Abstract
BACKGROUND Tenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against sexual HIV transmission. Because this is a new prevention strategy targeting large numbers of healthy people, we broadly assessed its effects on the mucosa.
METHODS AND FINDINGS In MTN-007, a phase 1, randomized, double-blinded rectal microbicide trial, we used systems genomics/proteomics to determine the effect of tenofovir 1% gel, nonoxynol-9 2% gel, placebo gel or no treatment on rectal biopsies taken at baseline, after one application or after seven daily applications (15 subjects/arm). Experiments were repeated using primary vaginal epithelial cells from four healthy women. After seven days of administration, tenofovir 1% gel had broad-ranging biological effects on the rectal mucosa, which were much more pronounced than—but different from—those caused by the detergent nonoxynol-9. Tenofovir profoundly suppressed anti-inflammatory mediators such as interleukin 10; increased T cell densities; caused mitochondrial dysfunction, possibly by blocking PNPT1 expression; and altered regulatory pathways of cell differentiation and survival. Except for leukocyte-derived factors, all these effects were replicated in primary vaginal epithelial cells, which also proliferated significantly faster in tenofovir's presence.
CONCLUSIONS Tenofovir's suppression of anti-inflammatory activity could diminish its prophylactic efficacy over time. The breadth of mucosal changes, including mitochondrial dysfunction and epithelial proliferation, raises questions about its safety for long-term topical use. These findings suggest that a systems biology evaluation of mucosal effects may be beneficial before advancing to large-scale efficacy trials with topical HIV prevention agents that achieve high, long-lasting local drug concentrations.
Introduction
The HIV prevention field has invested considerable resources in testing the phosphonated nucleoside reverse transcriptase inhibitor (NRTI) tenofovir as a mucosally-applied topical microbicide to prevent sexual HIV transmission. In a phase 2B trial, CAPRISA 004, pericoital tenofovir 1% gel was 39% efficacious in preventing vaginal HIV acquisition1. However, in another phase 2B trial, the VOICE study (MTN-003), the daily vaginal tenofovir 1% gel arm was discontinued for futility2. Adherence to product use was low in VOICE, likely explaining the differences in findings between the two studies. However, more frequent tenofovir gel exposure in VOICE (daily), as compared to pericoital use in CAPRISA 004, may also have contributed to this discrepancy.
A reduced glycerin formulation of the vaginal tenofovir 1% gel for use as a rectal microbicide appears safe when evaluated by epithelial sloughing, fecal calprotectin, inflammatory cytokine mRNA/protein levels and cellular immune activation markers3. However, because topical application of an antiretroviral drug to the mucosa is a novel prevention strategy without clinical precedent, we conducted a comprehensive systems biology assessment of tenofovir gel's effects on the mucosa.
Results
Tenofovir 1% gel induces broad and pronounced gene expression changes in the rectum
We measured mRNA expression changes across the complete human transcriptome by microarray (Illumina HumanHT12 v4 Expression BeadChips) in rectal biopsies taken at 9 and 15 cm proximal to the anal margin. Biopsies were obtained before treatment, after a single and after seven consecutive once-daily applications of reduced glycerin tenofovir 1% gel, nonoxynol-9 (N-9) 2% gel, hydroxyethyl cellulose (HEC) placebo gel, or no treatment (8 participants per arm were tested by microarrays). The primary results of the clinical study, MTN-007, a phase 1, randomized, double-blind, placebo-controlled trial at three US sites were reported elsewhere3. Relative to enrollment biopsies, after seven days of treatment, tenofovir 1% gel suppressed 505 genes and induced 137 genes in the 9 cm biopsies, whereas the detergent N-9, a transient mucosal toxin, suppressed 56 genes and induced 60 genes (log2 fold expression change ≥0.5 for induction or ≤-0.5 for suppression, FDR≤0.05) (Figure 1A,B). Fifteen suppressed and four induced genes were common to tenofovir and N-9 (Figure 1B). In the HEC gel and no treatment arms, 16 and 23 genes changed (Figure 1B). Tenofovir 1% gel affected more genes after seven days of treatment than after a single application and more genes in 9 cm than in 15 cm biopsies (Figure 1B), with significant correlations between expression changes at 9 cm and 15 cm (suppression: Spearman rho=0.4775; p<0.0001, Figure 1C; induction: Spearman rho=0.427; p<0.0001, Figure S1). Tenofovir suppressed genes more strongly than N-9 (median 0.505 vs 0.627 fold; p<0.0001), but induced genes less strongly (1.58 vs 1.69 fold; p=0.0002) (Figure S2).
Confirmatory RT-ddPCR quantification and in situ immunostaining in additional study participants
To independently confirm the microarray results, we selected nine induced and six suppressed genes and performed reverse transcription digital droplet PCR (RT-ddPCR) assays with RNA from the 9 cm biopsies in the remaining seven individuals enrolled in the tenofovir 1% gel arm (Figure 2). The mRNA copy numbers of all 15 genes increased or decreased as predicted from the microarray data between baseline and time point VII (Figure 2A,B). Next, we combined the microarray and RT-ddPCR expression data, normalized fluorescence and copy number values over their respective baselines, and compared the fold change after seven days of treatment with baseline (Figure 2C). Expression changes for all 15 genes assessed by microarray and RT-ddPCR were statistically significant (p<0.01 for all genes except TRIM5 [p=0.02]).
