A pilot study on expression of toll like receptors (TLRs) in response to herpes simplex virus (HSV) infection in acute retinal pigment epithelial cells (ARPE) cellsS Moses, M Jambulingam, HN Madhavan
L and T Microbiology Research Center, Kamal Nayan Bajaj Research Centre, Vision Research Foundation, Chennai, Tamil Nadu, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.138720
Source of Support: None, Conflict of Interest: None
Introduction: Toll like receptors (TLRs) have been proven to play an important role in mounting the innate immune response in an infected host. The expression of TLRs against herpes simplex virus (HSV) have not been studied in retinitis. Therefore, the current study was undertaken to determine the same using the retinal pigment epithelial (ARPE-19) cell line. Materials and Methods: APRE cells cultured in vitro were challenged with HSV 1 and 2 standard strains and 20 other clinical isolates. The cells were observed for cytopathic changes. The cell culture harvest was subjected to RNA extraction using a Total RNA mini kit. The RNA was subjected to reverse transcriptase polymerase chain reaction (PCR) for the amplification of TLRs 3, 4 and 9 and GAPDH housekeeping gene. The amplified products were subjected to electrophoresis on a 2% agarose gel and viewed under a transilluminator. Results: TLR 3 and 4 were expressed by ARPE treated with all the 22 isolates. TLR 9 expression was seen in 16 of the 22 isolates. Bacterial contamination was ruled out by subjecting the harvests to PCR amplification of 16sRNA gene amplification of the eubacterial genome. Conclusions: The expression of TLR 4 has been reported for the first time in HSV infection. TLR 4 along with TLR 3 and 9 is responsible for the antiviral response in HSV infections.
Keywords: Antiviral response, HSV, TLR
Human cells are equipped with a range of pattern recognition receptors (PRRs) that recognize microbial pathogen-associated molecular patterns (PAMPs) and mount innate immune responses following infection. Antiviral sensors can be broadly classified into two groups: Toll like receptor (TLR) family members and retinoid-inducible gene 1 (RIG-I) like receptors (RLRs). 
The viral protein sensors include the TLRs, which have been shown to recognize viral lipopeptides. The major trigger for cellular recognition of a viral infection appears to be viral nucleic acid that can be recognized at multiple cellular locations. , Extracellular double stranded ribonucleic acid (dsRNA) is detected at the cell surface by the class A scavenger receptors. Recent data suggest that these membrane-bound receptors recognize extracellular dsRNA and serve as chaperones to bring viral dsRNA into the cell for cytosolic recognition. In the endosomal compartment, TLR3 serves as a receptor for viral dsRNA and TLR9 recognizes CpG-rich DNA.  The most frequent cause of acute retinal necrosis (ARN) is herpes simplex virus (HSV) 1 and 2, associated with a history of encephalitis and meningitis in patients older and younger than 25 years, respectively. HSV-1 is the most commonly diagnosed cause of sporadic (nonepidemic) encephalitis in humans. ,, Triggering events such as periocular trauma, neurosurgery and high-dose corticosteroids have been reported. T lymphocyte infiltration of the brain and cytokine production cannot be detected until 1-2 days after virus infection. The necrotic process seems to be driven by CD4+ cells, macrophages, polymorphonuclear cells, B cells and the inflammatory cytokines tumor necrosis factor-α and interferon (IFN)-γ. HSV-1 tegument proteins have been characterized as major targets for T cells within the vitreous fluid. , Since TLRs have been known to play an important role in the immune response to various infectious diseases, this preliminary in vitro study on the expression of TLR in ARPE-19 cells on challenging with HSV was undertaken. TLR 3 and 9 have been described to play an important role in viral infections. TLR 4 has been described to be expressed only in certain viral infections like respiratory syncitial virus, H5N1 virus and influenza virus. The expression of TLR 4 in HSV infections has not so far been reported. Hence, the expression of these TLRs in HSV infections has been focused in our study.
To determine the host immune response to the HSV infections affecting the posterior part of the eye, especially ocular ARN that causes devastating effects such as blindness, an in vitro study on immune response to HSV infections was designed using ARPE. In this study, we have determined the immune response through TLRs on ARPE-19 cells.
ARPE-19 (ATCC) cells obtained from the National Center for Cell Science (NCCS), Pune, India, maintained in Advanced DMEM with F12 (Gibco, Grand Island, NY) with 20% FCS (Hi-media, Mumbai, India) were obtained at a passage of 23. The cells were trypsinized and cultured on to 24-well plates. The plates were incubated at 37°C with 10% CO 2 . Once the cells formed a monolayer, further inoculations of HSV was carried out.
