Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia

Abstract

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.

Fig 1

Detection of tight junction proteins in adult, infant, and fetal oral and fetal intestinal epithelia. (A) Representative immunofluorescence images of ZO-1 tight junction protein by confocal microscopy. Buccal tissues from a 41-year-old adult, 3-month-old infant, and 22-week-old fetus, as well as intestinal tissue from a 22-week-old fetus, were immunostained for ZO-1 (in red). Nuclei are stained in blue. Dashed white lines separate epithelium from the lamina propria. EP, epithelium; GR, granulosum; SP, spinosum; LP, lamina propria. Original magnification, ×600. (B, C, and D) Detection of tight junctions by electron microscopy. Adult buccal (B), fetal buccal (C), and fetal intestinal (D) epithelia were examined by electron microscopy. (B) Images are taken from the granulosum and spinosum layers. Insets in panel B and insets and red arrows in panels C and D show the electron-dense tight junction regions of lateral membranes. (E) Detection of insoluble tight junction proteins in adult and fetal buccal epithelia. The presence of tight junction proteins claudin-1 and occludin was examined by Western blotting of Triton X-100-soluble (S) and -insoluble (P) fractions of buccal epithelial extracts.

Fig 2

Penetration of HIV-1 into adult buccal mucosal epithelia. (A) A schematic model of the polarized oriented tissue explant system for studying cell-free HIV transmission. To establish polarized oriented mucosal epithelia, tissue explants were placed with the mucosal side facing up in the top chamber of a Millicell filter insert. The lateral edges of the explants were sealed with 3% agarose, and GFP-labeled HIV virions were added to the apical surfaces. After 4 h, tissues were fixed, sectioned, and examined for virus penetration by confocal immunofluorescence microscopy. (B) Representative fluorescence microscopy images of adult oral tissues exposed to GFP-labeled HIV-1. Adult buccal explants exposed to HIV-1_NL4-3 (X4) virions for 4 h at 37°C were immunostained for rabbit anti-GFP (green) and goat anti-HIV-1 p24 (red, upper panels) or normal goat IgG (red, lower panels). Yellow in merged panel indicates colocalization of HIV-Gag with GFP-labeled (green) virions. GR, granulosum; SP, spinosum; LP, lamina propria; BL, basal. White lines indicate the border between the lamina propria and the mucosal epithelium. Original magnification of images, ×400. Insets show GFP signals from virions at higher magnification. (C) To quantify HIV penetration into the adult buccal epithelium, GFP-labeled HIV-1_NL4-3 (X4) and HIV-1_81-A (R5) virions were added to the apical surfaces of polarized oriented matching adult tissue explants obtained from 3 independent donors. Tissues were sectioned and immunostained for HIV p24 and occludin. Quantitative evaluation of HIV penetration into epithelia was performed by counting HIV-GFP- and p24-positive cells within the granulosum layers (1 to 5 layers), where virus penetration was detected. Cells were counted in a minimum of 3 sections and 10 fields of each section. Error bars show ± standard errors of the means (±SEM). (D) For detection of HIV penetration into adult oral epithelium by electron microscopy, adult buccal tissues exposed to HIV-1_81-A (R5) virions were examined by electron microscopy. Mature HIV virions are visible in the cytoplasm of granulosum layers of oral epithelium (red arrowheads and inset).

Fig 3

Paracellular penetration of HIV into adult oral epithelia. (A) Adult buccal explants from 3 independent donors were incubated in media containing 10 mM EDTA for 2 h (disrupted junctions). One matching explant was not treated and served as a control (intact junctions). GFP-labeled HIV-1_81-A (R5) was added to the surfaces of explants for 4 h. Tissues were fixed, sectioned, and immunostained for GFP and ZO-1 (red). Cells were analyzed by confocal microscopy, and representative images from three independent tissues are shown. Green indicates GFP-labeled virions. Nuclei are stained in blue. GR, granulosum; SP, spinosum; LP, lamina propria; EP, epithelium; BL, basal. Original magnification, ×600. Insets show GFP-labeled virions at higher magnification. (B) For quantitative evaluation of HIV paracellular penetration, HIV-containing epithelial cells were counted within the granulosum, spinosum, and parabasal layers of untreated and EDTA-treated explants. Average numbers of HIV-containing epithelial cells from explants of 3 independent donors are presented. Cells were counted in a minimum of 10 fields. Error bars show ±SEM. (C) To examine the infectivity of HIV that had penetrated into adult oral epithelium, polarized oriented explants were propagated from buccal biopsy specimens of three independent adult donors. One of the matching explants was treated with EDTA, and one was left untreated. R5-tropic HIV-1_SF170 or X4-tropic HIV-1_92UG029 viruses were applied to the surfaces of intact and EDTA-disrupted tissue explants for 4 h. Explants were homogenized, and 200 ml of supernatant from tissue homogenate was used for infection of 10⁶ PBMCs. After 2 and 3 weeks, HIV-1 p24 was detected in PBMCs by the use of an ELISA p24 assay. *, not detected.

