Conceived and designed the experiments: AN CK. Performed the experiments: AN OC. Analyzed the data: AN OC ALA CK. Contributed reagents/materials/analysis tools: WNDB MO MJT SDGO CK. Wrote the paper: AN CK.
The authors have declared that no competing interests exist.
While several clinical studies have shown that HIV-1 infection is associated with increased permeability of the intestinal tract, there is very little understanding of the mechanisms underlying HIV-induced impairment of mucosal barriers. Here we demonstrate that exposure to HIV-1 can directly breach the integrity of mucosal epithelial barrier, allowing translocation of virus and bacteria. Purified primary epithelial cells (EC) isolated from female genital tract and T84 intestinal cell line were grown to form polarized, confluent monolayers and exposed to HIV-1. HIV-1 X4 and R5 tropic laboratory strains and clinical isolates were seen to reduce transepithelial resistance (TER), a measure of monolayer integrity, by 30–60% following exposure for 24 hours, without affecting viability of cells. The decrease in TER correlated with disruption of tight junction proteins (claudin 1, 2, 4, occludin and ZO-1) and increased permeability. Treatment of ECs with HIV envelope protein gp120, but not HIV tat, also resulted in impairment of barrier function. Neutralization of gp120 significantly abrogated the effect of HIV. No changes to the barrier function were observed when ECs were exposed to Env defective mutant of HIV. Significant upregulation of inflammatory cytokines, including TNF-α, were seen in both intestinal and genital epithelial cells following exposure to HIV-1. Neutralization of TNF-α reversed the reduction in TERs. The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers. Collectively, our data shows that mucosal epithelial cells respond directly to envelope glycoprotein of HIV-1 by upregulating inflammatory cytokines that lead to impairment of barrier functions. The increased permeability could be responsible for small but significant crossing of mucosal epithelium by virus and bacteria present in the lumen of mucosa. This mechanism could be particularly relevant to mucosal transmission of HIV-1 as well as immune activation seen in HIV-1 infected individuals.
Clinical studies have shown that HIV-1 infected patients have increased intestinal permeability. In chronically infected patients that progress to AIDS, there is activation of immune cells consistent with leakage of microbes via the gut. However, the mechanism by which this occurs is not clear. Here, we show that direct exposure of intestinal and genital epithelial cells to HIV leads to breaching of the mucosal barrier and increased leakage of both bacteria and virus across the epithelium. The mechanism of this breakdown appears to be due to inflammatory factors produced by epithelial cells themselves, in response to HIV-1 exposure, that destroy the tight junctions between epithelial cells, thereby allowing microbes access to the inside of the body. Interestingly, we found that treatment of epithelial cells with just the surface glycoprotein from HIV could lead to similar breakdown of the barrier. This implies that when mucosal epithelial cells come in direct contact with large amounts of HIV-1, the virus can cross into the inside of the body and cause direct infection of target cells. The crossing of the bacteria by similar mechanism can lead to chronic inflammation and activation of immune cells of the body.
The mucosa presents a primary barrier against a multitude of micro-organisms present on the mucosal surfaces of the human body
Various pathogenic organisms have developed strategies to either infect or traverse through the epithelial cells at mucosal surfaces, as part of the strategy to establish infection in the host. In fact, mucosal transmission account for majority of infections in humans
HIV-1 infection is initiated primarily on mucosal surfaces, through heterosexual or homosexual transmission
In the present study, we investigated the direct effects of HIV-1 exposure on intestinal and genital mucosal epithelia, where primary HIV-1 infection is frequently initiated. We show that in fact the impairment of epithelial barrier function can be a direct result of exposure to HIV-1. Using ex-vivo cultures of pure primary genital epithelium as well as an intestinal epithelial cell line, we show significantly decreased barrier functions and enhanced permeability that is not unique to the intestinal epithelium; similar increase in permeability was seen in the genital epithelium as well. Small amounts of both bacterial and viral translocation were seen following HIV-1 exposure. The mechanism appears to be mediated by increased production of inflammatory cytokines directly from the epithelial cells following exposure to HIV-1, including TNF-alpha, known to disrupt barrier functions. Further, we show that HIV-1 envelope protein gp120 was able to impair barrier functions in epithelial cells on its own. Neutralization of gp120 or exposure to HIV-1 lacking gp160 surface envelope glycoprotein did not have any effect on epithelial cells. These results provide strong evidence that exposure to HIV-1 may lead to impairment in barrier function of mucosal epithelium which could result both in translocation of HIV-1 and/or luminal bacteria that could serve as the source of immune activation during HIV-1 infection.