For additional confirmation, we selected three induced (CD7, CD3 and ubiquitin D) and one suppressed gene (IL-10) for immunohistochemical staining of the respective proteins in 9 cm rectal biopsies from 10 subjects (Figure 2D, Figure S3 and Figure S4). Consistent with the gene expression studies, infiltrating T lymphocytes increased three-to six-fold in the mucosa (p=0.0006 for CD7+ and p=0.023 for CD3+), whereas IL-10+ columnar epithelial cells decreased by more than half (p=0.017), between baseline and following seven days of tenofovir 1% gel use. Ubiquitin D was widely expressed in all biopsies, but tenofovir treatment increased the intensity of its expression (p=0.007), as predicted by the microarrays.
Gene expression patterns and functional pathways
Tenofovir 1% gel was more suppressive than stimulatory, with a ratio of induced to suppressed genes in the 9 cm rectal biopsies of 0.116 (17 genes up-regulated to 146 down) for nuclear products. However, genes encoding secreted proteins were more often induced than suppressed (Figure 3A), with a ratio of 2.33 (35 genes up-regulated to 15 down; χ2 p<0.0001). Noteworthy among induced genes for secreted products were the chemokines CCL2, CCL19, CCL21, CCL23, CXCL9 and CXCL13 (Figure 3A). Correspondingly, transcripts of a number of leukocyte-specific cell surface markers increased, specifically CD2, CD3D, CD7, CD8A, CD19, CD52, CD53, CCR6 and CCR7. The kinetics of gene induction is depicted in Figure S5.
Among suppressed genes with products localizing to the cell nucleus, we identified a large number of known or putative transcription factors and their co-factors, including CREB1 (CAMP responsive element-binding protein 1) and CREBBP (CREB-binding protein), both activators of IL-10 transcription26,27, NFAT5 (nuclear factor of activated T cells 5)28, and many zinc finger proteins (Figure 3A). Tenofovir 1% gel also suppressed genes important for regulation of transcription and translation, as well as biological processes involving transforming growth factor beta (TGFβ), epithelial structure organization, regulation of cell proliferation, and apoptosis (Figure S6). Lastly, tenofovir 1% gel suppressed genes important for mitochondrial function, including PNPT1 (polyribonucleotide nucleotidyltransferase 1)29 and OPA3 (optic atrophy 3)30. Among the few genes with nuclear products that were strongly induced, KIAA0101 and ubiquitin D were notable31–33.
Similarities between vaginal keratinocytes and rectal biopsies
Tenofovir 1% gel is most advanced as a candidate product for vaginal HIV prevention. We were therefore interested to extend our findings from the rectum to the vaginal mucosa. We isolated epithelial cells from vaginal tissues donated by three healthy women and tested their response to 50 and 500 μM tenofovir in vitro for up to 14 days of culture using RNA expression analysis. Preliminary tests showed that these dosages give roughly similar intracellular concentrations of the active drug (tenofovir diphosphate) as measured in the vagina after tenofovir 1% gel use (not shown)7. Tenofovir's effects on the purified vaginal epithelial cells (Figure 3B) were similar to its effects on the rectal mucosa (Figure 3A), but, as expected, changes likely driven by leukocytes in the biopsies were not seen in the purified epithelial cells, or were differently regulated. In the epithelial cells, tenofovir did not significantly change chemokine, chemokine receptor and cluster of differentiation (CD) genes, and it suppressed ISG 15 (interferon-stimulated gene 15) and MX1 (myxovirus resistance 1), which were induced in the biopsies.
The number of genes affected was initially higher with 500 μM than with 50 μM tenofovir, but equalized after 14 days of culture (Figure 4A). Next, we confirmed the expression changes of select genes by ddPCR with vaginal epithelial cells from four healthy women: mRNA copies of DSP (desmoplakin) and IL-10 significantly decreased, and KIAA0101 significantly increased during 7 days of tenofovir treatment, as seen in the microarray data (Figure 4B). In fact, IL-10 transcripts were virtually eliminated at 7 days (p<0.01), and KIAA0101 increased more than ten-fold at 500 μM tenofovir (p<0.01). By ELISA of cell lysates, IL-10 protein also decreased significantly (n=4 cell lines, p=0.0002) (Figure 4C).
Tenofovir treatment of primary vaginal epithelial cells in vitro mostly impacted the same biological processes as it did in the rectum in vivo (Figure 4D and Figure S6). Additionally, it suppressed genes important for keratinocyte differentiation and cellular innate immunity, and induced genes involved in DNA damage repair. Furthermore, tenofovir enhanced vaginal epithelial cell proliferation and/or cell survival in vitro (p=0.02) (Figure 4E).
Signs of mitochondrial toxicity
Our microarray results indicated that tenofovir suppresses PNPT1 (Figure 1C), which has been characterized as a master regulator of RNA import into mitochondria and whose deletion impairs mitochondrial function29. To explore tenofovir's effects on mitochondria, we first confirmed its inhibition of PNPT1 in the 9 cm and 15 cm rectal biopsies of all 15 study participants by RT-ddPCR. PNPT1 copy numbers decreased more than ten-fold at 9 cm (p<0.001) and by half at 15 cm (p<0.001) after 7 days of treatment (Figure 5A). To directly assess mitochondrial function, we picked one of the 13 genes encoded by mitochondrial DNA, ATP synthase F0 subunit 6 (ATP6), a key component of the proton channel34, and measured its transcription by RT-ddPCR in the 9 cm biopsies of all 15 study participants in the tenofovir arm (Figure 5B). Because mitochondrial genes are not included on the microarray chips, we had no preexisting information on its expression. ATP6 mRNA copy numbers decreased on average three-fold (p<0.01) after a single application and six-fold after seven days (p<0.001). In contrast, ATP6 copy numbers were stable in all 15 study participants treated with 2% N-9 gel (p=0.4911) (Figure 5B).