HSV - 1: ATCC 733 -VR (Chemicon, Inc Temecula, CA, USA)
HSV - 2: Species 753167 (NIV)
Twenty-two (n = 22) isolates along with two standard strains were used for the inoculation of 15 HSV- 1 and seven HSV-2 isolates. The source of isolates has been described in [Table 1]. Twenty microliters (20 μL) of the stock was inoculated onto the monolayer of ARPE 19 cell line after aspirating out the growth medium from the plate, and the cell lines were kept for rocking for 30 min. Following this, sterile advanced DMEM with F12 (Gibco, USA) containing 2% FBS, was added on to the wells, including the uninoculated cell controls. The initial morphological changes were observed after 24 h of incubation. The plates were then incubated at 37°C for 48 h and harvested.
The RNA was extracted from 200 μL of culture isolates harvested from ARPE cells and uninoculated cell controls by using a Total RNA mini kit (Qiagen, Germany) as per the protocol given by the supplier. The Total RNA was eluted in 60 μL of the AVE buffer and was stored at -80°C. The RNA was used for amplification of the GAPDH and TLR genes.
The primer sequences used for the amplification of TLRs 3, 4 and 9 and GAPDH genes and the amplified product size are described in [Table 2]. ,
Reverse transcriptase polymerase chain reaction (RT-PCR)
The RT-PCR was standardized according to our laboratory conditions. The enzyme mix facilitated both reverse transcription and PCR (USB Corporation, Cleveland, OH, USA). The reaction mix was incubated in a thermal cycler. The thermal reaction followed is as follows: 50°C for 30 min for reverse transcription followed by the activation of Hot star Taq DNA polymerase enzyme and the PCR amplification was then proceeded for 40 cycles by denaturing the cDNA template at 95°C for 60 s, annealing at 55°C for 60 s and extension at 72°C for 60 s following final extension at 72°C for 7 min for the amplification of the TLR 3 and 9 genes. The TLR 4 and GAPDH genes were amplified simultaneously in the same reaction condition except for the annealing temperature, which was standardized at 58°C for 60 s. The amplified products were visualized in 2% agarose gel incorporated with 0.5 g/mL of ethidium bromide.
The DNA was extracted from 200 μL of culture isolates harvested from ARPE cells and uninoculated cell controls by using the QIAamp DNA mini kit (Qiagen, Germany) as per the protocol given by the supplier. The DNA was eluted in 200 μL of the AE buffer and stored at -80°C. The DNA was used for the amplification of 16srRNA.
As bacteria can trigger the TLR expression, the possible role of the same was ruled out by performing PCR against the 16srRNA of bacterial genome. The RNA extract was converted to cDNA and nested PCR targeting the 16srRNA of bacteria was carried out as reported earlier by Therese et al. in 1998. The thermal profile consisted of an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 2 min and a final extension at 72°C for 7 min. The second round of PCR was carried out with the same thermal profile for 15 cycles.
Mycoplasmal contamination in the cell line has been ruled out by subjecting it to PCR using a ready-to-use kit procured from Sigma (Cat no: MP0035) (St. Louis, Missouri, USA).
Spectrophotometeric analysis band intensity of the amplified products
The gel images were analyzed for the intensity of the bands of the amplified products using the Image J software. The intensity of the bands of TLRs and GAPDH genes of the 22 isolates and the control (uninoculated cell control of ARPE) were compared for the relative expression of the genes.
Bacterial culture was performed to rule out bacterial contamination using Brain Heart Infusion broth and culture.
The standard strains and the isolates showed cytopathic effect at 24 h. [Figure 1] shows the cytopathic effect of HSV standard stains 1 and 2 and the uninoculated ARPE cell control. All clinical isolates and standard strains showed amplification of the house keeping gene corresponding to 600 bp of the 100 bp molecular weight marker [Figure 2]. Of the 22 isolates, 16 showed amplification of the TLR 3 gene corresponding to 304 bp of the 100 bp molecular weight marker [Figure 3]. The other six isolates did not show any expression of TLR 9. All clinical isolates and standard strains showed amplification of the TLR 9 gene corresponding to 259 bp of the 100 bp molecular weight marker [Figure 4]. The standard strains and all 20 isolates of HSV showed amplification of the TLR 4 gene corresponding to 267 bp of the 100 bp molecular weight marker [Figure 5].