In order to study HIV-1 induced barrier defect in epithelial monolayers, HIV-1 (106 infectious viral units/ml) was added apically to confluent monolayers of differentiated primary female genital epithelial cells (ECs) or T84 intestinal epithelial cells grown in transwells. Transepithelial resistance (TER), a measure of epithelial monolayer integrity, was measured before and 24h post-infection and calculated as a percentage of pretreatment TER. Transepithelial resistances of primary endometrial epithelial monolayers exposed to various strains of HIV-1 were significantly reduced (p<0.001 with all HIV-1 strains) by 30–60%, 24h post-exposure (
Corresponding p24 values were as follows for R5 tropic: Bal (0.6ng/ml), ADA (two different viral stocks 0.3 ng/ml and 1040 ng/ml), 4648 (49ng/ml); X4 tropic: IIIB (0.7ng/ml and 161ng/ml), MN (103ng/ml and 1110ng/ml), NLH4-3 (93ng/ml), 7681 (773ng/ml), 11242 (342ng/ml); dual tropic 11249 (200ng/ml). Control include cultures that were mock-treated with medium without virus (mock) or virus-free supernatants (R5 and X4 control). Transepithelial resistance (TER), was measured prior to and 24 hours post- exposure to HIV-1. *p<0.001, n = 3–9. Viability of primary female genital epithelial cells and T84 intestinal epithelial cells was assessed by MTT assay 24 hours after exposure with two HIV strains IIIB (X4-tropic) and ADA (R5-tropic) and compared with mock-infected epithelial monolayers (C). The effect of apical and basolateral exposure was determined. (D). Differentiated T84 epithelial monolayers were mock infected or exposed apically or basolaterally to HIV (ADA strain, 106 infectious viral particles/ml). TER was measured at 0, 24 and 48 hours post exposure. *P<0.001, **P<0.0001, n = 3.
To exclude the possibility that changes in the barrier function resulted from any cytotoxic effect of HIV-1 exposure that would result in breach of the integrity of the monolayers, we tested the viability of both intestinal and endometrial epithelial cultures following HIV-1 exposure by an MTT assay. The results indicated that exposure to HIV-1 for 24h did not have any affect on cell viability relative to controls (
We also determined if exposure to HIV on the basolateral side of the epithelial cell monolayer decreased TERs of epithelial monolayers. T84 intestinal EC monolayers were exposed to HIV on both apical and basolateral surface and TERs were compared 24 hours later. Basolateral exposure to HIV led to no significant decrease in TER (
We next examined the effect of HIV-1 exposure on gene and protein expression of tight junctions in genital and intestinal epithelial monolayers. Confluent monolayers of primary endometrial epithelial cells were mock-treated or exposed to HIV-1 for 8h and total mRNA was extracted and subjected to quantitative real time RT-PCR using primers specific to different tight junction genes including Claudin 1, 2, 3, 4, 5, Occludin and ZO-1 (
Total RNA was extracted and cDNA was synthesized. Quantitative Real-time RT-PCR was conducted for tight junction gene expression by measuring mRNA for Claudin 1–5, Occludin and ZO-1. GAPDH, a house keeping gene, was measured for internal control (A). * p<0.01; ** p<0.001; *** p<0.0001). Immunofluorescent staining of tight junction proteins following HIV-1 exposure compared to mock-treated epithelial monolayers. Representative staining is shown for claudin-2 (B), Occludin (C), and ZO-1 (D) at 24 hours post-exposure. Magnification: 1260×. Data shown is representative of 3 separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. For RNA extraction, 6–8 replicate cultures were pooled.