Next, we evaluated changes in mitochondrial number and size between baseline and after seven days of treatment in two study participants chosen for exhibiting pronounced PNPT1 suppression by tenofovir. The number of mitochondria per µm2 decreased by more than half after seven days of tenofovir treatment (p<0.001) (Figure 5C). In the second participant, mitochondria also increased in size by 1.4-fold (p<0.0001) (Figure 5D) and developed dysmorphic cristae during treatment (Figure 5E). In parallel, tenofovir also caused statistically significant inhibition of PNTP1 and ATP6 mRNA expression in vaginal epithelial cells (Figure 5F).
Proteomics of rectal secretions corroborates enhancing effect of tenofovir on cell survival
Extensive protein studies were not an intended part of MTN007 and samples were not preserved optimally for this purpose. However, 483 proteins were detected consistently at baseline and time point VII by mass spectrometry. 382 proteins increased in the tenofovir arm versus 112 in the no treatment arm (5 participants/arm). Among these, significant increases in individual protein expression were only seen in the tenofovir arm (Figure S7A). The top 100 proteins exhibited an average fold increase of 3.8 (median 3.2, range 1.6-128.7) in the tenofovir arm, listed by p value in Figure S7B. Enrichment analysis primarily indicated enhancement of cell survival and induction of leukocyte migration by tenofovir (Figure S7C), which is consistent with our other findings.
Discussion
Our findings indicate that reduced glycerin rectal tenofovir 1% gel affects expression of a different and much broader range of genes than N-9 2% gel, potentially impairing mucosal immune homeostasis, mitochondrial function, and regulation of epithelial cell differentiation and survival. These results make bio-logical sense given that tenofovir is a DNA chain terminator, with possible off-target effects in human cells35, and that topical application achieves at least one hundred-fold higher active drug concentrations in the mucosa than oral administration of 300 mg tenofovir disoproxil fumarate7,36. Moreover, tenofovir caused similar changes in primary vaginal epithelial cells cultured from several healthy women.
We did not find evidence that tenofovir directly causes inflammation. Rather, tenofovir dampened anti-inflammatory factors. Most prominently, it strongly inhibited IL-10 gene and protein expression, likely via blocking of CREB1 and its coactivator CREBBP26,27,37,38. In addition, it compromised signaling pathways downstream of TGF-β, a central anti-inflammatory mediator in the gut39,40. Consequently, a number of chemokines were induced, such as the B lymphocyte chemo-attractant CXCL1341, and CCL19 and CCL21, both ligands of CCR7 on T lymphocytes and dendritic cells42. Correspondingly, CCR7, the B cell marker CD19, and the T cell markers CD2, CD3D and CD7 increased. In keeping with this, we observed higher densities of CD3+ and CD7+ T lymphocytes in the rectal mucosa following seven days of tenofovir 1% gel use. In concert, these changes suggest that tenofovir creates a state of potential hyper-responsiveness to external inflammatory stimuli but does not itself cause inflammation. In populations with a high incidence of mucosal infections and associated immune activation, this could potentially negate the antiviral protective effect of topical tenofovir prophylaxis43. Intriguingly, recent findings in CAPRISA 004 participants support this notion: while pericoital vaginal tenofovir was overall protective1, inflammation increased the risk of HIV infection much more in the tenofovir arm than in the placebo arm (Passmore JA et al.; submitted).
Mitochondrial toxicity of nucleotide/nucleoside reverse transcriptase inhibitors such as tenofovir is well described but the mechanism remains unclear35. We found that tenofovir strongly and consistently inhibited expression of PNPT1, which encodes polynucleotide phosphorylase (PNPASE). PNPASE regulates nucleus-encoded RNA import into mitochondria29. In PNPT1 knock-out mice, mitochondrial morphology and respiratory capacity is disrupted in a manner quite similar to the disruption in renal proximal tubular cells in patients with tenofovir-induced nephrotoxicity29,44. Strikingly, in our study, just one week of daily tenofovir 1% gel application greatly lowered transcription of mitochondrial ATP6 and caused visible ultrastructural mitochondrial changes. These findings suggest that tenofovir's suppression of PNPT1 expression may underlie its reported, but heretofore unexplained, mitochondrial toxicity.
A number of changes in rectal biopsies and primary vaginal epithelial cells also suggested that tenofovir can cause epithelial hyper-proliferation. Furthermore, tenofovir's negative effect on mitochondrial function could lead to impairment of tumor progenitor cell apoptosis45,46, as has been specifically reported for loss-of-function mutations of mtATP6, a mitochondrial gene strongly suppressed by tenofovir in our study47. Neoplastic pressure could also arise from the strong induction of KIAA0101 and UBD (ubiquitin D). KIAA0101 is important for regulation of DNA repair48, is increased in tumor tissues31, and enhances cancer cell growth32,49. UBD appears to increase mitotic non-disjunction and chromosome instability50,51, and is highly up-regulated in gastrointestinal cancers33,50,51. Notably, though, these findings remain circumstantial, as there is no actual clinical evidence for carcinogenicity. Nevertheless, they raise the question of whether the relatively high concentrations of tenofovir achieved in the mucosa during topical use could potentially lead to neoplastic lesions with continuous and long-term use. According to Viread's Product Monograph, gastrointestinal tumorigenicity has been observed in mice after high oral dosing of tenofovir disoproxil fumarate. Vaginal tumorigenicity has been documented for azido-thymidine, an NRTI and DNA chain terminator like tenofovir, which induced vaginal hyperplasia and carcinomas when delivered to mice intravaginally as a 2% solution (∼25% carcinoma rate)52.