The bacterial contamination of the culture harvest was ruled out by performing PCR for the detection of eubacterial genome targeting the 16srRNA subunit and inoculation on to liquid media (Brain Heart Infusion broth) and checked for sterility for 12 days. The eubacterial PCR was negative [Figure 6], and the bacterial cultures did not show any bacterial growth. Mycoplasmal contamination in cell lines has been ruled out by amplification of the DNA extracted from the cell line using PCR [Figure 7].
The OD values of the TLR4 and GAPDH gene amplifications are tabulated in [Table 3]. The ratio of the OD values of TLRs revealed the relatively higher expression of the TLR genes with that of the GAPDH genes. In the uinoculated cell control the GAPDH expression alone was observed. The non- expression of TLR 4 gene in the control cells further confirms beyond doubt that the expression of TLR4 is only due to the challenging of the cells with the virus. The TLR 4 expression was confirmed with experiments carried out in duplicates and consistent results were obtained.
Host resistance to HSV infections includes nonspecific mechanisms involving IFNs, complement, macrophages, humoral (antibody) immunity, T cell-mediated immunity (such as cytotoxic T cells and T helper cell activity) and cytokine release. The relative importance of these various mechanisms is different in initial or recurrent HSV disease. Animal studies have suggested that activated macrophages, IFNs and, to a lesser extent, natural killer cells are important in limiting initial HSV infection, whereas humoral immunity and cell-mediated immunity are important in controlling both initial and recurrent infections. 
The pathogenesis of ARN was provided by an early animal model in which HSV inoculation into the anterior chamber of rabbits was followed by retinal necrosis of the uninoculated eye,  which was later confirmed in mice.  The virus spreads through synaptically connected nerve nuclei and neurons to the contralateral, but not ipsilateral, optical nerve and retina. ,, Hence, retinal pigment epithelial cells (ARPE-19) serve as the best model to study the expression of TLRs, which has been used in this study.
TLRs have been shown to recognize both nucleic acid and protein derived from HSV. In mice, four TLRs have been found to play a role in the resistance to HSV-1: TLR 3, TLR 9 and the TLR 2/6 heterodimer, which recognize dsRNA, CpG-rich DNA and lipopeptides, respectively. ,,,, HSV-1 infection via intranasal delivery causes 100% mortality in mice deficient in the TLR adaptor protein MyD88, suggesting that innate control via TLRs is important in limiting HSV-1 infection. Indeed, it has been shown in vivo that HSV-1-mediated production of IFN requires TLR 9 and MyD88. 
A number of groups have revealed that mucosal delivery of ligands for TLR 3 and 9, but not TLR 2 or 4, are protective against genital infection with HSV-2. Similarly, intranasal application of a ligand for TLR 3, but not for TLR 4 or 9, protected against HSV encephalitis in mice. ,,,,, The therapeutic approach of using TLR ligands to treat HSV is an area that requires further investigation as the downstream consequences of TLR engagement in vivo remain to be clarified.
In our study, the expression of TLR 9 and 4 was seen in all 22 isolates and TLR 3 was expressed in 16 of 22 isolates. The nonexpression of TLR 3 in the rest of the six isolates might probably be due to the low expression of TLR 3 in these isolates. The unusual fining in our study is that TLR 4 has been expressed in response to HSV infection in ARPE-19 cells.
TLR 4 expression has usually been reported in response to Lipopolysaccharide (LPS); in other words, in response to bacterial infection. Different human intestinal epithelial cell lines have been shown to have all three types of LPS responsiveness and TLR 4 expression: (1) relative hyporesponsiveness to LPS with a low level of TLR 4, (2) hyporesponsiveness to LPS with intracellular TLR 4 localization and (3) highly LPS-responsive with surface expression of TLR 4, suggesting that these cells may comprise different subpopulations with distinct roles in innate immune responses.  TLR 4 has also been shown to be involved in sensing various viruses, including respiratory syncytial virus, mouse mammary tumor virus, murine leukemia virus and Coxsackievirus B4 virus via different viral proteins. ,,,,,,,,, The nonexpression of TLR 4 in the uninoculated cell control has also strongly proven the fact that TLR 4 has been expressed only because of challenging the cells with HSV. Studies have reported low-level expression of TLR 4 in ARPE-19 cell lines, although it could not be detected in our study. , The TLR 4 expression was enhanced over the natural expression level after viral exposure.
In conclusion, this is the first study wherein we have observed that TLR 4 is expressed on HSV exposure and that it could play a protective role in HSV infection. Therefore, we conclude that TLR 3, 4 and 9 have been expressed in HSV infections in ARPE cells, which has a protective role to play in the immunity of ocular HSV infection. Further studies are required to study the production of IFNs and cytokines.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]