Tight junction genes | Primer names | Primer sequence 5′ - 3′ | References |
ZO-1 | ZO-1F | Pu H, Tian J, Andras AS, Hayashi IK, et al, (2005) HIV-1 Tat protein-induced alterations of ZO-1 expression are mediated by redox-regulated ERK 1/2 activation. J Cerebral Blood Flow & Metabolism 25:1325–1335 | |
ZO-1R | |||
Occludin | Occlu F | Bai L, Zhang Z, Zhang H, et al. (2008) HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study. BMC Infec. Dis..8:77–89 | |
Occlu R | |||
Claudin 1 | Claudin 1F | Bai L, et al (2008) BMC Infec. Dis..8:77–89 | |
Claudin 1R | |||
Claudin 2 | Claudin 2F | Bai L. et al (2008) BMC Infec. Dis..8:77–89 | |
Claudin 2R | |||
Claudin 3 | Claudin 3F | Bai L. et al (2008) BMC Infec. Dis..8:77–89 | |
Claudin 3R | |||
Claudin 4 | Claudin 4F | Bai L. et al (2008) BMC Infec. Dis..8:77–89 | |
Claudin 4R | |||
Claudin 5 | Claudin 5F | Bai L. et al (2008) BMC Infec. Dis..8:77–89 | |
Claudin 5R | |||
GAPDH | GAPDH F | Bai L. et al (2008) BMC Infec. Dis..8:77–89 | |
GAPDH R |
To correlate the decreased mRNA levels with tight junction protein expression, intestinal and endometrial monolayers were stained for different tight junction proteins 24h after HIV-1 exposure and compared with mock-treated monolayers by confocal microscopy. The protein expression correlated well with real-time quantitative RT-PCR results, showing distinct decrease in localization of Claudin 2 and Occludin and ZO-1 (
Next, we conducted a time course study to determine if HIV-1 mediated decrease in TER correlated with alterations in permeability of the monolayers. Polarized, confluent monolayers of primary endometrial and T84 intestinal epithelial monolayers were exposed to HIV-1 for different lengths of time. The TERs were measured prior to treatment and at 2h, 4h, 6h, 8h, 16h, 24h and 48h post-exposure. A reduction in TER was first evident after 2h of HIV-1 exposure of genital epithelial cells (
To measure barrier functions, TER was measured prior to and post exposure, ZO-1 staining was done and Dextran Blue dye leakage was measured across the monolayers at all time points. (A) TER values. p<0.001. (B) Paracellular permeability measured by addition of Blue Dextran dye on the apical side of monolayers. At different time intervals post-exposure, basolateral supernatants were sampled and absorbance was measured and compared to apical absorbance at initial time point (Time “0”). Blue Dextran leakage was calculated as a percentage of apical values. Data shown is representative of 3–4 separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.
A–C. Series of stack planes (XY) taken through the apical extent of the monolayer. D–F. Quantification of ZO-1 (dark line) and nuclear (grey bars) staining shown as graphs. Each cell layer (1–20) corresponds to series of images from Z-stack sections taken at 1µm thickness through the cell monolayer shown on the right. X-axis illustrates cell layers from apical to basolateral. Y-axis illustrates the number of pixels present over the entire area of image. A, D. Control mock infected monolayer, 24 hours post-treatment. B,E. HIV-1 exposed monolayer, 4 hours post-treatment. C,F. HIV-1 exposed monolayer, 24 hours post-treatment. Results shown are representative of 3 separate Z-stacks collected and analyzed from each replicate, each treatment group had 3–5 replicates and the experiment was repeated 3 times. (Magnification :1260×).
