HIV transcytosis across polarized oral epithelial cells
To study HIV transepithelial transmission across well-developed adult oral and developing fetal oral epithelia, we established monostratified polarized epithelial cells originating from adult tongue and tonsil, as well as fetal tongue and oropharyngeal mucosal epithelia. To compare HIV transcytosis of oral epithelial cells to that of genital epithelial cells, we also used polarized adult endometrial and cervical epithelial cells. Cells were grown on microporous filter inserts, and their polarity was confirmed by immunodetection of tight junction proteins and measurement of paracellular permeability and transepithelial resistance (TER). The tight junction proteins occludin () and ZO-1 (data not shown) were both found to be localized to the lateral membranes of polarized cells, consistent with the presence of tight junctions. To confirm the functional status of the tight junctions, paracellular permeability and TER were measured in polarized cells that were either untreated or treated with EDTA to dissociate the tight junctions. A substantial increase in [3H] inulin passage from the apical surface to the basolateral compartment and a decrease in TER in EDTA-treated cells in comparison to control, EDTA-untreated cells were observed. These findings indicate the formation of a tight, polarized, epithelial monolayer ().
Figure 1 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 (more ...)
HIV transcytosis was examined using cell-free and cell-associated dual (R5/X4)-tropic HIV-1SF33
virus, which was added to the apical membranes of polarized cells. For cell-associated virus, we used HIV-infected peripheral blood mononuclear cells (PBMCs). Previous reports have shown that 3 h incubation of either cell-free or cell-associated virus with polarized epithelial cells is sufficient for HIV transcytosis across these cells (Bomsel, 1997
; Carreno et al., 2002
; Hocini et al., 2001
). Therefore, this time period was used for our transcytosis experiments. HIV transcytosis was evaluated by detection of HIV-1 p24 in the lower chamber of the filter inserts using an ELISA p24 detection assay. We compared transcytosis of cell-free and cell-associated HIV-1SF33
virions in polarized endometrial (HEC-1A), adult tongue (HSC-3sort
, ATNG#1), fetal tongue (FTNG#1), and adult cervical (CERV#1) epithelial cells (). Transcytosis of cell-free HIV-1SF33
virus in all polarized cells was at a low level, i.e., about 0.005% of viruses (from the original inoculum) transmigrated the polarized monolayer. In contrast, transcytosis of cell-associated HIV-1SF33
virus was about 10-fold higher. No PBMCs were detected in the lower chambers, indicating that PBMCs did not migrate into lower chambers through polarized cells.
To determine whether the interaction of cell-free virus or HIV-infected PBMCs from the apical surface of epithelial cells altered the integrity of polarized epithelial cells, we examined the TER and paracellular permeability of the adult and fetal polarized monolayers during transcytosis. As a control, cell junctions were disrupted by incubation of polarized cells with 10 mM EDTA. We found that TER and paracellular permeability of polarized epithelial cells were not changed by transcytosis of either cell-free or cell-associated HIV-1SF33 virions ().
Expression of HIV coreceptors in adult and fetal oral epithelial cells
HIV co-receptors CXCR4, CCR5, galactosyl ceramide (GalCer), and heparan sulfate proteoglycans (HSPG) may play a critical role in HIV transcytosis (Alfsen and Bomsel, 2002
; Alfsen et al., 2005
; Bobardt et al., 2007
; Bomsel, 1997
; Yahi et al., 1994
). It has been well documented that polarized endometrial and vaginal epithelial cells do not express CD4, but may express one or more of these HIV co-receptors (Bobardt et al., 2007
; Bomsel, 1997
; Hocini et al., 2001
; Hocini and Bomsel, 1999
; Saidi et al., 2007
). We used FACS and immunofluorescence assays to determine whether CD4, CXCR4, CCR5, GalCer, and HSPG are expressed in polarized oral epithelial cells. Analysis of CD4 expression in adult and fetal epithelial and cervical and endometrial cells showed that CD4 was not detectable in any of these cell lines (data not shown). Detection of the HIV co-receptors GalCer, HSPG, CXCR4, and CCR5 in polarized oral epithelial cells by FACS revealed that primary adult and fetal oral epithelial cells, as well as cervical epithelial cells expressed all of these receptors (). Expression of CXCR4 and CCR5 in immortalized adult HSC-3sort
tongue cells was not detectable, and only low-level expression of these receptors was found in immortalized HEC-1A endometrial cells. Confocal microscopy showed that these receptors were present on the apical surfaces ().