This is the first time that a systems biology approach has been applied to a clinical trial of mucosal pre-exposure prophylaxis and our study shows the value of using these technologies for comprehensive mucosal safety assessment. Our findings raise concerns regarding the safety of topical tenofovir 1% gel in the rectum. Tenofovir's effects on vaginal epithelial cells suggest similar activities in the vagina, which we will verify in MTN-014, an upcoming phase I clinical trial comparing vaginal and rectal tenofovir 1% gel in a cross-over format. Further studies are required to gauge whether tenofovir, which has become a valuable cornerstone drug in treating HIV infection, can also be safely and effectively used as a vaginal or rectal microbicide.
Methods
Design of the Clinical Study
MTN-007 (ClinicalTrials.gov registration NCT01232803) was a phase 1, double blind, placebo-controlled trial in which participants were randomized to receive rectal reduced glycerin tenofovir 1%, nonoxynol-9 (N-9) 2% or hydroxyethylcellulose (HEC) gels, or no-treatment (1:1:1:1), at three clinical research sites (Pittsburgh, PA; Birmingham, AL; and Boston, MA). The study protocol was approved by IRBs at all three sites. All participants gave written informed consent. The study details, and general safety and acceptability data, have been published elsewhere and showed that rectal tenofovir 1% gel was well tolerated and appeared safe by established safety parameters3. Each gel was administered as a single dose and then, after at least a one-week recovery period, once daily for seven consecutive days. The first dose of study product was self-administered under supervision by the clinic staff at the Treatment 1 Visit. Subsequent administrations occurred at home, and study participants were instructed to insert one dose of gel into the rectum once daily throughout the 7-day period in the evening or before the longest period of rest.
Study Participants and Products
A total of 65 study participants were enrolled and randomized in the study, 62 of whom completed it (tenofovir, n = 15; N-9, n = 16; HEC, n = 15; and no treatment, n = 16). Forty-three (69%) were male. Microarray studies were performed on eight randomly selected male participants in each group, and confirmatory gene expression studies were done on the remaining participants. The study population consisted of healthy, HIV-uninfected adults aged 18 or older who were required to abstain from receptive anal intercourse during the course of the clinical trial. Female participants were required to use effective contraception. Individuals with abnormalities of the colorectal mucosa, significant gastrointestinal symptoms (such as a history of rectal bleeding or inflammatory bowel disease), evidence of anorectal Chlamydia trachomatis or Neisseria gonorrhea infection, hepatitis B infection, or who used anticoagulants were excluded from the study. Reduced glycerin tenofovir 1% gel and HEC gel, known as the “Universal Placebo Gel”4, were supplied by CONRAD (Arlington, VA, USA). 2% N-9 gel was provided as Gynol II® (Johnson & Johnson). All study products were provided in identical opaque HTI polypropylene pre-filled applicators (HTI Plastics) containing 4 mL of study product.
Mucosal Biopsy Procedures
Rectal biopsies for the microarray studies were obtained before treatment at enrollment (time point “0”), 30–60 minutes following application of the single gel dose (time point “I”), and again on the day following the last dose of the seven once-daily gel applications (time point “VII”). Following an enema with Normosol-R pH 7.4, a flexible sigmoidoscope was inserted into the rectum and biopsies were collected at 15 cm from the anal margin. Following the sigmoidoscopy, a disposable anoscope was inserted into the anal canal for collection of rectal biopsies at 9 cm from the anal margin. Immediately after harvest, biopsies were immersed in RNAlater (Qiagen), stored at 4°C overnight and transferred to a −80°C freezer for long-term storage until shipping to Seattle and processing.
Primary Vaginal Keratinocyte Cultures
Tissues routinely discarded from vaginal repair surgeries were harvested from four otherwise healthy adult women, placed in ice-cooled calcium-and magnesium-free phosphate-buffered saline containing 100 U/ml penicillin, 100 μg/ ml streptomycin and 2.5 μg/ml Fungizone (Life Technologies), and transported to the laboratory within 1 hr of removal from the donor. Tissue harvesting and experimental procedures were approved by the Institutional Review Boards of the University of Washington and the Fred Hutchinson Cancer Research Center. The deep submucosa was removed with surgical scissors and the remaining vaginal mucosa was cut into 5 x 5 mm pieces, which were incubated at 4°C for 18 hr in 5 ml of a 25 U/ml dispase solution (354235; BD Biosciences). The epithelial sheets were dissected off under a stereoscope and incubated for 10-12 min at 37°C in 2 ml 0.05% trypsin while gently shaking. The dispersed cells were poured through a 100 μm cell strainer into a 50 ml tube, pelleted by centrifugation, and resuspended in F medium (3:1 [v/v] F12 [Ham]-DMEM [Life Technologies], 5% fetal calf serum [Gemini Bio-Products], 0.4 μg/ml hydrocortisone [H-4001; Sigma], 5 μg/ml insulin [700-112P; Gemini Bio-Products], 8.4 ng/ml cholera toxin [227036; EMD Millipore], 10 ng/ml epidermal growth factor [PHG0311; Life Technologies], 24 μg/ml adenine [A-2786; Sigma], 100 U/ml penicillin, and 100 μg/ml streptomycin [Life Technologies]). The keratinocytes were plated into culture flasks in the presence of ∼12,500/cm2 irradiated (6000 Rad) 3T3-J2 feeder fibroblasts (a kind gift by Cary A. Moody) and 10 μM of Rho kinase inhibitor Y27632 (1254; Enzo Life Sciences) was added5,6. Keratinocytes were fed every 2-3 days and passaged when around 80% confluent by 1 min treatment with 10 ml versene (Life Technologies) to remove the feeder cells, followed by 5 min treatment with trypsin/EDTA (Life Technologies). Dislodged keratinocytes were washed and re-plated at ∼2,500 keratinocytes/cm2 with irradiated 3T3-J2 feeder fibroblasts.