The decrease in TER was correlated with leakage of Blue Dextran dye into basolateral compartment. Blue Dextran dye (mol. wt. 2000 kDa), added to the apical side of intact epithelial monolayers, normally cannot pass through the tight junctions between epithelial cells that prevent its paracellular transport
Among the tight junction proteins, the normal pattern of ZO-1 localization was very prominent in both primary genital ECs and intestinal T84 cells. Equally striking and apparent was its disruption following HIV-1 exposure. Therefore, ZO-1 was localized and quantified in confluent EC monolayers post-HIV-1 treatment and compared with control cultures to determine the disruption of tight junctions. The results are shown for an earlier time point (4 hour) and a later time point (24 hour) in control and HIV-1 exposed monolayers (
Next we determined whether the impairment of barrier function of the epithelium was dependent on the exposure dose of HIV. Various concentrations of HIV-1 strain ADA (102–107 infectious viral units/ml corresponding with MOI of 1∶10−4 to 1∶10 and p24 values of 0.2ng–2800ng/ml) were added on apical side of confluent EC monolayers. The TER values were measured 24h post-exposure and expressed as percent pretreatment TER (
TER values were measured, starting at viral concentration of 103 up to 107 infectious viral units/ml (equivalent to p24 values of 0.2ng–2800 ng/ml) (A).* p<0.001 **p<0.05. ZO-1 staining following exposure to different concentration of virus (B). Data shown is representative of three separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.
To determine if productive viral replication was required for increased permeability, polarized T84 cells were treated apically with infectious HIV or an equivalent amount of UV-inactivated virus (
Differentiated T84 cells were mock infected or exposed to live or UV inactivated HIV (IIIB strain, 106 infectious viral particles/ml). (A) TER values following exposure to both live and UV-inactivated HIV compared to mock treated monolayer (P<0.001). (B) ZO-1 staining in epithelial cells exposed to live and UV-inactivated HIV, but not mock infected cultures. The corresponding Z-stack series below each panel clearly shows majority of ZO-1 staining (green) in mock treated cultures on the apical side of the monolayer, while the nuclei are seen more basolaterally (red). Magnification:1260×. Primary endometrial EC monolayers (C) or intestinal T84 cells (D) were treated with either gp120 (0.1µg/ml, 0.8nM) or Tat (1.4ug/ml, 100nM) or a combination of both, for 24 hours. TER values were measured prior to and post-treatment. p<0.001, n = 6. (E) ZO-1 staining after gp120 treatment and Tat treatment. Magnification: 2520×. Data shown is representative of two (A,B) and six (C,D) separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.
Previous studies examining effect of HIV-1 on blood brain barrier demonstrated that HIV-1 gp120, a surface envelope glycoprotein and tat, an HIV regulatory protein that is produced by infected cells, could directly increase permeability of endothelial cells
To confirm that the barrier defect was mediated by HIV-1 viral envelope glycoprotein, we neutralized gp120 on HIV prior to exposure to epithelial cells (
(A) Epithelial monolayers were treated with medium alone, HIV-1 (IIIB, 106 infectious viral particles/ml), HIV-1 in combination with gp120 neutralizing antibody (35µg/ml) or isotype control antibody (35µg/ml), gp120 or isotype antibody alone. TER measurements were taken as a measure of change in permeability and presented as percent of pre-treatment TER. p<0.01. (B) Confluent T84 epithelial cell cultures were mock infected or exposed to NL4-3 (p24, 79 ng/ml) or NL4-3 Env− mutant (p24, 79 ng/ml) and TER measurements were taken prior to and 24 hours post-exposure. P<0.001 (C) ZO-1 localization after exposure to wildtype HIV-1 NL4-3 or Env− NL4-3 mutant. Data shown is representative of four separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.
The final confirmation that viral surface glycoprotein was critical for changes in epithelial cell permeability was obtained by treating epithelial cells with an Env-defective mutant of HIV (Env−) (
Epithelial cells are known to secrete a variety of cytokines at constitutive levels. Many of these are upregulated or induced
*p<0.01, **p<0.001. Data shown is representative of three separate experiments from different tissues, each experiment had 3–5 replicate cultures for each experimental condition.