Figure 2 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-3sort and ATNG#1 tongue, and (more ...)
To determine whether the expression of HIV co-receptors in established, polarized cells in vitro correlates with their expression in oral mucosal epithelia in vivo, we immunostained adult and fetal biopsy tissue sections for the HIV receptors CD4, CXCR4, CCR5, GalCer, and HSPG. Expression of CD4 was not detected in either adult or fetal oral epithelia. CXCR4 and CCR5 were detected mostly within basal and parabasal cells () in 6 of 7 (85.7%) buccal tissue samples; the more superficial layers of the adult buccal epithelia were negative. GalCer and HSPG were both detected within all layers of the adult buccal epithelia (). In fetal oral and intestinal tissues, CXCR4 and CCR5 were detected in 5 of 10 (50%) buccal, 7 of 9 (55.5%) oropharyngeal, and 4 of 8 (50%) intestinal tissue samples (). All were positive for GalCer and HSPG, primarily in the more superficial cell layers.
HIV transmission across adult oral epithelial cells leads to viral inactivation, whereas HIV transcytosed across fetal epithelial cells retains infectious activity
In the above experiments, we showed that the amount of HIV that transmigrated across fetal and adult oral epithelia was about similar (). We next sought to determine if the levels of infectious virus were similar. To determine the infectivity of transmigrated cell-free virions, we performed transcytosis assays with cell-free HIV-1SF33 in polarized adult tongue and tonsil, fetal tongue and oropharyngeal, and adult endometrial and cervical epithelial cells. The infectious activities of transmigrated virions were examined in target PBMCs using a reverse transcriptase (RT) assay after 7 days. In this analysis, no RT activity was detected in target PBMCs infected with cell-free virions transcytosed through any of these cells (data not shown). Similar data were obtained from 4 independent experiments. This observation may have reflected the low levels of cell-free HIV transcytosis via polarized epithelial cells ().
To examine the infectious activity of transmigrated, cell-associated virions, HIV-1SF33 - infected PBMCs were added to the apical surfaces of polarized adult and fetal tongue epithelial cells, and adult endometrial cells. After 3 h one third of the culture medium from the lower chamber was collected for detection of HIV-1 p24 by ELISA. The remaining media, along with the target PBMCs, were cultured for the next 7 days, and the PBMCs were used for RT assays. Detection of HIV-1 p24 using an ELISA assay showed that the rates of HIV transcytosis through adult and fetal tongue epithelial cells and adult endometrial cells were comparable. These findings indicate that about the same numbers of virions transmigrated through each cell type. However, virions that transmigrated across adult tongue epithelial cells showed no RT activity in target PBMCs (). In contrast, virions that passed through fetal tongue epithelial cells and adult endometrial cells did show RT activity in target PBMCs. These data indicate that the cell-associated virions that had transmigrated across adult oral epithelial cells were not infectious in the target PBMCs, but that those that had passed through fetal oral epithelial and adult endometrial cells retained infectious activity.
Figure 3 Analysis of infectious activity of HIV transcytosed through adult and fetal oral epithelial cells. (A) Transcytosis of cell-associated HIV-1SF33 virus across adult (ATNG#1) and fetal tongue (FTNG#1) epithelial cells and adult endometrial cells was examined. (more ...)
Analysis of the RT activity of PBMCs infected with transmigrated, cell-associated HIV-1SF33 virions in multiple experiments using several different adult and fetal oral epithelial cell lines () showed that virions that had transmigrated across adult oral epithelial cells had a strong tendency to lose their infectious activity for target PBMCs. In contrast, the infectious activity of virions that had traversed fetal oral epithelial cells was well preserved. HIV-1SF33 virions that transmigrated through adult endothelial and cervical epithelial cells were also infectious ().
