Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and

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Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating #4#7 Integrin CD4+ T cells: Implications for HIV Microbicides Indrani Mukhopadhya, Graeme I Murray, Linda Duncan, Raif Yuecel, Robin Shattock, Charles Kelly, Francesco Iannelli, Gianni Pozzi, Emad M El-Omar, Georgina L. Hold, and Karolin Hijazi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00351 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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Molecular Pharmaceutics

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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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 ARVmicrobicides. 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 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 5 ACS Paragon Plus Environment


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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 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


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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+ 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



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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). Singlets

Lymphocytes

CD45+ CD3+ CD4+

CD45+

CD45+ CD3+ CD4+ α4β7+

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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 twotailed t-test with significance at P2

++

Moderate expression, Mean RQ 1-2

+

Low expression, Mean RQ

Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and

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