Of the cytokines that showed increased production in genital and intestinal epithelial cells following HIV-1 exposure, TNF-α is well known to disrupt epithelial cell tight junction assembly and increase intestinal cell permeability
TER measurements were taken as a measure of change in permeability and presented as percentage of pre-treatment TER. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.
To correlate barrier dysfunction with increased permeability to luminal antigens, we examined bacterial and viral translocation across the epithelial monolayers post-exposure to HIV-1. Intestinal epithelial monolayers grown to confluence were exposed to HIV-1. TNF-α was used as a positive control since it is known to disrupt tight junctions and increase permeability. Because direct exposure to TNF-α for prolonged period of time causes irreversible damage to epithelial cells, TNF-α treatment was limited to 6 hours prior to addition of non-pathogenic
(A) Bacterial translocation was measured in T84 intestinal monolayers. Confluent monolayers were left untreated or treated for 6 hours with TNF-α (20ng/ml),
In a separate experiment, lipopolysaccaride (LPS) leakage in HIV-1 exposed endometrial monolayers was determined. LPS was added on apical side of HIV-1 exposed and control monolayers and one hour later basolateral supernatants were collected and LPS leakage was measured. The LPS levels in basolateral supernatants were increased by 47.3±0.922% in HIV-1 exposed monolayers in comparison with LPS leakage in mock-treated control monolayers.
We also measured translocation of HIV-1 through the primary endometrial monolayers (
To summarize, we were able to demonstrate that exposure to HIV-1 directly decreased the transepithelial resistance across intestinal and genital epithelial monolayers. The reduction in TER correlated with significant decrease in tight junction protein expression and increased permeability, indicating functional impairment of the barrier. The effect was specific for HIV-1 and reached significant levels within 2–4 hours following HIV-1 exposure. Similar reduction in tight junction functioning was observed following treatment of ECs with HIV-1 envelope protein gp120 but not tat, a regulatory protein. Neutralization of gp120 and exposure to an Env− HIV significantly abrogated the impairment of epithelial barrier, indicating that the effect was mediated by HIV-1 envelope glycoprotein. We further determined that exposure of the epithelial monolayers to HIV-1 led to enhanced production of a number of inflammatory cytokines, including TNF-α, by both intestinal and genital epithelial cells. When epithelial cells were exposed to HIV-1 in presence of anti-TNF antibody, there was no significant decrease in TER, indicating that TNF played a major role in impairing the barrier functions. In experiments designed to determine whether the disruption of epithelial barrier function could be directly associated with microbial leakage across the mucosa, we found evidence for small but significant bacterial and viral translocation across epithelial monolayers following HIV-1 exposure.
To the best of our knowledge, this is the first study to demonstrate that HIV-1 can directly disrupt mucosal epithelial barrier functions that can lead to enhanced microbial translocation. Previous clinical studies have documented that in HIV-1 infected patients intestinal permeability is altered, characterized by diarrhea-induction
That viral exposure could directly lead to compromised barrier function has been shown before
In our study, both the intestinal cell line and primary genital epithelial cells showed similar response to different strains of HIV-1: disruption of tight junctions, and increased permeability. However, we found the profile of cytokines produced constitutively by intestinal and genital epithelial cells was quite distinct. While the intestinal cell line T84 did not constitutively produce TNF-α, IL-6, IL-8 and MCP-1, there was significant induction of these cytokines following HIV-1 exposure. Primary genital epithelial cultures, on the other hand, constitutively produced TNF-α, IL-6, IL-8 and MCP-1 and production of TNF-α and IL-6 was significantly upregulated following HIV-1 exposure. Both types of ECs secreted minimal levels of IL-10 and IL-1β which was upregulated following HIV-1 exposure only in primary genital epithelial cells. The differences in the constitutive cytokine profile between genital and intestinal epithelial cells could be due to distinct characteristics of primary cells compared to cell lines. Alternatively, intestinal epithelial cells are likely to be more quiescent in terms of baseline cytokine production given their microenvironment where a variety of commensal organisms are always present in the lumen
