Establishment of polarized adult and fetal oral epithelial cells and transcytosis of HIV. (A) Formation of tight junctions in polarized adult and fetal oropharyngeal cells. Cells were grown under polarizing conditions on Transwell filter inserts for 7–14 days. Expression of occludin, a marker for tight junctions, was examined by immunostaining. Cell nuclei were stained in blue. Cells were analyzed by confocal microscopy and images were obtained approximately 3 µm below the apical surface of polarized cells in the x-y (horizontal) plane. (B) Transepithelial resistance (TER) and paracellular permeability of polarized adult and fetal oral epithelial cells were examined by measuring electrical resistance of monolayers and passage of radioactive ³H-inulin through paracellular spaces. As a positive control, cell junctions were disrupted by incubating cells with 10 mM EDTA before measurement. Error bars show ± standard error of the mean (s.e.m.) (n = 3). (C) Transcytosis of cell-free and cell-associated HIV. Cell-free (20 ng p24/insert) and cell-associated HIV-1_SF33 (10⁶ PBMCs) were added to the apical membranes of polarized endometrial (HEC-1A), adult tongue (HSC-3_sort, ATNG#1), fetal tongue (FTNG#1), and adult cervical (CERV#1) epithelial cells. After 3 h, culture media from the lower chambers were examined for HIV-1 p24 using an ELISA. (D) Analysis of the integrity of polarized oral epithelial cells after HIV transcytosis. For transcytosis assays, cell-free and cell-associated HIV-1_SF33 were added to the apical membranes of polarized adult (ATNG#1) and fetal tongue (FTNG#1) epithelial cells for 3 h. One set of filter inserts was without virus, and the next set of inserts was without virus and treated with EDTA for 30 min prior to measurement. TER and paracellular permeability of polarized cells were examined by Millicell-ERS voltohmmeter and radioactive ³H-inulin passage, respectively.

Expression of HIV-1 co-receptors in adult and fetal oral and fetal intestinal epithelia. (A and B) Expression of HIV-1 co-receptors in polarized epithelial cells. (A) Expression of CXCR4, CCR5, GalCer, and HSPG in adult (HSC-3_sort and ATNG#1 tongue, and ATSL#1 tonsil) and fetal (FTNG#1 tongue and FORPH#1 oropharyngeal) oral epithelial cells, as well as in adult endometrial (HEC-1A) and cervical (CERV#1) epithelial cells, was examined by flow cytometry. The mean fluorescence intensity is shown. *, not detected. (B) Localization of HIV co-receptors on the apical surfaces of polarized oral epithelial cells. Polarized adult and fetal oral epithelial cells were fixed with 4% paraformaldehyde, and non-permeabilized cells were immunostained for HIV co-receptors (green). Nuclei were counterstained in blue. Cells were analyzed by confocal microscopy and images are shown in the x-z (vertical) plane. Original magnification is ×600. (C and D) Expression and localization of HIV-1 co-receptors in adult (C), fetal oral (D, upper panels), and fetal intestinal (D, lower panels) epithelia. Tissue sections were immunostained with antibodies to GalCer, HSPG, CCR5 or CXCR4 (all green) or their isotype controls. Cell nuclei were stained in blue. Only merged panels are shown. GR, granulosum; SP, spinosum; BL, basal; LP, lamina propria; EP, epithelium. White lines indicate the border between the lamina propria and the mucosal epithelium. Original magnification is ×600 (A, upper and middle panes, B, all panels). Original magnification is ×400 (A, lower panels).

Analysis of infectious activity of HIV transcytosed through adult and fetal oral epithelial cells. (A) Transcytosis of cell-associated HIV-1_SF33 virus across adult (ATNG#1) and fetal tongue (FTNG#1) epithelial cells and adult endometrial cells was examined. After 3 h one third of culture media with PBMCs from the lower chambers was collected and used for an ELISA p24 assay. The rest of the media were maintained with target PBMCs for the next 7 days, and the infectious activity of the virus was determined using an RT assay. Data shown are mean ± sem from 3 filter inserts. Similar results were obtained in 3 independent experiments. (B) Transcytosis of cell-associated HIV-1_SF33 through adult endometrial HEC-1A, cervical (CERV#1), tongue (HSC-3_sort, ATNG#1, ATNG#2), and tonsil (ATSL#1, ATSL#2) epithelial cells, as well as fetal tongue (FTNG#1, FTNG#2, FTNG#3) and oropharyngeal (FORPH#1, FORPH#1) epithelial cells. Infectious activity of transcytosed virions was examined using an RT assay after 7 days of culturing PBMCs from the lower chambers. Each data symbol (circle) indicates one experiment, and each experiment used three separate filter inserts. n, number of filter inserts. Horizontal lines represent median values. (C) Cell-associated R5-tropic HIV-1_SF170 and X4-tropic HIV-1_92UG029 were used for transcytosis through endomethrial (HEC-1A), adult tongue (HSC-3_sort, ATNG#1), tonsil (ATSL#1), and fetal oropharyngeal (FORPH#1, FORPH#2, FORPH#3) epithelial cells. After 1, 2, and 3 weeks of transcytosis, the infectious activity of virions in PBMCs was examined using an RT assay. Results shown are from one representative experiment out of two experiments. Error bars show ± s.e.m. (n = 3).

