Human Immunodeficiency Virus Infects Human Seminal Vesicles in Vitro and in Vivo
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.08.005
ISSN1525-2191
AutoresClaire Deléage, Marina Moreau, N. Costedoat‐Chalumeau, Annick Ruffault, Bernard Jégou, Nathalie Dejucq‐Rainsford,
Tópico(s)Sex work and related issues
ResumoSemen represents the main vector of HIV dissemination worldwide, yet the origin of HIV in semen remains unclear. Viral populations distinct from those found in blood have been observed in semen, indicating local viral replication within the male genital tract. The seminal vesicles, the secretions of which constitute more than 60% of the seminal fluid, could represent a major source of virus in semen. This study is the first to investigate the susceptibility of human seminal vesicles to HIV infection both in vitro and in vivo. We developed and characterized an organotypic culture of human seminal vesicles to test for target cells and HIV infection, and, in parallel, analyzed the seminal vesicle tissues from HIV-infected donors. In vitro, in contrast to HIV-1 X4, HIV-1 R5 exposure induced productive infection. Infected cells consisted primarily of resident CD163+ macrophages, often located close to the lumen. In vivo, HIV protein and RNA were also detected primarily in seminal vesicle macrophages in seven of nine HIV-infected donors, some of whom were receiving prolonged suppressive highly active antiretroviral therapy. These results demonstrate that human seminal vesicles support HIV infection in vitro and in vivo and, therefore, have the potential to contribute virus to semen. The presence of infected cells in the seminal vesicles of treated men with undetectable viremia suggests that this organ could constitute a reservoir for HIV. Semen represents the main vector of HIV dissemination worldwide, yet the origin of HIV in semen remains unclear. Viral populations distinct from those found in blood have been observed in semen, indicating local viral replication within the male genital tract. The seminal vesicles, the secretions of which constitute more than 60% of the seminal fluid, could represent a major source of virus in semen. This study is the first to investigate the susceptibility of human seminal vesicles to HIV infection both in vitro and in vivo. We developed and characterized an organotypic culture of human seminal vesicles to test for target cells and HIV infection, and, in parallel, analyzed the seminal vesicle tissues from HIV-infected donors. In vitro, in contrast to HIV-1 X4, HIV-1 R5 exposure induced productive infection. Infected cells consisted primarily of resident CD163+ macrophages, often located close to the lumen. In vivo, HIV protein and RNA were also detected primarily in seminal vesicle macrophages in seven of nine HIV-infected donors, some of whom were receiving prolonged suppressive highly active antiretroviral therapy. These results demonstrate that human seminal vesicles support HIV infection in vitro and in vivo and, therefore, have the potential to contribute virus to semen. The presence of infected cells in the seminal vesicles of treated men with undetectable viremia suggests that this organ could constitute a reservoir for HIV. Every year, approximately 2.5 million persons become newly infected with HIV, most through heterosexual transmission (World Health Organization and United States estimates). Although semen represents the foremost vector of HIV-1 dissemination,1Royce R.A. Sena A. Cates Jr, W. Cohen M.S. Sexual transmission of HIV.N Engl J Med. 1997; 336: 1072-1078Crossref PubMed Scopus (909) Google Scholar the precise origins of the infected leukocytes and free viral particles contaminating the seminal plasma remain unclear. Phylogenetic studies have established that HIV in semen arises from local sources within the male genital tract and/or from passive diffusion via the blood2Anderson J.A. Ping L.H. Dibben O. Jabara C.B. Arney L. Kincer L. Tang Y. Hobbs M. Hoffman I. Kazembe P. Jones C.D. Borrow P. Fiscus S. Cohen M.S. Swanstrom R. HIV-1 populations in semen arise through multiple mechanisms.PLoS Pathog. 