Objective
The pathway by which herpes simplex virus 2 (HSV2) triggers the innate immune system in the urogenital system has not as yet been fully elucidated. In this study, we aimed to determine which pattern recognition receptors (PRRs) recognize HSV2 in primary vaginal epithelial cells. Once we deciphered the receptors involved, we aimed to target them to immunomodulate innate responses as a prophylactic or therapeutic intervention for early HSV2 infection.
Study Design
To determine which PRRs are involved, receptor silencing as well as confocal microscopy was utilized. For immunomodulation, PRR agonists were utilized to induce a strong, local response to limit the infection, and we used 2 quantitative methods, flow cytometry and plaque assays, to determine their effect on HSV2 replication.
Results
Our results show that HSV2 is detected by a plethora of PRRs: Toll-like receptors (TLR) 2 as well as deoxyribonucleic acid (DNA) sensors TLR9, DNA-dependent activator of interferon regulatory factors, and to a lesser extent interferon-inducible 16, which trigger cytokine secretion to protect the host. Using PRR agonists, such as lipoproteins, CpG DNA, and cyclic dinucleotides, we could significantly limit HSV2 replication.
Conclusion
Different PRRs are strategically placed in different cell locations to detect virus invasion. Use of agonists that target and activate these PRRs appeared to be effective in preventing primary HSV2 infection in vaginal cells and could provide new insights in defense against HSV2 urogenital infections.
Genital infections caused by herpes simplex virus (HSV) 2 are the most frequent cause of genital ulcerations. HSV is a double-stranded deoxyribonucleic acid (DNA) virus composed of a central genome, surrounded by an icosahedral capsid, and an outer lipid and glycoprotein envelope, which facilitates an invasion and evasion of the host and its defences. HSV infection has a high prevalence, and humans are its primary host; consequently, it represents a large economic burden.
In industrialized countries, the prevalence rate for HSV2 infection in adults is up to 20%. In a subset of people with more than 10 lifetime partners, the rate of genital herpes was 50%. Although some infections are self-limiting, HSV2 genital infection has a relapsing pattern of illness (staying latently in sensory ganglia before reactivation), which has an impact on patients’ quality of life both psychologically and socially as well as increasing susceptibility to other infections such as HIV.
There is currently no vaccine available, and although many antivirals such as acyclovir have been developed to reduce duration and severity of infection, there are no therapies that prevent initial infection. Innate immune responses are crucial during the period of acute infection to limit early virus replication and to facilitate the development of an appropriate specific acquired immunity. Knowledge of these innate immune mechanisms is therefore vital if a new therapeutic approach is to be developed.
Studies have shown that HSV glycoproteins bind and activate Toll-like receptor (TLR) 2 on the cell membrane which triggers nuclear factor-κB activation and cytokine production. Furthermore, endosomal TLR9 has been discovered to be important in the detection of the HSV1 DNA demonstrating a TLR9-dependent proinflammatory cytokine response by specialized plasmacytoid dendritic cells.
However, many viruses induce the production of type 1 interferon (IFN) independent of TLRs. The first cytoplasmic DNA receptor for HSV DNA was identified in 2007. The DNA-dependent activator of IFN regulatory factors (DAI) was found to mediate the recognition of HSV DNA in a murine fibroblast cell line. Since the initial publications relating to DAI, accumulating evidence has suggested that additional DNA receptors exist. Within the past 2 years, major breakthroughs have been achieved, and an increasing number of DNA receptors have been associated with innate sensing of HSV, including the pyrin and HIN domain-containing protein family member IFN-inducible 16 (IFI16). IFI16 recognizes HSV-1–derived DNA in murine macrophages and Hela cells and induces IFN-β production.
However, the pathway by which HSV2 triggers the innate immune system in the urogenital system has not as yet been fully elucidated. In this study, we show that HSV2 is detected by a plethora of pattern recognition receptors (PRRs) to trigger innate immune responses. TLR2 recognizes virus lipoproteins, and TLR9 as well as DAI and to a lesser extent IFI16 recognize the viral DNA and mount a strong inflammatory response. By utilizing TLR2 and TLR9 agonists as well as agonists for stimulator of IFN genes (STING), which is a crucial signaling adaptor for DAI and IFI16, we limit HSV2 infection in vaginal cells, indicating that these compounds could have therapeutic utility for urogenital HSV2 infection.