Tenofovir Treatment of Primary Vaginal Keratinocytes
Tenofovir (CAS 147127-20-6; T018500, Toronto Research Chemicals) was dissolved in phosphate-buffered saline, 7% dimethyl sulfoxide and 5% 5N sodium hydroxide to result in a 767 mM stock solution, and further diluted in culture media for addition to keratinocyte cultures in concentrations ranging from 0.05 to 32 mM. Based on initial titration experiments in which we measured the intracellular concentration of tenofovir diphosphate, the active cellular metabolite of tenofovir, by liquid chromatography-tandem mass spectrometry (performed in the laboratory of Dr. Craig Hendrix, Johns Hopkins University), concentrations in the culture media at the lower end (0.05 to 0.5 mM range) were estimated to be equivalent to the active concentrations likely achieved by topical tenofovir 1% gel (35 mM) in mucosal epithelial cells in vivo7,8. In all experiments, control keratinocytes were cultured in parallel without tenofovir. Keratinocytes were harvested at pre-determined time points, split into several aliquots, and pelleted. Pellets were frozen and stored at-80°C either as dry pellets for protein assays or after suspension in RNAprotect Cell Reagent (Qiagen) for RNA assays.
AlamarBlue Cell Proliferation and Cell Viability Assay
Primary vaginal keratinocytes were cultured in 6-well plates (Corning) with or without various concentrations of tenofovir for up to 14 days. At day 1, day 4, day 7 or day 14 the alamarBlue reagent (BUF012A; AbD Serotec) was added. After 5 hours, absorbance was measured at 570 and 600 nm on a Varioskan Flash Multimode reader (Thermo Scientific). Percentage reduction of alamar-Blue, corresponding to the level of cell proliferation, was calculated as specified in the manufacturer's technical datasheet. This was converted to percentage of maximal alamarBlue reduction by dividing each well by the well with the highest amount of alamarBlue reduction.
Interleukin-10 ELISA Assay
The IL-10 concentration in primary vaginal keratinocytes was measured after lysis of frozen cell pellets in Cell Lysis buffer (R&D Systems) using a commercial human IL-10 immunoassay (Quantikine HS, HS100C; R&D Systems) according to the manufacturer's specifications.
RNA isolation, cRNA Preparation and Whole Genome BeadArray Hybridization
RNA was isolated from the biopsies using the RNeasy Fibrous Tissue Mini Kit (Qiagen), and from the vaginal keratinocytes using the Direct-zol RNA MiniPrep kit (Zymo Research), according to the manufacturer's instructions, treated with 27 Kunitz units of DNAse (Qiagen) to remove genomic DNA contamination, and evaluated for integrity using the Agilent RNA 6000 Nano Kit (Agilent, Palo Alto, CA) on an Agilent 2100 Bioanalyzer. All samples had an RNA Integrity Number of 7 or greater. 500 ng of total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit (Ambion). cRNA from a total of 192 rectal biopsy samples (8 men per study arm, 4 study arms, 3 time points and 2 biopsy sites [9 cm and 15 cm]), and from a total of 36 primary vaginal keratinocyte cultures (3 tissue donors, 3 arms and 4 time points), was hybridized to HumanHT12 v4 Expression BeadChips (Illumina) according to the manufacturer's protocols. Each chip contains 47,323 probes, corresponding to 30,557 genes.
Unbiased Mass Spectrometry of Rectal Sponge Eluates
Protein concentration of rectal eluates was determined by BCA assay (Novagen). Equal amounts of total protein from each sample (100 μg) were then denatured in pH 8.0 urea exchange buffer (8 M Urea [GE HealthCare], 50 mM HEPES [Sigma]) for 20 minutes at room temperature, and then placed into 10 kDa Nanosep filter cartridges. After centrifugation, samples were treated with 25mM dithiothreitol (Sigma) for 20 minutes, then 50mM iodoacetamide (Sigma) for 20 minutes, followed by a wash with 50mM HEPES buffer. Trypsin (Promega) was added (2 μg/100 μg protein) and samples were incubated at 37°C overnight in the cartridge. Peptides were eluted off the filter with 50 mM HEPES, and the digestion was stopped with 1% formic acid. Peptides were dried via vacuum centrifugation and cleaned of salts and detergents by reversed-phase liquid chromatography (high pH RP, Agilent 1200 series micro-flow pump, Water XBridge column) using a step-function gradient. The fractions were then dried via vacuum centrifugation. Equal amounts of peptides were re-suspended in 2% acetonitrile (Fisher Scientific), 0.1% formic acid (EMD Canada) and injected into a nano-flow LC system (Easy nLC, ThermoFisher) connected in-line to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher). Mass spectrometry instrument settings were the same as described previously9.
Quality Control and Processing of Microarray and Mass Spectrometry Data
Microarray Data
All chips used in the analysis passed standard quality control metrics assessed by GenomeStudio (Illumina) as well as visual inspection for anomalies and artifacts. GenomeStudio calculates a detection p value for each probe, which represents the confidence that a given transcript is expressed above back-ground defined by negative control probes. Further processing and statistical analysis of data was done using the R/Bioconductor software suite10. Data were pre-processed by robust spline normalization and variance stabilizing transformation using the Bioconductor lumi package11,12. The pre-processed data were filtered to include only probes with detection threshold p values of < 0.05 in 100% of biopsies in at least one of the four study arms, or in 100% of vaginal epithelial cell cultures, and to remove probes that had a low standard deviation (≤ 0.5) across all arrays, using the Bioconductor genefilter package13. Finally, probes without an Entrez ID were removed, leaving 1,928 probes in the rectal biopsies, and 6,699 probes in the vaginal epithelial cell cultures, for further statistical analysis.