2010; 6: e1001053Crossref PubMed Scopus (105) Google Scholar, 3Pillai S.K. Good B. Pond S.K. Wong J.K. Strain M.C. Richman D.D. Smith D.M. Semen-specific genetic characteristics of human immunodeficiency virus type 1 env.J Virol. 2005; 79: 1734-1742Crossref PubMed Scopus (93) Google Scholar, 4Ghosn J. Viard J.P. Katlama C. de Almeida M. Tubiana R. Letourneur F. Aaron L. Goujard C. Salmon D. Leruez-Ville M. Rouzioux C. Chaix M.L. Evidence of genotypic resistance diversity of archived and circulating viral strains in blood and semen of pre-treated HIV-infected men.AIDS. 2004; 18: 447-457Crossref PubMed Scopus (81) Google Scholar (previous references in Le Tortorec and Dejucq-Rainsford5Le Tortorec A. Dejucq-Rainsford N. HIV infection of the male genital tract: consequences for sexual transmission and reproduction.Int J Androl. 2010; 33: e98-e108Crossref PubMed Scopus (47) Google Scholar). The existence of productive sources in the male genital tract is further substantiated through observations of several differences between blood and semen, including i) detection of persistent infectious HIV in the semen of 5% to 30% of men with undetectable blood viral load receiving fully suppressive antiretroviral therapy5Le Tortorec A. Dejucq-Rainsford N. HIV infection of the male genital tract: consequences for sexual transmission and reproduction.Int J Androl. 2010; 33: e98-e108Crossref PubMed Scopus (47) Google Scholar, 6Halfon P. Giorgetti C. Khiri H. Penaranda G. Terriou P. Porcu-Buisson G. Chabert-Orsini V. Semen may harbor HIV despite effective HAART: another piece in the puzzle.PLoS One. 2010; 5: e10569Crossref PubMed Scopus (42) Google Scholar, 7Sheth P.M. Kovacs C. Kemal K.S. Jones R.B. Raboud J.M. Pilon R. la Porte C. Ostrowski M. Loutfy M. Burger H. Weiser B. Kaul R. 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Detection and quantification of HIV-1 in semen: identification of a subpopulation of men at high potential risk of viral sexual transmission.AIDS. 1999; 13: 823-831Crossref PubMed Scopus (140) Google Scholar; iii) different rates, kinetics of emergence, and diversity of drug-resistant strains4Ghosn J. Viard J.P. Katlama C. de Almeida M. Tubiana R. Letourneur F. Aaron L. Goujard C. Salmon D. Leruez-Ville M. Rouzioux C. Chaix M.L. Evidence of genotypic resistance diversity of archived and circulating viral strains in blood and semen of pre-treated HIV-infected men.AIDS. 2004; 18: 447-457Crossref PubMed Scopus (81) Google Scholar, 11Eron J. Vernazza P.L. Johnston D.M. Seillier-Moiseiwitsch F. Alcorn T.M. Fiscus S.A. Cohen M.S. Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission.AIDS. 1998; 12: F181-F189Crossref PubMed Scopus (160) Google Scholar, 12Kroodsma K.L. Kozal M.J. Hamed K.A. Winters M.A. Merigan T.C. Detection of drug resistant mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene: differences in semen and blood HIV-1 RNA and proviral DNA.J Infect Dis. 1994; 170: 1292-1295Crossref PubMed Scopus (61) Google Scholar; and iv) different ratio of infected versus noninfected leukocytes.13Kiessling A.A. Fitzgerald L.M. Zhang D. Chhay H. Brettler D. Eyre R.C. Steinberg J. McGowan K. Byrn R.A. Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir.AIDS Res Hum Retroviruses. 1998; 14: S33-S41PubMed Google Scholar At present, the nature of the sources of HIV in the male genital tract remains unclear. This knowledge is crucial to the understanding of the biology of HIV sexual transmission and to the design of targeted therapies for eradicating HIV from semen. Semen is composed of secretions and cells from the testes, epididymides, prostate, seminal vesicles, and bulbo urethral glands. Vasectomy has little effect on seminal shedding of HIV-1 RNA,14Krieger J.N. Nirapathpongporn A. Chaiyaporn M. Peterson G. Nikolaeva I. Akridge R. Ross S.O. Coombs R.W. Vasectomy and human immunodeficiency virus type 1 in semen.J Urol. 1998; 159: 820-825Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar which suggests that the testes and epididymides are not the primary sources of HIV particles in semen. The seminal vesicles, the secretions of which