Materials and Methods
Viruses
Prototype strains of HSV2 were obtained from the American Type Culture Collection (Manassas, VA). Viral genomic DNA was isolated from purified virus according to the protocol described by Ling et al in 1996. All procedures for DNA extraction were performed in a class II cabinet, using sterile endotoxin free plasticware and endotoxin-free water. Endotoxin content using the LAL assay was also performed. The test showed low levels of endotoxin less than 0.1 EU/μg DNA (considered endotoxin free). Viral-purified DNA was used to stimulate cells at 20 μg/mL, whereas 5 multiplicities of infection (MOIs) were used when whole virus was used for stimulations.
Cells
Human vaginal epithelial primary cells from human vaginal epithelial primary tissue from healthy donors were obtained from Celprogen (Torrance, CA). The cells were positive for epithelium specific antigen (ESA), cytokeratin-4, -8, and -18. No ethical approval was required because the cells were commercially available.
The cells were maintained in Celprogen’s human vaginal epithelial primary cell culture complete growth medium and subcultured every 24-48 hours on human vaginal epithelial primary cell culture extracellular matrix. They were grown at 37°C in a 5% CO 2 humidified incubator. Primary culture media were replaced after 24 hours of culture and subsequently changed every 48 hours. Cells were used up to 8 passages.
Ribonucleic acid interference
Vaginal cells were plated in a 24-well plate at 10 6 cells/mL and grown to yield 70% confluency. The culture medium was changed to Opti-MEM medium (Life Technologies, Paisley, UK) prior to transfection, and the plasmid expressing small hairpin RNA (pshRNAs) (final concentration 300 nM/well) combined with Lipofectamine 2000 (Invitrogen, Paisley, UK) were added to each well and incubated for 8 hours at 37°C. The target sequence of short hairpin ribonucleic acid (shRNA) used were: TLR2, 5′G-TCAATTCAGAACGTAAGTCA-3′; TLR4, 5′-GCUUAUAUCCUUAAAGAAATT-3′; IFI16, 5′-GGUGCUGAACGCAACAGAAUCAUUU-3′; DAI 5′-GGCCACCUUGAACAAAGAAtt-3′; STING, 5′-GCAUCAAGGAUCGGGUUU-3; and TLR9, 5′-CCGCATCGTCAAACTGGCG-3′.
After transfection, 1 mL fresh culture medium with 2 μg/mL puromycin for selection were added to each well, and the cells were grown for an additional time up to 72 hours. Knockdown of the specified gene was confirmed by indirect immunofluorescence and Western blot analysis for protein expression.
Transfections with the specific shRNAs resulted in an approximately 70% decrease in receptor expression as determined by Western blotting, whereas transfection of cells with the scrambled shRNA did not show any decrease in TLR or IFI16, STING, and DAI expression.
Chemicals
All fine chemicals were obtained from Sigma (Dorset, UK). TLR2-, TLR4-, TLR9-, IFI16-, and DAI-specific polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc (Heidelberg, Germany). Isotype control normal rabbit IgG and normal goat IgG as well as normal mouse IgG 2 were also obtained from Santa Cruz Biotechnology. The anti-HSV2 ICP5 antibody as well as the rabbit anti-STING antibody was obtained from Abcam (Cambridge, UK). Secondary antibodies immunoglobin (Ig) Alexa 546 and Ig-Alexa 633 were obtained from Molecular Probes (Leiden, The Netherlands). Viral DNA was labeled with the Alexa-Fluor-488-ulysis reagent according to the manufacturer’s instructions (Molecular Probes Inc, Cambridge Biosciences, Cambridge, UK). Pam 2 CSK 4 , Pam 3 CSK 4 , FSL-1, CpG DNA (type C), cyclic diguanylate monophosphate (c-di-GMP), and cyclic diadenylate monophosphate (c-di-AMP) were obtained from Invivogen (San Diego, CA).