Mass Spectrometry Data
All spectra were processed using Mascot Distiller v2.3.2 (Matrix Science), against the UniProtKB/SwissProt (2012-05) Human (v3.87) database using the decoy database option (2% false discovery rate), along with the following parameters: carbamidomethylation (cysteine residues) (K and N-terminus) as fixed modifications, oxidations (methionine residues) as a variable modification, fragment ion mass tolerance of 0.5 Da, parent ion tolerance of 10 ppm, and trypsin enzyme with up to 1 missed cleavage. Mascot search results were imported into Scaffold (v4.21) (Proteome Software) and filtered using 80% confidence for peptides, 99% confidence for proteins, and at least 2 peptides per protein. Label-free protein expression levels based on MS peak intensities were calculated using Progenesis LC-MS software (v4.0 Nonlinear Dynamics). Feature detection, normalization and quantification were all performed using default settings in the software. Retention time alignment was performed using automatic settings and manually reviewed for accuracy on each sample. Only charge states between 2+ and 10+ were included. Protein abundances were further normalized by total ion current.
Statistical Analysis of Microarray and Mass Spectrometry Data
Microarray Data
A Bayesian statistical framework, Cyber-T14,15, was run from the CyberT/ hdarray library in R to test for the effect of tenofovir treatment on gene expression. The effect of tenofovir 1% gel on the rectal mucosa in vivo was tested in paired comparisons between time point I or VII and time point 0. Each study arm was considered separately. The effect of tenofovir on vaginal epithelial cell cultures in vitro was tested in paired comparisons between treated and untreated cultures. Each tenofovir dosage (50 and 500 μM) and time point (1, 4, 7 and 14 days) was considered separately. The Benjamini & Hochberg method for estimating false discovery rates (FDR) was used to control for multiple comparisons16. Criteria for significance and relevance were an estimated FDR ≤ 0.05 and a log2 fold expression change of ≥ 0.5 (induction) or ≤ −0.5 (suppression), respectively. For confirmation and to control for the potential time effect in this longitudinal study, the normalized but unfiltered data (all 47,323 probes) were also analyzed for significant treatment effects using a double subtraction strategy, where the paired differences between time point 0 and time points I or VII within each treatment arm were first calculated for each probe and study participant. In a second step, the mean paired differences for each probe within each of the three treatment arms were compared to mean paired differences for each probe within the notreatment arm. Analysis for significance was performed via a linear fit model using the limma package17, with the same significance criteria used for Cyber-T. The resulting gene lists greatly overlapped with the Cyber-T results. For simplicity, the numbers of induced and suppressed genes reported in Results are based on the Cyber-T analysis. Heat maps of differentially regulated genes were generated using MeV 4.8 within the TM4 Microarray Software Suite18, and hierarchically clustered according to selected gene ontologies found in the databases DAVID 6.719 and InnateDB20. All microarray data were deposited into the GEO database (accession numbers GSE57025 and GSE57026).
Mass Spectrometry Data
Protein intensity values were log transformed to mean expression values across all samples. To evaluate time-induced effects in the tenofovir gel arm, paired protein abundance values in each subject were normalized to time point 0 from time point VI. Statistical analysis for differences between the two time points was done by paired t tests.
Pathway and Network Analysis of Microarray and Mass Spectrometry Data
Microarray Data
Entrez ID designations, assigned to the array probes by Illumina, were uploaded to the Innate DB database and a Gene Ontology (GO) over-representation analysis was performed for gene groups signifying a particular molecular function or biological process, or occurring in specific cellular compartments20. The following strategy was used to determine which gene groups were enriched in the data set: separately for suppressed and induced genes, ratios of the number of genes in a particular GO group to the total number of genes detected in our data set were compared to the ratios in the same GO group reported for the complete human genome using Fisher's exact test. Ingenuity Pathways Analysis (IPA) (Ingenuity Systems) was used to visualize direct and indirect relationships between individual gene products and map their cellular localizations.
Mass Spectrometry Data
Proteomic data were uploaded into the IPA software package (Ingenuity Systems). Associations between protein groups in the dataset and canonical pathways were measured similarly to the over-representation analysis described for the microarray data above. IPA software was also used to compare protein expression patterns against the IPA biological function database. IPA software calculates a Benjamini & Hochberg-adjusted p-value for the association of protein abundance changes with each biological function, and a weighted z-score, which estimates whether the protein abundance changes found associated with a specific biological function are likely to enhance (positive z values) or inhibit (negative) the function.
Quantitative Confirmation of Microarray Results by RT-ddPCR
A two-step reverse transcription (RT) droplet digital PCR (ddPCR) was used to confirm microarray transcriptome data for selected genes of interest21,22. In a ddPCR assay, each sample is partitioned into ∼20,000 droplets representing as many individual PCR reactions. The number of target DNA copies present per sample can be quantified based on Poisson distribution statistics, because each individual droplet is categorized as positive or negative for a given gene. For rectal biopsies, cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Life Technologies) and the ddPCR was carried out using the 2X ddPCR Supermix for Probes (BioRad). For epithelial cell lines, these steps were combined using the 2X One-Step RT-ddPCR Kit for Probes (BioRad). ddPCR was performed on the QX100 droplet digital PCR system (BioRad). Reactions were set up with 20X 6-carboxyfluorescein (FAM)-labeled target gene-specific qPCR assay (Integrated DNA Technologies or Life Technologies) and 20X VIC-labeled housekeeping hemoglobin B (HBB) gene-specific Taqman gene expression assay (Life Technologies). Each assembled ddPCR reaction mixture was loaded in duplicate into the sample wells of an eight-channel disposable droplet generator cartridge (BioRad) and droplet generation oil (BioRad) was added. After droplet generation, the samples were amplified to the endpoint in 96-well PCR plates on a conventional thermal cycler using the following conditions: denaturation/enzyme activation for 10 min at 95°C, 40 cycles of 30 sec denaturation at 94°C and 60 sec annealing/amplification at 60°C, followed by a final 10 min incubation step at 98°C. After PCR, the droplets were read on the QX100 Droplet Reader (BioRad). Analysis of the ddPCR data was performed with QuantaSoft analysis software version 1.3.1.0 (BioRad).