represent more than 60% of the seminal fluid, could be an important source of seminal HIV. We recently demonstrated that the seminal vesicles of asymptomatic macaques are productively infected by simian immunodeficiency virus (SIV) in vivo and, together with the prostate, exhibit the highest level of infection among the male genital tract organs in this animal model.15Le Tortorec A. Le Grand R. Denis H. Satie A.P. Mannioui K. Roques P. Maillard A. Daniels S. Jegou B. Dejucq-Rainsford N. Infection of semen-producing organs by SIV during the acute and chronic stages of the disease.PLoS One. 2008; 3 (e1792)Crossref PubMed Scopus (50) Google Scholar To date, infection of human seminal vesicles by HIV has not been reported. To test the hypothesis that the human seminal vesicles may represent a source of virus in semen, we developed an organotypic culture of human seminal vesicle tissue to assess whether the resident immune cells or other cell types present in this organ are susceptible to in vitro infection by HIV-1 strains with different cell tropism, and searched for the presence of infected cells in the seminal vesicles in HIV-infected men and analyzed the nature of the infected cell types. The following reagents were used: Dulbelcco's modified Eagle's medium, and RPMI 1640 medium, fetal calf serum (FCS), and glutamine (all three from Gibco-BRL, Life Technologies, Cergy-Pontoise, France), 5α-dihydrotestosterone and phytohemagglutinin (both from Sigma-Aldrich Chimie SNC, St. Quentin Fallavier, France), penicillin-streptomycin (Eurobio Laboratories, Les Ulis, Courtaboeuf, France), IL-2 (Boerhinger-Mannheim GmbH, Mannheim, Germany), and human serum (Jackson ImmunoResearch Europe, Ltd., Newmarket, Suffolk, England). Primary antibodies, isotypic controls, and concentrations used were as follows: mouse monoclonal antibodies against HIV p24 (2 μg/mL, clone Kal-1), CD3 (T-lymphocyte marker, 7.3 μg/mL, clone F.7.2.38), CD1a (Langerhans/immature dendritic cell marker, ready to use, clone 010), CD8 (20 μg/mL, C8/144B), HLA-DR α-chain (1.5 μg/mL, clone TAL.1B5), α-actin (smooth muscle) antibody (smooth muscle cells and myofibroblasts marker, 0.1 μg/mL, clone 1A4), cytokeratin (epithelial cell marker, ready to use, clone AE1/AE3), and Ki-67 (0.55 μg/mL, clone MIB-1) (all from Dako SA, Trappes, France), CD4 (2.5 μg/mL, clone 4B12, Novocastra Laboratories, Ltd., Newcastle-upon-Tyne, England), CD83 (mature dendritic cell marker, 1:20, clone 1H4b, Leica Biosystems Newcastle, Ltd., Newcastle-upon-Tyne, England), CCR5 (25 μg/mL, clone 183, R&D Systems, Inc., Minneapolis, MN), CXCR4 (10 μg/mL, clone 12G5, Dr. J Hoxie, NIBSC [National Institute for Biological Standards and Control] Centralised Facility for AIDS Reagents, Potters Bar, Hertfordshire, England); and matching mouse isotypic controls IgG2a and IgG2b (R&D Systems, Inc.), IgG1 kappa (Dako SA); rabbit polyclonal anti-CD3 (20 μg/mL, Sigma-Aldrich Chimie SNC), CD163 (monocyte/macrophage marker,16Lau S.K. Chu P.G. Weiss L.M. CD163: a specific marker of macrophages in paraffin-embedded tissue samples.Am J Clin Pathol. 2004; 122: 794-801Crossref PubMed Google Scholar, 17Nguyen T.T. Schwartz E.J. West R.B. Warnke R.A. Arber D.A. Natkunam Y. Expression of CD163 (hemoglobin scavenger receptor) in normal tissues, lymphomas, carcinomas, and sarcomas is largely restricted to the monocyte/macrophage lineage.Am J Surg Pathol. 2005; 29: 617-624Crossref PubMed Scopus (174) Google Scholar 2 μg/mL, K20T, Novus Biologicals, LLC, Littleton, CO), and control IgG rabbit (Jackson ImmunoResearch Europe, Ltd.). Mounting medium with DAPI was used for immunofluorescence (Vectashield; Vector Laboratories, Ltd., Peterborough, England). Secondary antibodies were either biotinylated (anti-mouse or anti-rabbit IgG, 1:500, Dako SA) or fluorescently labeled [goat anti-mouse Alexa Fluor 488 (4 μg/mL) or goat anti-rabbit Alexa Fluor 594 (4 μg/mL)], Invitrogen BP, Cergy-Pontoise, France). HIV-1 clade B R5SF162 and X4IIIB strains were obtained from the NIBSC Centralised Facility for AIDS Reagents. They were grown in peripheral blood mononuclear cells (PBMCs) stimulated