Flow cytometric determination of PRR expression
To investigate PRR expression before and after HSV2 infection, vaginal cells (2 × 10 5 ) were infected in serum-free medium with HSV2 (5 MOI) or stimulated with viral DNA (20 μg/mL) for different time points before fixation with 4% paraformaldehyde. The cells were subsequently scrapped, washed, and permeabilized using phosphate-buffered saline (PBS)/0.02% bovine serum albumin (BSA)/0.02% saponin. After permeabilization, the cells were incubated with antibodies against different TLRs, as well as DAI, and IFI16 and the appropriate secondaries conjugated to fluorescein isothiocyanate (FITC). Appropriate isotype controls were also used. The cells were washed twice in PBS/0.02% BSA/0.02% Saponin and resuspended in 500 mL of PBS. Fluorescence was detected using a FACSCalibur counting 10,000 cells not gated (Becton Dickinson, Oxford, UK).
Confocal microscopy
Human primary vaginal epithelial cells on microchamber culture slides (Lab-Tek; Gibco, Life Technologies Corporation, Carlsbad, CA), were transfected with 20 μg of HSV2 DNA using Lipofectamine 2000 (Invitrogen) and incubated for different time points. Endosomal compartments were labeled with early endosome antigen 1 (EEA-1)–specific antibody followed by goat-antimouse Ig-Alexa 546. Calreticulin-specific antibody followed by goat-antimouse Ig-Alexa 546 was used to label the endoplasmic reticulum (ER), whereas a TOPRO nuclear stain was used to stain the cell nucleus.
To disrupt endosomal pathways, cells were stimulated with DNA in the presence of monensin (10 μm) or in the presence of Brefeldin A (BFA) (10 μg/mL), which blocks transport between the rough ER and the Golgi complex, with 1 hour of pretreatment. No cellular toxicity was observed. Cells were rinsed twice in PBS, prior to fixation with 4% formaldehyde for 15 minutes to prevent potential reorganization of the proteins during the course of the experiment. Cells were permeabilized using PBS/0.02% BSA/0.02% saponin and labeled with antibodies for TLR9, IFI16, and STING, followed with the appropriate secondary conjugated to Alexa 546.
Cells were imaged on a LSM510 META confocal microscope (with an Axiovert 200 fluorescent microscope; Carl Zeiss, Inc, Jena, Germany) using a 1.4 NA ×63 Zeiss objective. The images were analyzed using LSM 2.5 image analysis software (Carl Zeiss, Inc).
To quantify the degree of colocalization, we used Costes’ approach. Costes’ approach, Pearson’s correlation coefficients, and P values were calculated using MBF ImageJ with JACoP (Just Another Colocalisation Plugin, http://macbiophotonics.ca/ ).
Cytokine analysis
Vaginal cells (2×10 6 ) were stimulated with no stimulus or with HSV2 (5 MOI). The cultures were incubated for the designated times. The supernatants were collected and frozen until the cytokine assays were performed. The Becton Dickinson bead array system was used according to the manufacturer’s instructions to determine the level of multiple cytokines at the same time.
Statistical analysis
Statistical analysis was performed using the program SigmaStat 2.03 (SSPS; IBM, Armonk, NY). Data were evaluated by an analysis of variance, and where appropriate (comparison of 2 groups only) 2-tailed Student t tests were performed. Results were considered statistically significant if the value was P < .05.
Determination of the effect of agonists on HSV2 replication
Two quantitative methods were utilized to determine the effects of different PRR agonists on HSV2 replication. Pa 3 CSK 4 was used at 100-300 ng/mL, Pa 2 CSK 4 was used at 50-150 ng/mL, and FSL-1 was used at 50-150 ng/mL. CpG DNA was used at 100-300 nM and c-di-GMP as well as c-di-AMP at 2-6 μg/mL. Vaginal cells were seeded at a density of 1.5 × 10 6 /well in a 6-well plate and incubated overnight until cells were confluent. Cells were cultured in media containing different concentrations of agonists for 60 minutes. Control cells were cultured in media alone. Then the cells were challenged with HSV2 at 5 MOI.
After adsorption for 1 hour at 37°C in 5% CO 2 humidified incubator, the free virus was removed and replaced with fresh medium, and the cells were incubated for 18 hours. HSV2 replication was determined by flow cytometry staining using anti-anti-HSV2 ICP5 antibody and the appropriate secondary conjugated to FITC. Isotype controls were also used. Fluorescence was detected using a FACSCalibur counting 10,000 cells not gated (Becton Dickinson).