Primary HIV isolates lose their infectious activity after transmigration in adult oral epithelial cells but not in fetal oral epithelial cells
To determine the infectious activity of clinical HIV-1 isolates after their transmigration across adult and fetal oral epithelial cells, we performed transcytosis assays with cell-associated X4-tropic HIV-192UG029 and R5-tropic HIV-1SF170 primary isolates in adult endometrial, tongue and tonsil, and fetal tongue and oropharyngeal polarized epithelial cells (). The infectious activity of transmigrated virions was examined after 1, 2, and 3 weeks in target PBMCs. These data showed that virions that traversed adult oral epithelial cells were not infectious, but those transmitted through adult endometrial and fetal oral epithelial cells were infectious in PBMCs and that their RT values increased with time. Transmigration of infectious virions was also detected through polarized primary cervical epithelial cells (data not shown). Thus, transmission of HIV-1 clinical isolates through adult oral epithelial cells caused their complete inactivation, whereas viral transmigration through fetal and cervical epithelial cells did not affect their infectious activity.
Analysis of anti-HIV innate protein expression and secretion in polarized cells
To understand the mechanisms of HIV inactivation in adult oral epithelial cells and the preservation of infectious activity in fetal oral epithelial cells, we examined the expression of anti-HIV innate proteins beta-defensins (HBD) 1, 2 and 3 and secretory leukocyte protease inhibitor (SLPI) (Dale et al., 2001
; Dunsche et al., 2002
; Jana, Gray, and Shugars, 2005
; Moutsopoulos et al., 2007
; Quinones-Mateu et al., 2003
; Sun et al., 2005
) in both cell types. In an immunofluorescence assay, we found that about 70% of adult and fetal oral epithelial cells and about 80% of cervical epithelial cells were positive for HBD1 (data not shown). We also found that 60–80% of adult oral epithelial cells were positive for HBD2, HBD3, and SLPI (). However, the fetal oral epithelial cells had an approximately 5- to 10-fold lower level of expression of these molecules than did the adult cells. Furthermore, the expression of HBD2, HBD3, and SLPI in adult cervical epithelial cells was 2- to 5-fold lower than that in adult oral epithelial cells. None of these proteins was detected in HEC-1A endometrial cells (data not shown).
Figure 4 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 (more ...)
Secretion of HBD2, HBD3, and SLPI was measured for the polarized and non-polarized adult and fetal oral and adult cervical cells using an ELISA assay. These data revealed that all three proteins were secreted at the highest levels only from polarized adult tongue and tonsil epithelial cells (). Their secretion from non-polarized adult tongue and tonsil epithelial cells was about 5–10 fold lower than from polarized adult oral epithelial cells. Secretion of HBD2 and HBD3 from fetal oropharyngeal and adult cervical epithelial cells was about 10–15 fold lower than that of adult oral epithelial cells. High-level SLPI secretion was detected only in adult tongue and tonsil epithelial cells, and SLPI secretion from fetal oropharyngeal and adult cervical epithelial cells was not detectable.
We next examined the expression of anti-HIV proteins in adult and fetal oral biopsy tissues. Immunofluorescence analysis of 5 adult buccal and 6 fetal oropharyngeal () and 8 buccal (data not shown) tissue explants for HBD2, HBD3, and SLPI showed that HBD2, HBD3, and SLPI were all strongly expressed in the adult oral epithelium (). All three proteins were detected both within cells and in the extracellular environment, consistent with their being secreted. In contrast, expression of these proteins in fetal buccal and oropharyngeal epithelia was mainly negative, and only weak positive signals were observed in only small areas of fetal epithelia (). We also examined HBD2, HBD3, and SLPI expression in 3 infant tonsil epithelia and found that they were all negative or only weakly positive for these proteins.