using phytohemagglutinin (3 μg/mL) and IL-2 (5%) or in C8166 cells (for X4IIIB) to provide viral stocks. The study protocol was approved by the local ethics committee, and informed consent was obtained from all donors. Normal seminal vesicles were obtained at Rennes University Hospital from patients seronegative for HIV-1 who underwent radical prostatectomy and had not received hormone treatment. Immediately after surgery, seminal vesicle tissues were placed at 4°C in fresh medium supplemented with antibiotics and processed within 1 hour. The absence of disease was assessed at histologic analysis. Seminal vesicles were dissected into 2 × 2 × 6-mm longitudinal sections, and each section was transferred onto a polyethylene terephthalate insert in a well of a 12-well plate (Falcon Labware; Becton Dickinson & Co., Lincoln Park, NJ) containing 1 mL medium (RPMI 1640 with antibiotics, 10% fetal calf serum, and 800 ng/mL 5α-dyhydrotestosterone). For each experimental condition, two wells were tested. The culture was maintained for 15 days in a humidified atmosphere containing 5% CO2 at 37°C, and the medium was changed every 2 days and stored frozen at −80°C. Every 3 days, seminal vesicle explants were either fixed in neutral buffered 4% formaldehyde or frozen and stored at −80°C. Immunohistochemistry using the avidin-biotin-peroxidase complex technique was performed on formaldehyde-fixed, paraffin-embedded (FFPE) tissues as previously described.18Roulet V. Satie A.P. Ruffault A. Le Tortorec A. Denis H. Guist'hau O. Patard J.J. Rioux-Leclerq N. Gicquel J. Jegou B. Dejucq-Rainsford N. Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro.Am J Pathol. 2006; 169: 2094-2103Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar In brief, after antigen retrieval [1 mmol/L EDTA (pH 9.0) or 10 mmol/L citrate (pH 6.0) for 20 minutes] and blockage of unspecific sites with 4% normal human serum in PBS, tissues were reacted at 4°C overnight using the appropriate antibody diluted in PBS containing 2% normal human serum. The primary antibody was replaced with the appropriate isotopic control at the same concentration in control sections. Sections were washed and reacted with a biotinylated secondary antibody at room temperature for 1 hour. After subsequent washes, the sections were incubated using the avidin-biotin-peroxidase complex (Vectastain ABC Kit; Vector Laboratories, Ltd.) for 30 minutes, and bound antibodies were visualized using aminoethyl carbazole substrate or 3,3′-diaminobenzidine (Dako SA). The nuclei were counterstained using Masson's hemalum. The sections were observed and photographed using an AxioImager M1 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Cell staining was never observed for istotypic controls. Cell counting was performed on tissues from a minimum of three donors using Cast software (Olympus, Lille, France). Cells that stained positive were counted at 40× magnification in the total surface of one or several tissue sections per individual (minimum surface, 30 mm2). Total RNA was extracted from the seminal vesicle explants at days 1, 9, 13, and 15 of culture using the RNeasy isolation kit (Quiagen SA, Courtaboeuf, France) and depleted of contaminating DNA via DNase treatment (Quiagen SA). cDNA was generated from 2 μg total RNA using M-MLV reverse transcriptase (SuperScript II; Gibco-BRL, Life Technologies). PCR was performed on 40-ng equivalent RNA with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA) using commercially available master mix and target probes (Applied Biosystems, Inc.): Hs00181217_m1 (CD4), Hs00607978_s1 (CXCR4), Hs00152917_m1 (CCR5), Hs99999901_s1 (18 S). For steroid dehydrogenase (accession No. NCBI GenBank: NM_003104.4), the following primers and probes were designed: forward, 5′-AGGATGCATTCTGTTGGAATC-3′; reverse, 5′-GCAACACGATCACCTGGTT-3′; and probe, 5′-TGGGACATGAAGCTTCGGGA-3′. The CT value of each gene was calculated using the ABI sequence detection system 1.9 program (Applied Biosystems, Inc.). The relative gene expression in a minimum of three independent cultures at various times was normalized to 18S