Fig 4

Distribution of HIV-susceptible lymphocytes, macrophages, and dendritic/Langerhans cells in infant and fetal oral and fetal intestinal epithelium. (A) Tonsil tissue from a 3-month-old infant and oropharyngeal tissue from a 22-week-old fetus were immunostained for CD3, CD68, and CD1a markers for T lymphocytes, macrophages, and Langerhans cells, respectively. Infant tissues were also stained with isotype control antibodies for each marker. Intestinal tissues from a 22-week-old fetus were immunostained for CD3 and CD68, as well as for DC-SIGN, which is a marker for dendritic cells. All cell markers are in red, and nuclei are in blue. Representative images are shown. EP, epithelium; LP, lamina propria. A dashed yellow line indicates the boundary between epithelium and lamina propria. Original magnification, ×400. (B to E) Quantitative evaluation of HIV target cells within infant and fetal oral and fetal intestinal epithelia. (B, C, and D) CD3-, CD68-, and CD1a-positive cells in infant and fetal buccal mucosa were counted. (E) In the fetal intestinal mucosae, CD3-, CD68-, and DS-SIGN-positive cells were counted. (B to E) WKS, weeks. Cells were counted in a minimum of 10 separate fields, and results are presented as the average number of cells per square millimeter. Error bars show ±SEM.

Fig 5

Penetration of HIV-1 into fetal oral and intestinal mucosal epithelia. (A) Representative confocal immunofluorescence images of fetal oropharyngeal and intestinal tissues exposed to cell-free HIV-1. GFP-labeled HIV-1_NL4-3 (X4) virions were added to the apical surfaces of polarized oriented fetal oropharyngeal and intestinal tissue explants. Tissues were incubated at 37°C or 4°C for 4 h. In parallel experiments, fetal tissues were pretreated with 10 μM colchicine for 2 h at 37°C and then used for virion penetration at 37°C. Tissue sections were coimmunostained for GFP and occludin. Original magnification, ×600. (B) For quantitation of HIV penetration into fetal oropharyngeal and intestinal epithelia exposed to HIV-1_NL4-3 (X4) or HIV-1_81A (R5) virions, tissue sections were immunostained for HIV-p24 and CD45⁺, which is a marker for white blood cells, including HIV-susceptible immune cells. Virus penetration into epithelia was evaluated by counting p24-positive CD45⁺ cells. Cells were counted in a minimum of 10 fields. Error bars show ±SEM. Average numbers of HIV-infected CD45⁺ cells per square millimeter from 3 independent tissues are presented. (C and D) For electron microscopy detection of HIV penetration into fetal oral and intestinal epithelium, oropharyngeal and intestinal tissues were exposed to HIV-1_81-A (R5) virions for 4 h and then fixed and examined by electron microscopy. Mature virions are present in the cytoplasm of fetal oral and intestinal epithelium (red arrowheads and inset). (E) To examine the role of HIV-1 coreceptors in HIV transmission through fetal oral mucosal epithelia, polarized oriented oropharyngeal tissue explants from two independent donors were exposed to antibodies against GalCer, HSPG, CXCR4, or CCR5. After the explants were washed, R5-tropic HIV-1_SF170 or X4-tropic HIV-1_92UG029 viruses were applied to the surfaces of tissues for 4 h. Explants were fixed and immunostained for HIV by the use of pooled antibodies against gp41, gp120, and p24. Quantitative analysis was performed by counting HIV-positive epithelial cells in the explants treated with specific and control antibodies. HIV-positive epithelial cells were counted in at least 10 fields. Inhibition of viral penetration by antibodies is expressed as the percentage of HIV-positive epithelial cells in the tissues exposed to specific antibodies relative to HIV-positive cells in the tissues exposed to the isotype control antibodies. The average percentage of inhibition determined for two tissue explants obtained from two independent donors is presented. Error bars show ±SEM.

