Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T cells: Implications for HIV Microbicides Indrani Mukhopadhya †, § , Graeme I Murray ǁ , Linda Duncan § , Raif Yuecel § , Robin Shattock ǂ , 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 1 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20 21 22
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kclpure.kcl.ac.uk · Web viewPurified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4β7 integrin were
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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,
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
Decreased intracellular drug concentration
PIs ( ritonavir, lopinavir, atazanavir, saquinavir, and indinavir)20, 21