Among the cytokines that were upregulated, the direct effect of TNF-α on disruption of intestinal epithelial tight junction and increased permeability has been extensively characterized
The finding that disruption of barrier function can result in small but significant amount of both viral and bacterial translocation across ECs following exposure to HIV-1 has profound implications. Although previous studies have demonstrated presence of LPS in serum of HIV infected patients and correlated it with immune activation in North American cohorts, the inference that microbial flora in the intestines was the source of LPS was indirect
While the viral-epithelial interactions described here are novel, further investigation is needed to determine what role increased barrier permeability plays in initiating HIV-1 infection. HIV-1 transmission across intestinal and genital mucosa occurs predominantly via infected semen; currently the role of seminal plasma in HIV-1 transmission is far from clear. Recent studies indicate that seminal plasma can lead to inflammatory responses and facilitate HIV-1 transmission
In conclusion, the current study provides evidence for the first time that HIV-1 exposure at the mucosal surface leads to direct response by the mucosal epithelium, seen by production of inflammatory cytokines. This response is rapid, independent of viral infection and likely plays a key role in initiation of mucosal damage. This information will be critical for strategies to target control of mucosal damage.
Reproductive tract tissues were obtained from women aged 30–59 years (mean age 42.9+7.2) undergoing hysterectomy for benign gynecological reasons at Hamilton Health Sciences Hospital. Written informed consent was obtained from all patients, with the approval of Hamilton Health Sciences Research Ethics Board. The most common reasons for surgery were uterine fibroids and heavy bleeding. Tissues were first examined by pathologists and if they were deemed free from any malignant or other clinically observed disease, coin-sized pieces were collected for further processing.
Detailed protocol for isolation and culture of genital epithelial cells (GEC) has been described previously
The human colon-derived crypt-like T84 epithelial cell line was maintained and cultured as described previously
HIV-1 R5 and X4 -tropic laboratory strains were prepared by one of two methods. R5-tropic ADA and X4-tropic laboratory strain IIIB viral stocks were prepared by infection of adherent monocytes from human PBMCs (ADA) or from chronically infected H9 cell line (IIIB), followed by virus concentration by Amicon Ultra-15 filtration system (Millipore, Billerica, US). Virus stock preparations were checked for possible contamination by cellular factors by multiplex bead-based sandwich immunoassay (Luminex Corporation, Austin, TX, USA). TNF-α, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IL-1α, IL-1β were not detected in any viral stock (standard range of detection limit for different factors: 0.1–4.5 pg/ml). Laboratory strains of HIV-1 virus were also prepared by ultracentrifugation method. HIV-1 R5 laboratory strains ADA, Bal, and X4 strains IIIB, MN and NL4-3 and four clinical strains 11242 (dual), 11249 (R5), 4648 (R5), 7681 (X4) (Dr. Donald R. Branch, University of Toronto) were prepared in human PBMC preparations and concentrated by ultracentrifugation over 20% sucrose for 1 hour at 19,000 rpm (33,000g). All HIV-1 stocks were titered for infectious viral count/ml by TZMb-1 indicator cell assay as described previously
For HIV-1 exposure, primary epithelial cells, isolated from human female genital tract tissues or intestinal T84 cells were grown to confluence. Epithelial cell cultures were exposed apically or basolaterally to HIV-1 virus (105 infectious viral units/well/100µl, final concentration 106 infectious viral units/ml), corresponding to MOI of 1.0 or other viral doses as mentioned in individual experiments. The p24 values corresponding to this standard concentration of virus (106 infectious viral units/ml) varied, depending on the viral strain, as determined by p24 ELISA (Zeptometrix Corp., Buffalo, NY, USA); it corresponded to p24 concentration between 0.7–1110 ng/ml for X4 tropic lab strains and between 0.3–790 ng/ml for R5 tropic viruses. For clinical strains p24 concentrations used for HIV exposures were between 49–773 ng/ml. Mock infection controls included exposure to same volume of media without HIV-1 (media control, mock) or exposure to same volume of virus (and gp120) free supernatant from PBMC (for R5 HIV-1) or H-9 (X4 HIV-1) cell line cultures (R5 and X4 controls).