Expression of anti-HIV innate proteins in adult, fetal, and infant oral epithelial cells. (A) Expression of anti-HIV innate proteins in polarized epithelial cells. Adult and fetal epithelial cells were grown under polarizing conditions and immunostained for HBD2, HBD3 and SLPI. Cells were analyzed by confocal microscopy and positive cells were counted. Error bars show ± s.e.m. (n = 3). (B) Secretion of HBD2, HBD3, and SLPI from polarized and non-polarized adult tongue and tonsil, fetal oropharyngeal, and adult cervical epithelial cells. Culture media were collected separately from apical and basolateral membranes of polarized cells. In parallel experiments, culture media were collected from non-polarized cells. Secretion of HBD2, HBD3, and SLPI was measured using an ELISA detection kit for each protein. Error bars show ± s.e.m. (n = 3). (C) Expression of anti-HIV proteins in adult buccal, fetal oropharyngeal, and infant tonsil epithelia. Tissue sections were immunostained with antibodies to HBD2, HBD3, or SLPI (all green) or their isotype controls. Cell nuclei are counterstained in blue. Cells were analyzed by confocal microscopy. Original magnification is ×600. GR, granulosum; LP, lamina propria; EP, epithelium.

The role of anti-HIV innate proteins in infectious activity of transcytosed HIV-1 through adult oral epithelial cells. (A) Polarized adult tonsil (ATNSL#1) epithelial cells were transfected with siRNA against HBD1, HBD2, HBD3 and SLPI. Control cells were transfected with irrelevant siRNAs or were untreated. After 3 days cells were fixed and immunostained for HBD1, HBD2, HBD3 and SLPI. Expression of innate proteins in cells treated with siRNA and control siRNA, and in untreated cells, was analyzed by confocal microscopy and positive cells were counted in 10 randomly chosen fields. Error bars show ± s.e.m. (B) Polarized adult tonsil (ATNSL#1) epithelial cells were grown in 6-well Transwell inserts and transfected with anti-HBD-3 and SLPI siRNAs and control siRNA. After 3 days cells were extracted and HBD3 and SLPI were detected using a Western blot assay. (C) Polarized adult tonsil cells transfected with specific siRNA or control siRNA and untreated cells were used for transcytosis of cell-associated HIV-1_SF33. After 3 h filter inserts were removed and target PBMCs in the lower chambers were cultured for the next 10 days and RT activity was determined. Input virus, the RT value of HIV-infected PBMCs before transcytosis. Control, cells transfected with irrelevant siRNA. Untreated cells, cells were not treated with specific or control siRNA. Error bars show ± s.e.m. (n = 3). Data shown are from one representative experiment out of two experiments. (D) Antibodies to HDB2, HBD3, and SLPI were internalized into adult tongue epithelial cells (ATNG#1) using antibody delivery reagents. As a control, cells were treated with matched isotype antibodies. After 16 h, cell-associated HIV-1_SF33 transcytosis was performed, and after 7 days the RT activity of traversed virions was determined in PBMCs from the lower chamber. Input virus, the RT value of HIV-infected PBMCs before transcytosis. Control, transcytosis in the absence of antibodies. Error bars show ± s.e.m. (n = 3). Similar results were obtained in 4 independent experiments.

Localization of HIV in the HBD2 and HBD3 containing early endosomal vesicles. GFP-labeled HIV-1 81-A virus was added to the apical surfaces of polarized adult tonsil epithelial cells. Cells were incubated at 37°C for 2 h. Cells were then fixed and co-immunostained for EEA1 (red) and HBD2 or HBD3 (both in blue). Cells were analyzed by confocal microscopy and images were taken about 3 µm below the apical surface. The pink in merged panels indicates co-localization of EEA1 (red) with HBD2 or HBD3 (blue). The white in the merged panels indicates co-localization of HIV (green), HBD2 (blue), and EEA-1 (red), indicating co-localization of HBD2 and virions in the EEA1-positive early endosomes (red).