Immunohistochemistry of Formalin-Fixed Rectal Biopsies
Four-micron sections were cut on a Leica RM2255 Automated Rotary Microtome (Leica), mounted on positively charged EP-3000 slides (Creative Waste Solutions), dried for 1 hr at 60°C, and stored until staining at 4°C. Slides were deparaffinized in xylene and rehydrated in graded dilutions of ethanol in water. Antigen retrieval was performed by heating the slides in Trilogy™ Pretreatment Solution (Cell Marque) for 20 min at boiling temperature in a conventional steamer (Black&Decker). Slides were then cooled for 20 min, rinsed three times in Tris-buffered 0.15 M NaCl solution containing 0.05% Tween 20 (Wash Buffer; Dako), and stained at room temperature in an automated slide-processing system (Dako Autostainer Plus). Endogenous peroxide activity was blocked using 3% H2O2 for 8 min, followed by 10 min in Serum-Free Protein Block (Dako). The slides were then stained for one hour with the primary antibodies. Staining was performed with the following primary antibodies: anti-CD3 (RM9107S, rabbit clone SP7; Lab Vision Corporation), antiubiquitin D (NBP2-13498, rabbit polyclonal anti-FAT10 antibody; Novus Biologicals), anti-CD7 (M7255, mouse clone CBC.37; Dako), and antiinterleukin-10 (sc-8438, mouse clone E-10; Santa Cruz Biotechnology). Negative control primary immunoglobulins were either whole rabbit IgG (011-000-003; Jackson ImmunoResearch Laboratories) or mouse IgG (I-2000; Vector Laboratories).
For CD3 staining, the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25, washed in Wash Buffer, incubated for 30 min with sheep-anti-rabbit Dylight 649 (611-643-122; Rockland Immunochemicals), washed, and incubated for 30 min with donkey-anti-sheep Dylight 649 (613-743-168; Rockland). Sections were counter-stained for 20 min with the nucleic acid-binding dye Sytox Orange (S11368; Life Technologies) at a dilution of 1:20,000, and coverslipped in ProLong Gold antifade reagent (P36930; Life Technologies). For ubiquitin D (UBD), CD7 and interleukin-10 (IL-10) staining, the slides were incubated with 0.8 μg/ml (UBD and CD7) or 4 μg/ml (IL-10) of the primary antibody for 60 min. After washing, the anti-UBD stained slides were incubated for 30 min with Leica Power Vision HRP rabbit specific polymer (PV6119; Leica Biosystems); and the anti-CD7 and anti-IL-10 stained slides were incubated for 30 min with Leica Power Vision HRP mouse specific polymer (PV6114; Leica Biosystems). After washing, staining was visualized with 3,3’-diaminobenzidine (Liquid DAB+ Substrate Chromogen System; Dako) for 7 minutes, and the sections were counter-stained for 2 min with hematoxylin (Biocare). Controls for all antibodies were run with identical procedures but replacing the primary antibodies with either rabbit IgG or mouse IgG, as appropriate, at calculated matching concentrations.
Acquisition and Analysis of Stained Tissue Sections
Sequences of slightly overlapping 20X (for CD3, UBD or CD7) or 40X (for IL-10) images covering each stained tissue section in its entirety were acquired on a bright-field Aperio Scansope AT (for UBD, CD7 and IL-10) or a fluorescent Aperio Scansope FL (for CD3) (Aperio ePathology Solutions, Leica Biosystems). Overlapping images were stitched together using the Aperio Image Analysis Suite, and the entire tissue sections on each slide were analyzed using Definiens Tissue Studio (Definiens AG). Individual cells were identified based on the nuclear counter-stain. For CD3 and CD7, all positive cells were automatically counted and reported as number of positive cells per mm2 of tissue section. For IL-10 and UBD, tight regions were manually drawn around all areas of the tissue sections containing columnar epithelial cells. For IL-10, all positive columnar epithelial cells were automatically counted and reported as number of positive cells per mm2. For UBD, which stained all epithelial cells, the mean staining intensity (MSI) of each columnar epithelial cell was measured in arbitrary units and overall UBD staining intensity for each section was reported as the average MSI of all epithelial cells.
Electron Microscopy
Formalin-fixed paraffin-embedded rectal biopsies were de-paraffinized and fixed overnight in half-strength Karnovsky's fixative. Staining, embedding, cutting and viewing on a JEOL 1400 SX transmission electron microscope were performed as previously described23,24. Twenty images per sample were acquired at 5,000X magnification. Using ImageJ25, the 2-dimensional sizes in µm2 of all individual mitochondria with a circularity index of ≥0.9 were calculated (for standardization purposes, only mitochondria cut near perfectly along their minor axis were evaluated). Ten images per sample were also acquired at 2,000X magnification, always including the epithelial cell brush border. Using ImageJ, a grid of 1.32 µm2 squares of defined size was overlaid onto each image. All mitochondria in the images were counted, except those in the first row of squares falling on the brush border and in squares along the image rims which only partially covered tissue), and the mean numbers of mitochondria per µm2 were calculated for each of the acquired 2,000X images (range of counted squares per image: 23 to 50). Mitochondrial sizes and mean numbers per µm2 were compared between time point 0 and time point VII using unpaired two-sided t tests.