Env-defective mutant, Env− (kind gift of D. Johnson, NCI) was on an NL4-3 backbone (X4-tropic HIV-1 laboratory strain) and was compared to wildtype NL4-3 for its effect on epithelial cell permeability
HIV-1 R5-tropic strain ADA and X4-tropic strain IIIB were inactivated by UV exposure. 106 infectious units/ml of virus was subjected to 25–100mJ/cm2 UV with a UV cross-linker (Fisher Scientific, USA). UV inactivation of virus was confirmed by titration on TZMb-1 cells.
HIV-1 proteins gp120 envelope protein and soluble Tat protein were obtained from NIH AIDS Research & Reference Reagent Program. Epithelial cell cultures were treated with HIV-1 viral proteins gp120 (0.8nM, 0.1 µg/ml) or Tat (100 nM, 1.4ug/ml). A range of Gp120 concentration (50ng–1µg/ml) was tried based on those used in previous studies
To test the role of gp120, HIV-1 IIIB was incubated at 37C with a recombinant human monoclonal neutralizing antibody against HIV-1 gp120 (IgG1, clone 2G12, Polymun Scientific, Austria) at a concentration of 35µg/ml or an isotype control antibody (Southern Biotechnology, Birmingham, USA) at same concentration for 1 hour. TERs were measured prior to and post-exposure.
Quantitation of tight junction gene expression in epithelial cells post-HIV-1 exposure and comparison with unexposed control epithelial cells was done by real time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) with Syber Green. The tight junction genes examined were Claudin 1, 2, 3, 4, 5, ZO-1, and Occludin. ECs were lysed by Trizol reagent, total RNA was extracted by RNeasy mini kit (Qiagen Inc., ON, Canada) and treated with DNase column (RNase-free DNase set, Qiagen Inc., ON, Canada) to remove DNA contamination. The cDNA was synthesized by qScript™ cDNA supermix (Quanta Bioscience Inc., Gaithersburg, MD, US) according to manufacturer's protocol. Real-time PCR was performed for each tight junction gene mRNA and GAPDH (internal control) in AB7700 SDS V1.7 (Applied Biosystems, Foster City, CA) with the program: 50°C 2 min, 94°C for 10 minutes and 40 cycles at 94°C for 15 s and 60°C for 1 minute. To validate the quantitative real-time RT-PCR protocol, melting curve analysis was performed to check for the absence of primer dimers. The sequence of primers targeting tight junction genes was taken from published studies (
Following treatment, EC monolayers were fixed in 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100 (Mallinckrodt Inc., Paris, KY), and blocked for 30 minutes in blocking solution (5% bovine serum albumin and 5% goat serum (Sigma-Aldrich, ON, Canada) in 0.1% Triton X-100]. Primary antibodies (rabbit anti-human claudin-2, rabbit anti-human Occludin, or rabbit anti-human ZO-1 from Zymed Laboratories, CA, USA) were diluted (2 µg/ml) in blocking solution and incubated with monolayers for 1 hour at room temperature. Normal rabbit serum was used as a negative control to check the specificity of primary antibodies. Following incubation with primary antibodies the monolayers were washed with PBS and secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (1.5 µg/ml, Molecular Probes, Eugene, OR) was added for 1 hour at room temperature. Nuclear counterstaining was done with Propidium Iodide (500nM, Molecular Probes, Eugene, OR). After extensive washing, filters were excised from the polystyrene inserts and mounted on glass slides in mounting medium (Vectashield mounting medium, Vector Lab, CA, USA). All samples were imaged on an inverted confocal laser-scanning microscope (LSM 510, Zeiss, Germany) using standard operating conditions (63× objective, optical laser thickness of 1µm, image dimension of 512×512, lasers: argon (450nm) and HeNe (543nm) for ZO-1 and nuclear staining, respectively. For each experiment, confocal microscope settings for image acquisition and processing were identical between control and treated monolayers and 3 separate, random images were acquired and analyzed for each experimental condition. Each experiment was repeated at least 3 times. Monolayers were scanned in an apical to basolateral sequence and sequential image sets were analyzed by image analysis software (Image J, NIH) to measure the areas of both fluorescently stained ZO-1 and cellular nuclei. Images are presented as either