The second method was a plaque assay and was performed in a 6-well plate format. Vaginal cells were seeded at density of 1.5 × 10 6 /well and incubated overnight until cells were 100% confluent. Cells were similarly treated with different agonists. Control cells were left untreated (without agonists). After treatment, cells were challenged with HSV2 at an MOI of 5. After adsorption for 1 hour at 37°C in a 5% CO 2 humidified incubator, the free virus was removed and replaced with a mixture of complete media and 1% agarose. Cells were incubated for 72 hours and then stained with 1% crystal violet and plaques.
Results
PRRs involved in HSV2 recognition
To determine which PRRs contribute to HSV2 recognition in vaginal cells, HSV2 (5 MOI) as well as purified DNA from HSV2 (20 μg/mL) was used to stimulate vaginal cells at different time points, and the PRR expersion levels were investigated by indirect immunofluorscence and flow cytometry ( Figure 1 , A). Isotype controls were also performed, which showed values similar to unstimulated samples ( Figure 1 , B). HSV infects its host through both lytic and latent infection, and replication of HSV occurs within 15-18 hours after infection ; therefore, we investigated PRR expression over an 18 hour period, which represents the time for a full infection.
From the data obtained, there was TLR2 up-regulation when cells were infected with HSV2. There was also an up-regulation in DAI and TLR9 in both HSV2 and HSV2 DNA stimulation, suggesting that DAI and TLR9 play a role in HSV2 DNA recognition in the first hours of the infection probably when the DNA is released from the endosomes in the cytoplasm. A small up-regulation in the IFI16 DNA sensor is also detected in both HSV2 and DNA stimulation ( Figure 1 ).
TLR2, TLR9, and DAI play a role in the innate immune response of vaginal cells
To determine the significance of TLR2 and DNA sensors in the host response to HSV2, we silenced TLR2, TLR9, DAI, and IFI16, and then we stimulated with either HSV2 or HSV2 DNA ( Figure 2 , A) and cytokine production was measured. TLR4 was silenced as a control because it is not involved in HSV2 recognition. Our data showed that interleukin (IL)-6 secretion was severely impaired when TLR2 was knocked down and cells were stimulated with HSV2. TLR9 silencing also affected IL-6 secretion when cells were stimulated with HSV2 or viral DNA. Furthermore, when both TLR9 and TLR2 were silenced, there was a significant inhibition in IL-6 secretion when cells were stimulated either with HSV2 or DNA ( Figure 2 , A). As expected, DAI and IFI16 silencing had no effect on IL-6 secretion because they trigger IFNα/β production.
IFNβ secretion was reduced by knocking down DAI and TLR9; however, it was only minimally affected when IFI16 was knocked down. TLR2 silencing had no effect on IFNβ secretion. When TLR9 and DAI were simultaneously silenced, there was a significant reduction, indicating that they are the main sensors which activate IFNβ production ( Figure 2 , B).
Activation of signal transduction in response to intracellular DNA sensing
To determine the activation of signal transduction in response to intracellular HSV2 DNA sensing, vaginal cells were infected with HSV2 and the presence of STING and interferon regulatory factor 3 (IRF3) was determined via Western blotting ( Figure 3 , A). STING is essential for the activation of the signaling pathway upstream of TANK-binding kinase 1 (TBK1) following HSV1 infection and has been shown to associate with IFI16 and to relay signals downstream of DAI. The data showed an increased expression of STING and IRF3 in vaginal infected cells.
To demonstrate the importance of STING in HSV2 innate immune responses, STING expression was knocked down by pshRNA in vaginal cells, and then cells were infected with HSV2, HSV2 DNA as well as c-di-AMP, which is a second-messenger molecule produced in bacteria but not in mammals. STING has been shown to directly sense this cyclic dinucleotide and induce type I interferon production ; therefore, cyclic dinucleotides were used as a positive control. The data showed a significant decrease in IFNβ production in the absence of STING, confirming the importance of STING mediation in HSV2 DNA detection and host response. Overall, these data verify that STING is essential for activation of the signalling pathway in response to HSV2 DNA and IFNβ production ( Figure 3 , B).
PRR trafficking in response to HSV2 infection
To verify the involvement of TLR2 in HSV2 infection, confocal microscopy was used. Colocalization of HSV2 with myeloid primary response gene (88) (MyD88), a signaling adaptor essential for TLR signaling, was observed when cells were stimulated with HSV2, whereas there was no TLR2 and MyD88 colocalization in unstimulated cells or when cells were stimulated with DNA, confirming TLR2 signaling in the presence of virus particles ( Figure 4 , A).