Fig 6

Detection of HIV-infected immune cells in fetal oral and intestinal epithelium. (A) For detection of HIV-infected immune cells, fetal oropharyngeal epithelia were exposed to GFP-labeled HIV-1_81-A (R5) virions (upper panels) and HIV-1_NL4-3 (X4) virions (lower panels) for 4 h at 37°C. Tissue sections were coimmunostained for GFP and CD3⁺, CD68⁺, or CD1a⁺ cells (all in red). (B) Fetal intestinal epithelium exposed to GFP-labeled HIV-1_81-A (R5) virions (upper panels) and HIV-1_NL4-3 (X4) virions (lower panels) for 4 h at 37°C was coimmunostained for GFP and CD3⁺ or CD68⁺ cells (all in red). (A and B) Insets show GFP signals from virions at higher magnification. Representative images from 3 fetal buccal and intestinal tissues are shown. Original magnification of images, ×600. EP, epithelium; LP, lamina propria. (C) For quantitative analysis of HIV-infected immune cells, tissue sections were immunostained for HIV-p24, as well as for CD3, CD68, and CD1a, which are markers for T lymphocytes, LCs, and macrophages, respectively. HIV-1 p24-positive immune cells expressing CD3, CD68, or CD1a were counted in at least 10 fields. Average data are presented from three tissue explants obtained from three independent donors. Error bars show ±SEM. (D) To determine the infectivity of HIV within the fetal oral and intestinal epithelium, polarized oriented oral and intestinal explants from three independent donors were exposed to R5-tropic HIV-1_SF170 or X4-tropic HIV-1_92UG029 viruses. After 4 h, explants were extensively washed and homogenized. PBMCs were infected with 200 ml of supernatant of tissue homogenate, and after 1 week, HIV-1 p24 was detected in PBMCs by the use of an ELISA p24 assay. Average data from three independent tissue explants are presented.

Fig 7

Cell-associated HIV-1 does not penetrate into the adult oral mucosal epithelium. (A) A schematic model of the polarized oriented tissue explant system for studying cell-associated HIV transmission. R5-tropic HIV-infected macrophages and X4-tropic HIV-1-infected lymphocytes were labeled with 10 μM CFSE for 10 min. HIV-infected and CFSE-labeled macrophages and lymphocytes were added to the mucosal surfaces of polarized oriented explants. After 4 h, tissue explants were fixed and sectioned, and cross-sections were examined for CFSE-labeled cells (green) coimmunostained with goat anti-HIV-1 antiserum (red). Colocalization of the red HIV signal with the green CFSE signal generates a yellow signal. Cells with yellow signals represent transmigration of HIV-infected cells through the mucosal epithelium. Cells containing only red signal represent HIV-infected fetal cells, which might have been infected by cell-free virions released from apically added adult lymphocytes or macrophages. (B) X4-tropic HIV-1_92UG029- and R5-tropic HIV-1_SF170-infected CD4⁺ lymphocytes and CD68⁺ macrophages, respectively, were labeled with CFSE (green) and added to the mucosal surfaces of adult buccal tissues. After 4 h, tissues were fixed, sectioned, and immunostained with goat anti-HIV-1 antiserum (red). Merged panels are shown. (C) To determine the integrity of cell junctions in adult oral epithelia exposed to HIV-infected lymphocytes and macrophages, sections of buccal explants incubated with HIV-infected lymphocytes and macrophages were immunostained for the tight junction protein ZO-1 (red). Cell nuclei are stained in blue. (B and C) Original magnification, ×400. Images are representative of explants from 3 independent donors.