Functional role of anti-HIV innate proteins in inactivation of HIV during transepithelial transmigration
To determine the functional role of innate proteins in the inactivation of HIV during transcytosis in adult oral epithelial cells, we interfered with innate protein function using two independent approaches: inhibition of innate protein expression and intracellular neutralization. For inhibition of innate protein expression, polarized adult tonsil epithelial cells were treated with siRNA against HBD1, HBD2, HBD3, or SLPI. As a control, we used an unrelated siRNA. Immunostaining of HBD1, HBD2, HBD3, and SLPI after 3 days showed that treatment of cells with specific siRNAs reduced expression of innate proteins by about 60–70% (). A Western blot assay also showed that siRNA-treatment reduced HBD-3 and SLPI expression below detectable levels (). These cells were then used for HIV-1SF33 cell-associated transcytosis assays. The TER of these epithelia was similar to the TER of untreated polarized cells (about 650 Ω/cm2 in both treated and untreated cells), indicating that the siRNA treatment did not disrupt tight junctions. Measurement of the RT activity of target PBMCs after 10 days showed that virions that had traversed polarized cells treated with siRNA against HBD2, HBD3, and SLPI were infectious (). In contrast, virions that had traversed cells treated with either anti-HBD1 siRNA or control siRNA did not cause infection in target PBMCs, i.e., no RT activity was detected in PBMCs after 10 days.
Figure 5 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 (more ...)
In the next set of experiments, we neutralized intracellular innate proteins in adult tongue epithelial cells using specific antibodies. Antibody-mediated intracellular neutralization of viruses and proteins has been shown (Bomsel et al., 1998
; Burns et al., 1996
; Devito et al., 2000
; Edelson and Unanue, 2001
; Krautz-Peterson et al., 2008
; Wright et al., 2006
). Antibodies against HBD2, HBD3, or SLPI and isotype control antibodies were added to polarized adult tongue epithelial cells with the lipid-based antibody-delivery reagent Ab-DeliverIN™ (OZ Biosciences) to promote their internalization. The TER in these epithelia was similar to the TER of untreated polarized cells (about 600 Ω/cm2
in both treated and untreated cells), indicating that the antibody-delivery reagent did not disrupt tight junctions. Measurement of SLPI secretion from cells treated with anti-SLPI antibodies showed that internalization of antibodies completely blocked the secretion of SLPI, indicating that antibodies in the cytoplasm may inhibit secretion of innate proteins. Antibody-internalized cells were then used for HIV-1SF33
cell-associated transcytosis. Measurement of the RT activity of target PBMCs after 7 days showed that virions that had traversed polarized cells treated with antibodies against HBD2, HBD3, and SLPI were infectious (). The RT values of these target PBMCs further increased about 3–5 fold by the 14-day time point (data not shown). In contrast, virions that had traversed cells treated with isotype control antibodies did not cause infection in target PBMCs, i.e., no RT activity was detected in PBMCs after 7 and 14 days. Thus, reduction of innate protein expression by siRNA and their intracellular neutralization by specific antibodies in adult tonsil and tongue epithelial cells allowed transcytosed virions to remain infectious. These findings strongly suggest that the innate proteins HBD2, HBD3, and SLPI are responsible for the loss of virion infectivity during transcytosis.
Co-localization of HIV with HBD2 and -3 in the vesicular compartment
HBD2 and HBD3 inactivate HIV by direct interaction with the virions (Cole et al., 2002
; Wang et al., 2003
; Wang et al., 2004
; Weinberg, Quinones-Mateu, and Lederman, 2006
), which may occur during viral transcytosis. To determine whether HBD2 and HBD3 co-localize with HIV in the vesicular compartment during HIV transcytosis, we performed HIV transcytosis assays in polarized adult tonsil epithelial cells. R5-tropic HIV-1 81-A virus labeled with GFP (HIV-GFP) was added to the apical surfaces of polarized cells and, after 2 h, cells were fixed and immunostained for early endosomal vesicles using antibodies against early endosomal antigen 1 (EEA1) (, red). The same cells were also co-stained for HBD2 or HBD3. Confocal microscopy revealed that the HIV-GFP signal (, green) colocalized with EEA1, indicating the presence of virions in the early endosomal compartment. Notably, HBD2 and HBD3 (, blue) also colocalized with EEA1, indicating that HBD2 and HBD3 were present in the vesicular compartment. Furthermore, HBD2 and HBD3 were also found to colocalize with both the GFP-virions and EEA1 (, merged panel, white), suggesting that HBD2 and HBD3 may interact with virions in the vesicular compartment, leading to inactivation of intracellular virus.
Figure 6 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 (more ...)