastor-Anglada, M.; Martinez-Picado, J. Expression and

functionality of anti-human immunodeficiency virus and anticancer drug uptake transporters

in immune cells. J Pharmacol Exp Ther 2008, 324, (2), 558-67.

8. Neyfakh, A. A.; Serpinskaya, A. S.; Chervonsky, A. V.; Apasov, S. G.; Kazarov, A.

R. Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug

selection. Exp Cell Res 1989, 185, (2), 496-505.

22

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

9. Legrand, O.; Perrot, J. Y.; Tang, R.; Simonin, G.; Gurbuxani, S.; Zittoun, R.; Marie, J.

P. Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in

normal peripheral blood and bone marrow haemopoietic cells. Br J Haematol 1996, 94, (1),

23-33.

10. Mukhopadhya, I.; Murray, G. I.; Berry, S.; Thomson, J.; Frank, B.; Gwozdz, G.;

Ekeruche-Makinde, J.; Shattock, R.; Kelly, C.; Iannelli, F.; Pozzi, G.; El-Omar, E. M.; Hold,

G. L.; Hijazi, K. Drug transporter gene expression in human colorectal tissue and cell lines:

modulation with antiretrovirals for microbicide optimization. J Antimicrob Chemother 2015.

11. Schmittgen, T. D.; Livak, K. J. Analyzing real-time PCR data by the comparative

C(T) method. Nature protocols 2008, 3, (6), 1101-8.

12. Abramson, O.; Qiu, S.; Erle, D. J. Preferential production of interferon-gamma by

CD4+ T cells expressing the homing receptor integrin alpha4/beta7. Immunology 2001, 103,

(2), 155-63.

13. Wang, X.; Xu, H.; Gill, A. F.; Pahar, B.; Kempf, D.; Rasmussen, T.; Lackner, A. A.;

Veazey, R. S. Monitoring alpha4beta7 integrin expression on circulating CD4+ T cells as a

surrogate marker for tracking intestinal CD4+ T-cell loss in SIV infection. Mucosal Immunol

2009, 2, (6), 518-26.

14. Farstad, I. N.; Halstensen, T. S.; Lien, B.; Kilshaw, P. J.; Lazarovits, A. I.;

Brandtzaeg, P. Distribution of beta 7 integrins in human intestinal mucosa and organized

gut-associated lymphoid tissue. Immunology 1996, 89, (2), 227-37.

15. Jones, K.; Hoggard, P. G.; Sales, S. D.; Khoo, S.; Davey, R.; Back, D. J. Differences

in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active

transport. AIDS 2001, 15, (6), 675-81.

23

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

16. Lee, C. G.; Gottesman, M. M.; Cardarelli, C. O.; Ramachandra, M.; Jeang, K. T.;

Ambudkar, S. V.; Pastan, I.; Dey, S. HIV-1 protease inhibitors are substrates for the MDR1

multidrug transporter. Biochemistry 1998, 37, (11), 3594-601.

17. van Gelder, J.; Deferme, S.; Naesens, L.; De Clercq, E.; van den Mooter, G.; Kinget,

R.; Augustijns, P. Intestinal absorption enhancement of the ester prodrug tenofovir

disoproxil fumarate through modulation of the biochemical barrier by defined ester mixtures.

Drug Metab Dispos 2002, 30, (8), 924-30.

18. Shaik, N.; Giri, N.; Pan, G.; Elmquist, W. F. P-glycoprotein-mediated active efflux of

the anti-HIV1 nucleoside abacavir limits cellular accumulation and brain distribution. Drug

Metab Dispos 2007, 35, (11), 2076-85.

19. Reid, G.; Wielinga, P.; Zelcer, N.; De Haas, M.; Van Deemter, L.; Wijnholds, J.;

Balzarini, J.; Borst, P. Characterization of the transport of nucleoside analog drugs by the

human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 2003, 63, (5), 1094-

103.

20. Kis, O.; Robillard, K.; Chan, G. N.; Bendayan, R. The complexities of antiretroviral

drug-drug interactions: role of ABC and SLC transporters. Trends Pharmacol Sci 2010, 31,

(1), 22-35.

21. Weiss, J.; Theile, D.; Ketabi-Kiyanvash, N.; Lindenmaier, H.; Haefeli, W. E.

Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by nucleoside, nucleotide,

and non-nucleoside reverse transcriptase inhibitors. Drug Metab Dispos 2007, 35, (3), 340-4.

22. Ritzel, M. W.; Yao, S. Y.; Ng, A. M.; Mackey, J. R.; Cass, C. E.; Young, J. D.

Molecular cloning, functional expression and chromosomal localization of a cDNA encoding

a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine.

Mol Membr Biol 1998, 15, (4), 203-11.

24

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

23. Ford, J.; Khoo, S. H.; Back, D. J. The intracellular pharmacology of antiretroviral

protease inhibitors. J Antimicrob Chemother 2004, 54, (6), 982-90.

24. Konig, S. K.; Herzog, M.; Theile, D.; Zembruski, N.; Haefeli, W. E.; Weiss, J.

Impact of drug transporters on cellular resistance towards saquinavir and darunavir. J

Antimicrob Chemother 2010, 65, (11), 2319-28.

25. Cost, M.; Dezzutti, C. S.; Clark, M. R.; Friend, D. R.; Akil, A.; Rohan, L. C.

Characterization of UC781-tenofovir combination gel products for HIV-1 infection

prevention in an ex vivo ectocervical model. Antimicrob Agents Chemother 2012, 56, (6),

3058-66.

26. Weiss, J.; Weis, N.; Ketabi-Kiyanvash, N.; Storch, C. H.; Haefeli, W. E. Comparison

of the induction of P-glycoprotein activity by nucleotide, nucleoside, and non-nucleoside

reverse transcriptase inhibitors. Eur J Pharmacol 2008, 579, (1-3), 104-9.

27. Storch, C. H.; Theile, D.; Lindenmaier, H.; Haefeli, W. E.; Weiss, J. Comparison of

the inhibitory activity of anti-HIV drugs on P-glycoprotein. Biochem Pharmacol 2007, 73,

(10), 1573-81.

28. Wang, X.; Nitanda, T.; Shi, M.; Okamoto, M.; Furukawa, T.; Sugimoto, Y.; Akiyama,

S.; Baba, M. Induction of cellular resistance to nucleoside reverse transcriptase inhibitors by

the wild-type breast cancer resistance protein. Biochem Pharmacol 2004, 68, (7), 1363-70.

29. Gupta, A.; Zhang, Y.; Unadkat, J. D.; Mao, Q. HIV protease inhibitors are inhibitors

but not substrates of the human breast cancer resistance protein (BCRP/ABCG2). J

Pharmacol Exp Ther 2004, 310, (1), 334-41.

30. Yao, S. Y.; Ng, A. M.; Sundaram, M.; Cass, C. E.; Baldwin, S. A.; Young, J. D.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,

nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins

produced in Xenopus oocytes. Mol Membr Biol 2001, 18, (2), 161-7.

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kclpure.kcl.ac.uk · Web viewPurified total CD4+ T cells were obtained from colorectal tissue and...

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