General Statistics
All p values reported were adjusted for multiple testing as appropriate, except for the exploratory protein mass spectrometry data. Correlations of log2 fold gene expression changes between 9 cm and 15 cm biopsies in Figure 1C and Figure S1 were tested by Spearman's rank correlation coefficient. Gene and protein expression changes over baseline were compared between study arms by two-tailed Mann-Whitney test (Figure S2 and S7). The combined expression data from the microarray and RT-ddPCR tests shown in Figure 2C were compared separately for log-fold induction or suppression over baseline using a one-tailed Wilcoxon signed-rank test with Bonferroni adjustment. Immuno-histology measurements in Figure 2D were compared between baseline and after seven days of treatment by two-tailed paired t tests. Due to high skewness of the CD7+, CD3+ and IL-10+ cell counts, these were tested after log10 transformation. Ratios of induced to suppressed genes in Figure 3A were tested for a difference between cellular compartments using Chi-square statistics. DSP, IL-10, KIAA0101, PNPT1 and ATP6 copy numbers or protein concentrations in Figure 4B, 4C, 5A, 5B and 5G were tested for significant change across three time points by repeated measures ANOVA, and exact Bonferroni-adjusted post-tests were run for comparisons between two time points. Due to high skewness of the KIAA0101 copy numbers, these were tested after log10 transformation. The effect of increasing tenofovir concentrations on the proliferation of primary vaginal epithelial cells in Figure 4D was assessed for significance by a linear model accounting for days and donors. Copy numbers between 9 cm and 15 cm biopsies in Figure 5A were compared by two-tailed paired t test. Copy numbers between baseline tenofovir and N-9 in Figure 5B were compared by a two-tailed unpaired t test. Mitochondria counts and sizes in Fig. 5D and D were compared between baseline and Day VII by two-tailed unpaired t tests. Microarray chip probe expression values were tested for correlation between the three primary vaginal cell cultures by computing pairwise Pearson correlation coefficients (Table S1). Statistics packages used were Prism 6 (Graphpad Software) and Bioconductor/Lumi in R.
Author Contributions
FH, LB, JD, MJM and IM designed the microarray study. FH and LB developed microarray technologies. LB carried out microarray and RT-ddPCR experiments. AB and TBB carried out the protein mass spectrometry experiments. LV, JS, LB and FH carried out the electron microscopy experiments. LB and LV established the primary epithelial cell cultures. CH, RDC, and KHM recruited study participants and acquired participant data. FH, LB, JD, SF, RG and MJC analyzed the gene expression data. CH, PA, SJ, RDC and KHM provided operational support for the clinical study. JP advised on all aspects of the study. DRF provided the study drug. IM designed the clinical trial. FH is the principal author of this manuscript, and LB, SH, MJM and IM contributed to writing the manuscript.
Funding
Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers U01AI068633 (to Sharon Hillier), U19AI082637 (to I. M.) and R01HD51455 (to F.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or CONRAD. The funders had no role in data collection and analysis, and decision to publish. Jeanna Piper, employee of the National Institute of Allergy and Infectious Diseases, participated in design of the clinical study and preparation of the manuscript.
Acknowledgements
We thank the study participants for their time and effort, and staff in the participating clinics for enrolling and following study participants. We thank Peter Wilkinson and Rafick-Pierre Sékaly from the Vaccine and Gene Therapy Institute of Florida and Sangsoon Woo from the Vaccine and Infectious Disease Division at the Fred Hutchinson Cancer Research Center for advice about microarray data analysis; Gustavo Doncel from CONRAD and the Eastern Virginia Medical School, David Fredricks and Stephen Voght from the Fred Hutchinson Cancer Research Center, and Anna Wald from the University of Washington, for critical reading and editing of the manuscript; Keith Jerome from the Vaccine and Infectious Disease Division at the Fred Hutchinson Cancer Research Center for assistance with ddPCR assays; Julie Randolph-Habecker and Kim R. Melton from the Experimental Histopathology and Bobbie Schneider from the Electron Microscopy Shared Resources Core Facility at the Fred Hutchinson Cancer Research Center for assistance with immunohistochemistry and electron micropscopy; Cary A. Moody from the Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, for providing the 3T3-H2 fibroblast feeder cell line; Gretchen Lentz and Michael Fialkow from the Department of Obstetrics and Gynecology at the University of Washington for procuring vaginal tissues for the isolation of vaginal keratinocytes; Aornrutai Promsong and Surada Satthakarn, currently at Prince of Songkla University, Thailand, for help with establishing the four primary vaginal keratinocyte cultures; Allison A. McBride from the Laboratory of Viral Diseases at the National Institute of Allergy and Infectious Diseases for advice regarding the culture of primary vaginal keratinocytes; and Craig W. Hendrix and Mark A. Marzinke from the Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University School of Medicine, for measuring the intracellular concentration of tenofovir diphosphate in primary vaginal keratinocytes treated with tenofovir in vitro; Kenzie Birse from the Department of Medical Microbiology, University of Manitoba, for support in proteomic data analysis; Max Abou, Garrett Westmacott and Stuart McCorrister of the Proteomic Core of the National Microbiology Laboratory at the Public Health Agency of Canada for proteomic sample preparation and technical assistance in mass spectrometry.
Footnotes
The authors have declared that no competing interests exist.