Human immunodeficiency virus infects human seminal vesicles in vitro and in vivo

Abstract

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.

Figure 1

Characterization of human seminal vesicles in organotypic culture. A–H: PFPE sections were examined morphologically (A–D) or immunostained for several seminal vesicle cell markers (E–J) before (E, G, and I) and after 15 days (F, H, and J) of culture. The overall architecture of the organ was preserved throughout the culture (A, day 0; B, day 5; C, day 11; D, day 15). The markers used for characterization of seminal vesicle cell types were cytokeratin for epithelial cells (E and F), α-actin (smooth muscle) and myofibroblastic cells (G and H), and Ki-67 for proliferating cells (I and J). K: The level of expression during the culture period of the transcript encoding SDH, an enzyme produced by seminal vesicle epithelial cells, was analyzed using real-time PCR and compared with control (day 1 of culture). The results represent the mean ± SD of a minimum of three independent cultures performed on seminal vesicles from three different donors (Kruskal-Wallis test, *P < 0.05; control, day 1). Scale bars = 50 μm.

Figure 2

Analysis of potential HIV target cells in uninfected seminal vesicles. A: Immunohistochemistry performed on uninfected seminal vesicle sections before and during culture (day 9) demonstrated the presence of scattered stromal cells staining positive for CD163, CD3, CD4, CCR5, and CXCR4. CD163⁺ cells were also observed in close contact with epithelial cells (arrows). CD8⁺ cells were found primarily at the level of the epithelium. Scale bars = 20 μm. B: Respective percentages of CD68⁺, CD3⁺, CD4⁺, CD8⁺, CCR5⁺, and CXCR4⁺ cells per surface unit before culture were evaluated in seminal vesicle sections from five donors. Results represent the mean number of positive cells ± SEM. C: HIV receptors bearing cells (CD4⁺, CXCR4⁺, and CCR5⁺) were characterized by double labeling with either CD3 (T-lymphocyte marker) or CD163 (macrophage marker). Nuclei labeled with DAPI are shown (blue). Large panel represents a merged image combining all channels. Side panels represent individual channels. Scale bars = 10 μm.

Figure 3

Real-time RT-PCR quantification of transcripts encoding HIV receptor CD4 and co-receptors CCR5 and CXCR4 in seminal vesicle explants during culture. The relative copy number of the cDNA of interest is standardized to the copy number of the ubiquitous 18S housekeeping gene cDNA and expressed relative to control (day 1 of culture). Results represent the mean ± SD of three independent experiments corresponding to three donors (Kruskal-Wallis test, *P < 0.05; control, day 1).

Figure 4

HIV-1 R5_SF162 and X4_IIIB infection of human seminal vesicles in organotypic culture. A: Reverse transcriptase activity measured in supernatants of human seminal vesicle explants demonstrated a significant increase between days 9 and 11 after exposure to HIV-1 R5_SF162 (circle, black line), whereas no increased reverse transcriptase activity was detected after exposure to HIV_IIIB (square, dotted line) (n = 6 donors in each case). B: Activated PBMCs exposed to supernatants of R5_SF162 infected explants collected at peak of reverse transcription demonstrated increased reverse transcriptase activity in the supernatants during culture (n = 3), whereas no increase was observed after exposure of activated PBMCs to X4_IIIB-infected seminal vesicle supernatants collected at different times during culture (n = 3; data shown for day 9). C: Accumulation of HIV-1 DNA in seminal vesicle explants between days 5 and 13 to 15 after exposure to either HIV-1 R5_SF162 or X4_IIIB, as assayed for LTR DNA using quantitative real-time PCR. For each virus, six different explants from six donors were tested. Results represent the mean ± SEM [Mann-Whitney test, *P < 0.05; control, day 7 (A), day 4 (B), and day 5 (C)].

Figure 5

Localization and characterization of HIV p24–positive cells in human seminal vesicles infected in vitro. A: Localization of cells positive for HIV-1 (red) within seminal vesicle explants infected for 9 days with R5_SF162, as detected using immunohistochemistry for the capsid protein p24. B: Double immunostaining for p24 (green) and cell markers CD163 (red) (B) or CD3 (red) (C) was performed to determine the nature of the infected cells. Nuclei labeled with DAPI are shown in blue. Large panel represents a merged image combining all channels. Side panels represent individual channels. Scale bars = 50 μm.

Figure 6

Localization and characterization of HIV-infected cells in seminal vesicles from HIV-positive donors. PFPE sections of seminal vesicles obtained at autopsy of HIV-infected donors were examined for the presence of HIV-infected cells using immunohistochemistry for HIV p24 (A) and in situ hybridization for HIV Gag RNA (B). Small arrows in A indicate infected p24⁺ cells. The open arrow in B indicates an infected HIV RNA⁺ cell co-localized with CD163 cell marker. Immunophenotyping of p24⁺ cells (green) using either CD163 (C) or CD3 (red) (D) demonstrated the presence of infected macrophages and T lymphocytes. Nuclei labeled with DAPI are shown (blue). Large panel represents a merged image combining all channels. Side panels represent individual channels. In C and D, open arrows indicate co-labeled cells; the small arrow in D indicates an infected cell in the lumen of the seminal vesicle epithelium (shown with dotted lines) that did not co-localize with CD3. Scale bars = 50 μm.

Figure 7

Quantification of CD163⁺, HLA-DR⁺, CD4⁺, and CD3⁺ cells in seminal vesicles from uninfected versus HIV-infected men. The respective percentages of CD163⁺, HLA-DR⁺, CD8⁺, CD4⁺, and CD3⁺ cells per surface unit were evaluated using immunohistochemistry in the seminal vesicles from a minimum of five uninfected donors and from nine HIV-infected men (Wilcoxon test, *P < 0.05; control, uninfected).