expression and calculated using the comparative CT method, as previously described.19Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123424) Google Scholar Immediately after dissection, each explant was immersed in 200 to 500 μL of a stock of HIV-1 R5SF162 or X4IIIB cell-free viral supernatant [corresponding to 8 to 13 ng of reverse transcriptase activity and 1 to 4 × 104 TCID50 (median tissue culture infective dose)] for 3 hours (R5SF162 and X4IIIB) or overnight (X4IIIB) at 37°C and then thoroughly rinsed three times in PBS. The explants were placed in culture as described previously, and the culture medium was collected and replaced every 2 days throughout the culture. HIV-1 reverse transcriptase activity in the seminal vesicle supernatants was assayed using the Lenti-RT activity assay (Cavidi AB, Uppsala, Sweden) as previously described.18Roulet V. Satie A.P. Ruffault A. Le Tortorec A. Denis H. Guist'hau O. Patard J.J. Rioux-Leclerq N. Gicquel J. Jegou B. Dejucq-Rainsford N. Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro.Am J Pathol. 2006; 169: 2094-2103Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar For infectivity assay, 500 μL seminal vesicle culture supernatants collected at the day of peak reverse transcriptase activity or 500 μL viral stock used for infection and maintained at 37°C for 9 days, used here as a negative control, was ultracentrifuged for 3 hours at 39,500 × g. Supernatants were discarded, and the pellets were dissolved in 500 μL RPMI 1640, which was added to 4 × 106 phytohemagglutinin-activated PBMCs for 3 hours at 37°C. PBMCs were resuspended in 2 mL RPMI 1640 supplemented with 5% IL-2 and maintained at 37°C for 15 days. The culture medium was changed at day 1 and then every 3 days, and was stored frozen at −80°C for subsequent reverse transcriptase assay. Total DNA was extracted using the QIAamp DNA Mini Kit (Quiagen SA) according to the manufacturer's instructions. Quantitative real-time PCR for HIV-1 LTR DNA and for the albumin gene used as a reference was performed on 250 ng DNA, as previously described.18Roulet V. Satie A.P. Ruffault A. Le Tortorec A. Denis H. Guist'hau O. Patard J.J. Rioux-Leclerq N. Gicquel J. Jegou B. Dejucq-Rainsford N. Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro.Am J Pathol. 2006; 169: 2094-2103Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar For each donor and each time point, two separate blocks of tissue were analyzed in duplicate. For co-localization experiments, the FFPE tissue sections were analyzed via double-color immunostaining using the antibodies described in Materials (polyclonal antibodies anti-CD163 or anti-CD3 and monoclonal antibodies against either p24 antigen, HLA-DR, CCR5, CXCR4, or CD4). Antigen unmasking was performed via 10 minutes of irradiation in a microwave oven of deparaffinized sections in 10 mmol/L citrate buffer (pH 6). Sections were incubated with primary antibodies or isotypic controls for 1 to 2 hours at room temperature or overnight at 4°C, washed with Tris-buffered saline solution, and sequentially incubated with fluorescently labeled secondary antibodies. Cell nuclei were visualized using nuclear chromatin staining (DAPI). Slides were examined under a fluorescence microscope (AxioImager M1; Carl Zeiss MicroImaging GmbH) equipped with a charge-coupled device camera. Individual channels were collected simultaneously using computer software (AxioVision, version 4.7.1; Carl Zeiss MicroImaging GmbH). The percentage of double staining was calculated by counting co-labeled cells in 200 positive cells for one marker in distinct areas of the seminal vesicle from a minimum of three donors. Data are given as the mean percentage ± SEM of co-labeled cells for the indicated number of donors. After approval of our research protocol by National Disease Research Interchange (Phildelphia, PA), seminal vesicle tissues were obtained at autopsy from HIV-infected men and underwent PFPE. Characteristics of the donors are given in Table 1.Table 1Characteristics of Donors Infected with HIV-1DonorPlasma CD4+ cells (count/μL)Plasma HIV-1 RNA (copies/mL)Duration