Fig 8

Penetration of cell-associated HIV-1 into fetal oral mucosal epithelium. (A) CFSE-labeled X4-tropic HIV-1_92UG029- and R5-tropic HIV-1_SF170-infected CD4⁺ lymphocytes and CD68⁺ macrophages, respectively, were added to the mucosal surfaces of fetal buccal tissues. After 4 h, tissues were fixed, sectioned, and immunostained with goat anti-HIV-1 antiserum (red). Merged panels are shown. CFSE-labeled lymphocytes and macrophages are in green. HIV-infected cells are in red. Yellow indicates colocalization of CFSE-labeled and HIV-infected cells. (B) For quantitative analysis of transmigration of HIV-infected lymphocytes and macrophages into fetal oral epithelia, HIV-infected and CFSE-labeled cells (HIV⁺/CFSE⁺ cells) were counted. HIV-infected and CFSE-unlabeled cells (HIV⁺/CFSE⁻ cells) were also counted. Cell numbers per square millimeter are presented. In each section, cells were counted in a minimum of 10 fields. Average numbers of HIV⁺/CFSE⁺ and HIV⁺/CFSE⁻ cells per square millimeter are presented from 3 independent tissues. *, not detected. Error bars show ±SEM. (C) To examine the tight junctions of fetal oral epithelium exposed to HIV-infected lymphocytes and macrophages, tissue sections were immunostained for the tight junction protein ZO-1 (red). Cell nuclei are stained in blue. (A and C) Original magnification, ×400. Images are representative of three independent experiments.

Fig 9

Penetration of cell-associated HIV-1 into fetal oral and intestinal mucosal epithelium in the presence of breast milk. (A) X4-tropic HIV-1_92UG029- and R5-tropic HIV-1_SF170-infected CD4⁺ lymphocytes and CD68⁺ macrophages, respectively, were labeled with CFSE (green) and resuspended in breast milk. Cells with breast milk were then added to the mucosal surfaces of fetal buccal and intestinal epithelia for 4 h. Tissues were fixed, sectioned, and immunostained with goat anti-HIV-1 antiserum (red). Merged panels are shown. Green shows CFSE-labeled lymphocytes or macrophages. Red indicates HIV-infected cells. Yellow indicates colocalization of CFSE-labeled and HIV-infected cells. Original magnification, ×400. Representative images from 3 independent tissues are shown. (B) For quantitative evaluation, HIV-infected and CFSE-labeled cells (HIV⁺/CFSE⁺ cells) and HIV-infected and CFSE-unlabeled cells (HIV⁺/CFSE⁻ cells) were counted. Cell numbers per square millimeter are presented. In each section, cells were counted in a minimum of 10 fields. Average numbers of HIV⁺/CFSE⁺ and HIV⁺/CFSE⁻ cells per square millimeter are presented from 3 independent tissues. *, not detected. Error bars show ±SEM.

Fig 10

Model of HIV transmigration in adult (A) and infant/fetal oral (B) and infant/fetal intestinal (C) epithelial cells. The adult oral mucosal epithelium is multistratified, with 10 to 30 epithelial layers, whereas the infant/fetal oral mucosal epithelium is incompletely stratified, with only about 2 to 7 epithelial layers. Transmigration of cell-free HIV across the intact adult oral epithelium may occur only to a limited extent (2 to 5 layers) within the stratum granulosum, preventing spread of virus into the stratum spinosum, stratum basale, and lamina propria. However, disruption of tight junctions of the adult oral epithelium diminishes its barrier function and therefore facilitates HIV penetration to the deeper parts of the epithelium. Cell-free HIV transmigration across the paucistratified fetal and infant mucosal epithelium leads to passage of virions into the intraepithelial and subepithelial virus-susceptible cells. Cell-free HIV also transmigrates across monostratified fetal intestinal epithelia. HIV-infected lymphocytes and macrophages do not penetrate into adult oral mucosae. HIV-infected lymphocytes also do not transmigrate across fetal oral epithelia; however, HIV-infected macrophages penetrate into fetal epithelia and reach the lamina propria. Both HIV-infected lymphocytes and macrophages transmigrate across fetal intestinal epithelia; however, macrophage transmigration is substantially higher than lymphocyte transmigration. Transcellular spread of cell-free and HIV-infected macrophages across fetal and infant oral and intestinal mucosal epithelia may lead to infection of intraepithelial and submucosal HIV-susceptible cells, including T lymphocytes, Langerhans/dendritic cells, and macrophages. Thus, fetal/infant oropharyngeal and intestinal mucosal epithelia may serve as key portals of entry for HIV MTCT. The increased rate of HIV transmission across fetal/infant versus adult oral epithelia may reflect a combination of reduced barrier function (associated with paucistratification) and lower levels of innate immune response proteins (54).