MTT assay was used to determine viability of HIV-1 exposed monolayers and compared to unexposed control monolayers. The assay was performed according to manufactures instructions (Biotium Inc., CA, USA). Briefly, human primary endometrial epithelial cells and T84 intestinal epithelial cells were seeded on 96-well plates at a density of 103 cells/well and allowed to attach to the plate and grown for 5 days. Triplicate wells were treated with media or exposed with laboratory strains of HIV-1 (104 infectious viral units/ml, MOI 1∶1) in 100 µl quantity. After 24 hours incubation, 10 µl of MTT solution was added and incubated for 4h at 37°C. After incubation, the medium was discarded and the purple blue sediment was dissolved in 200 µl DMSO. The relative optical density (OD)/well were determined at a test wavelength of 570 nm in a ELISA reader using a 630 nm reference wavelength. The MTT assay is based on the cleavage of the yellow tetrazolium salt (MTT) to purple formazan by metabolically active cells, based on their mitochondrial activity. Cell viability was expressed as a percentage of untreated cells, which served as a negative control group and was designated 100%; the results are expressed as % of negative control. All assays were performed in triplicate.
Blue Dextran dye was dissolved in primary medium (2.3 mg/ml,
Apical and basolateral supernatants were analyzed for multiple cytokines using the Luminex multianalyte technology (Luminex Corporation, Austin, TX, USA) as described before
Epithelial cells were grown to confluence and treated with TNF-α (20ng/ml) or HIV-1 (ADA, 106 infectious viral units /ml ) for 24 hours. To test the role of TNF-α, mouse anti-human TNF-α neutralizing antibody (25 µg/ml) (R&D Systems, USA) or normal mouse serum (25 µg/ml) was added to confluent monolayers for 1 hour at 37C prior to treatment with TNF-α (20ng/ml) or HIV-1. Barrier function was determined by TER measurements before and after treatment.
For bacterial translocation experiments, non-pathogenic
For viral translocation, HIV-1 was added to the apical surface of confluent EC monolayers at a concentration of 105 infectious viral units/well and basolateral supernatants were collected at different time intervals. Viral counts were determined using TZMb-1 indicator cell assay.
For assessment of LPS leakage, LPS (100ng/ml; from
GraphPad Prism Version 4 (GraphPad Software, San Diego, CA) was used to compare three or more means by 2 way analysis of variance (ANOVA). When an overall statistically significant difference was seen, post-tests were performed to compare pairs of treatments, using the Bonferroni method to adjust the
TNF-a (NCBI Accession number AAD18091), IL-8 (NCBI Accession number CAA77745), IL-6 (NCBI Accession number AAD13886), IL-10 (NCBI Accession number AAA63207), IL-1b (NCBI Accession number AAC03536), MCP-1 (NCBI Accession number AABB29926). ZO-1 (GeneBank Accession number NM_003257), Occludin (GeneBank Accession number NM_002538), Claudin-1 (Genebank Accession number NM_021101, Claudin-2 (Genebank Accession number NM_020384), Claudin-3 (Genebank Accession number NM_001306), Claudin-4 (Genebank Accession number NM_001305), Claudin-5 (Genebank Accession number NM_003277), GAPDH (Genebank Accession number NM_002046).
The authors would like to thank the Clinical Pathology Staff at Hamilton Health Sciences Center for their assistance in providing genital tract tissues. We thank the women who donated their tissues for this study. We would also like to thank Dr. A. Ashkar for useful discussions and Suzanna Goncharova for technical assistance with Luminex assays.