of HIV-1 infection (years)Co-infectionsARVCause of deathP24+ cells in SV⁎Number of cells/mm2.85NA139,000NAHCVNAAIDS2.20108<2051,681NAHCV/HBVCombivir, SustivaAIDS0.24111134<50NAHCVAtriplaLymphoma01 month†Time of measurement before death.1 month†Time of measurement before death.110219<5017NoneOxanadol, Itellence, Prezista, Viread, Isentress, NorvirEnd-stage renal disease0.121 week†Time of measurement before death.1 week†Time of measurement before death. (for 4 years)112256<5026HBVTenofovir, Epivir, Abacavir, TrizivirLiver cancer0.971 month†Time of measurement before death.1 month†Time of measurement before death.103269<50 (for 5 years)NAHCV/HBV/HTLVTenofovir, Sustiva, Ziagen, EpivirNephropathy0.61107437<5017HCV/HBVEpzicom, Reyataz, NorvirRespiratory failure due to pneumonia0.041 day†Time of measurement before death.1.5 month†Time of measurement before death. (previous AIDS)139495<5020NoneAtriplaMyocardial infarction02 months†Time of measurement before death.4 months†Time of measurement before death.921076<50NAHCV/HBVTruvada (stopped 1 week before death)Adenocarcinoma0.742 months†Time of measurement before death.4 months†Time of measurement before death.ARV, antiretroviral drugs; HBV, hepatitis B virus; HCV, hepatitis C virus; HTLV, human T cell leukemia virus; NA, not available; SV, seminal vesicles. Number of cells/mm2.† Time of measurement before death. Open table in a new tab ARV, antiretroviral drugs; HBV, hepatitis B virus; HCV, hepatitis C virus; HTLV, human T cell leukemia virus; NA, not available; SV, seminal vesicles. Localization and identification of cells expressing HIV-1 RNA was performed by combining radioactive in situ hybridization for HIV-1 Gag protein and immunohistochemical staining for cell markers, as previously described.18Roulet V. Satie A.P. Ruffault A. Le Tortorec A. Denis H. Guist'hau O. Patard J.J. Rioux-Leclerq N. Gicquel J. Jegou B. Dejucq-Rainsford N. Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro.Am J Pathol. 2006; 169: 2094-2103Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar The specificity of the hybridization signal was systematically checked by hybridizing sense probes on parallel sections and anti-sense probes on seminal vesicles from uninfected men. The significance of the differences or correlation between values was evaluated using the appropriate nonparametric test, as specified in the text or in the figure legends. P < 0.05 was considered statistically significant. Statistical analyses were performed using commercially available software (SAS version 9.1.3; SAS Institute, Inc., Cary, NC). The architecture and expression of seminal vesicle cell markers were compared in seminal vesicle explants before culture and throughout the culture period. Histologic examination of seminal vesicle fragments revealed that the tissue architecture was maintained throughout the 15-day culture period (Figure 1, A–D). Columnar epithelial cells retained their height up to day 5 (Figure 1, B versus A), and began to flatten therafter (Figure 1, C and D). Positive staining for cytokeratin observed for the pseudo-stratified epithelium before culture was maintained until the end of culture at day 15 (Figure 1, E and F). Similarly, quantification of the transcript-encoding steroid dehydrogenase, an enzyme produced by seminal vesicle epithelial cells, demonstrated expression of this cell marker throughout the culture period (Figure 1K). Stromal cells were still abundant at day 11 of culture (Figure 1, C versus A), with only a few patchy losses (data not shown), whereas a more generalized decrease was observed at day 15 (Figure 1D). Positive staining for α–actin, a marker of smooth muscle cells and myofibroblasts, was observed in the stroma before and at the end of the 15-day culture period (Figure 1, G and H). In the explants before culture, only a small number of cells were proliferating, as indicated by the detection of Ki-67 (Figure 1I). The number of cells increased only slightly during culture, primarily at the epithelial level (Figure 1J). Cells that stained positive for the monocyte/macrophage marker CD163, the T-lymphocyte marker CD3, and the HIV receptor CD4 were evident within the fixed seminal vesicle tissues from all donors before and during culture (Figure 2A). CD163+ cells were observed both in the stroma and inserted, or close to, the epithelium, whereas CD4+ cells were primarily concentrated within the stroma. In contrast, CD8+ cells were consistently observed, primarily inserted within the epithelium and rarely in the stroma (Figure 2A). Some rare cells stained positive for CD83, a marker of mature dendritic cells, whereas no staining was observed for CD1a, a marker of Langerhans/immature dendritic cells (data not shown). The HIV co-receptors CCR5 and CXCR4 (Figure 2A) were detected in cells with immune cell–type morphologic features, whereas no staining was present on epithelial cells. Quantitative immunohistochemistry revealed that the seminal vesicle contained primarily CD163+ macrophages and, to a lesser extent, CD3+ T lymphocytes and CD4+ cells, whereas stromal CD8+ cells were scarce (Figure 2B). Cells staining positive for CCR5 were far more abundant than those staining positive for CXCR4 (Figure 2B). To assess the nature of CD4+, CCR5+, and CXCR4+ cells, double labeling was performed (Figure 2C). CD4 staining co-localized with CD3+ T lymphocytes but not with CD163+ macrophages (Figure 2C), in line with the known low level of CD4 expression on macrophages.20Walter B.L. Wehrly K. Swanstrom R. Platt E. Kabat D. Chesebro B. Role of low CD4 levels in the influence of human immunodeficiency virus type 1 envelope V1 and V2 regions on entry and spread in macrophages.J Virol. 2005; 79: 4828-4837Crossref PubMed Scopus (51) Google Scholar CCR5 co-localized with CD163+ macrophages and co-labeled with 48.25% ± 27.8% (mean ± SEM; n = 4 donors) of CD3+ T lymphocytes (Figure 2C), whereas CXCR4 was exclusively detected in T lymphocytes (Figure 2C). Using real-time RT-PCR, we quantified the persistence during culture of the expression of transcripts encoding HIV receptors CD4, CCR5, and CXCR4. CD4 mRNA copy numbers were maintained throughout the culture period, and only a slight decrease was observed at day 15 (Figure 3). CCR5 transcript copy number also demonstrated good maintenance up to day 13, whereas a decrease was observed at day 15. In contrast, although expressed throughout the culture period, CXCR4 mRNA expression decreased from day 9 onward, and was significantly lower at days 13 and 15 compared with day 1 (Figure 3). After incubation of R5SF162 with seminal vesicle explants from six donors, a significant increase in reverse transcriptase activity was consistently observed between days 9 and 11 (Figure 4A). In contrast, in supernatants of seminal vesicle tissues from matched patients exposed to X4IIIB, no increase in reverse transcriptase activity was detected during the 15-day culture period (Figure 4A), irrespective of the duration of exposure (3 hours or overnight) and viral stock (X4IIIB grown in PBMCs or in the C8166 cell line). In line with this, the viral particles obtained by ultracentrifugation of R5SF162-exposed seminal vesicle supernatants productively infected activated PBMCs, whereas the ultracentrifuged supernatants from X4IIIB-exposed seminal vesicle cultures did not lead to any detectable PBMC infection (Figure 4B). The accumulation of HIV DNA within the seminal vesicle explants exposed to either R5SF162 or X4IIIB was quantified using real-time PCR (Figure 4C). After exposure to R5SF162, the HIV-1 DNA level increased almost 30-fold during culture, demonstrating productive infection. In contrast, no HIV DNA increase was observed in seminal vesicle explants exposed to X4IIIB. HIV-infected cells were localized in the explants by immunohistochemistry for the viral protein p24 (Figure 5A). HIV p24+ cells were found in the stroma and close to the epithelium of seminal vesicles exposed to HIV-1 R5SF162. In contrast, no infected cells were evident in explants exposed to HIV-1 X4IIIB (data not shown). Double staining for p24 and either the macrophage marker CD163 or the lymphocyte marker CD3 in HIV R5–infect
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