Herpes simplex virus type 1 induces simultaneous activation of Toll-like receptors 2 and 4 and expression of the endogenous ligand serum amyloid A in astrocytes
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- Villalba, M., Hott, M., Martin, C. et al. Med Microbiol Immunol (2012) 201: 371. doi:10.1007/s00430-012-0247-0
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Herpes simplex virus type 1 (HSV-1) is the most common pathogenic cause of sporadic acute encephalitis and it produces latent persistent infection lifelong in infected individuals. Brain inflammation is associated with activation of glial cells, which can detect pathogen-associated molecular patterns (PAMPs) through a variety of pattern-recognition receptors (PRR), including Toll-like receptors (TLRs). In this study, we evaluated the expression and activation of TLR2, TLR3, and TLR4 in HSV-1-infected astrocyte and neuronal primary cultures. Our results showed a clear induction in TLR2 and TLR4 expression in astrocytes as early as 1 h after HSV-1 infection, whereas no significant change was observed in neurons. In addition, infected astrocytes showed increased levels of interferon regulatory factors IRF3 and IRF7, interferon β (INFβ), interleukin 6 (IL6), and serum amyloid A (SAA3) transcripts, as well as phospho-IRF3 protein. These effects seemed to be dependent on viral replication since previous treatment of the cells with acyclovir resulted in low levels of TLRs expression and activation even after 4 h post-infection. These results suggest that reactivation of HSV-1 at the central nervous system (CNS) would likely induce and activate TLR2 and TLR4 receptors directly through interaction of astrocytes with the pathogen and also indirectly by endogenous ligands produced locally, such as serum amyloid protein, potentiating the neuroinflammatory response.
The pathogenic mechanisms of herpes simplex virus type 1 (HSV-1) at the central nervous system (CNS) are not well known; however, all the existing data suggest that HSV-1 is able to establish latency in the CNS for life in humans and that this condition would not be harmless, especially in people whose immune system is declined. In addition, it has been estimated that in approximately 70 % of the population over 50 years old, the virus enters the brain and infects neurons, where recurrent reactivations of HSV-1 in CNS of adult people could happen [1, 2]. This trait has been strongly suggested as a risk factor for the development of neurodegenerative pathologies such as Alzheimer’s disease (AD) [3–6]. In fact, our group previously demonstrated that in vitro HSV-1 neuronal infection is associated with several neurodegenerative features, such as tau hyperphosphorylation and cleavage, alteration of the microtubular dynamics and damage in axonal and dendritic processes [7–9]. Nevertheless, it is currently unclear whether a neuron that undergoes viral reactivation and produces infectious particles survives and resumes latency or is killed. These data highlight the need for more studies at cellular and molecular level to understand the pathophysiology of HSV-1 neuronal infection. Brain inflammation due to infection, aging, and other deleterious processes is associated with activation of the local innate immune system, including Toll-like receptors (TLRs), which are ubiquitous and their expression is rapidly altered in response to pathogens, cytokines, and environmental stressors . Also, considering that glial cells are the resident innate immune cells in the CNS responsible for the early control of infections through the release of proinflammatory mediators and recruitment of cells of the adaptive immune system, in the present study we sought to evaluate the effects of HSV-1 infection on the expression and activation of several TLRs in glial cells such as astrocytes and also in neurons. Although there is an increasing body of evidence that TLR signaling mediates beneficial effects in the CNS, it has become clear that TLR-induced activation of microglia and the release of proinflammatory molecules are responsible for neurotoxic processes in the course of various CNS diseases. Walter et al.  demonstrated that TLR4 and TLR2 may be relevant in chronic neuroinflammation in AD. Thus, the functional outcome of TLR-induced activation of microglia in the CNS depends on a subtle balance between protective and harmful effects.
In addition to microbial products, there are several endogenous TLR ligands that have been recently identified; one of them is the acute serum amyloid A protein (A-SAA) [12–14]. The presence of endogenous TLR ligands supports the notion that TLRs play an important role in the detection of danger signals. The acute phase proteins, such as A-SAA, correspond to danger-signaling molecules, which trigger tissue-controlled immune response. In fact, A-SAA have been proposed as endogenous ligand for TLR2  and TLR4  and have been shown to be up-regulated in the brain of AD patients and predominantly localized to neuritic plaques [15–17], supporting the neuroinflammation hypothesis of neurodegeneration. Therefore, in this study, we also analyzed the expression of 2 different isoforms of A-SAA, in primary mice astrocytes and neuronal cultures infected with HSV-1.
Our results showed that HSV-1 infection derived in a clear up-regulation of TLR2 and TLR4 expression and activation results in phosphorylation and nuclear translocation of interferon response factor 3 (IRF3) and induction of IRF7, which are transcription factors that have been implicated as the main regulators of type I IFN gene expression elicited by viruses . Although IRF3 is expressed ubiquitously and constitutively, IRF7 is expressed at low levels in most cells, but its expression is induced by viral infections. Furthermore, TLR2 and TLR4 induction were accompanied by an increased expression of the proinflammatory cytokine IL6 and also the extrahepatic isoform of the endogenous agonist SAA.
Isolation and culture of mouse astrocytes and neurons
Neurons and astrocytes were obtained from 17-day-old embryos and 3-day-old neonate mice, respectively [19, 20]. The animals were killed by lethal doses of intravenous sodium pentabarbitone (200 mg/kg of total weight). Death was confirmed observing cessation of heartbeat and respiration, and absence of reflexes, in agreement with international standards (www.lal.org.uk). Briefly, to prepare neuronal cultures, embryos were removed from the dams at E17 and placed into Hank’s balanced salt solution (1 mM HEPES, pH 7.4, 8 mM NaCl, 0.27 mM KCl, 0.28 mM glucose, 0.02 mM KH2PO4). Embryonic day 1 was defined as the day of conception established by the presence of a vaginal plug. Embryos or neonates were dissected and minced well with scissors. Tissue was dissociated with 0.25 % trypsin at 37 °C for 15 min and then by mechanical grinding with a sterile, fire-polished glass Pasteur pipette, in Minimum Essential Media (MEM) supplemented with 10 % FBS. For neurons, cells were plated at 0.3 × 106 cells/cm2 in plates coated with poly-l-lysine (mol. wt >350 kDa; Sigma-Aldrich Corporation, St Louis, MO, USA). After 20 min, floating cells were removed and attached cells cultured for 5 days in Neurobasal Medium (Gibco, NY, USA) supplemented with B27 (Gibco, NY, USA), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 293 mg/ml l-glutamine (Nalgene, Rochester, NY, USA) . Astrocyte cultures were established from five brains plated in 10 culture dishes (100 × 15 mm) and grown in Minimal Essential Medium containing 10 % fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 293 mg/ml l-glutamine . After 3–4 weeks, the cultured cells were shaken for 12 h, the floating cells were removed, and attached cells were cultured for an additional 2 weeks under the same conditions, obtaining cultures with over 98.9 % of astrocyte cells .
For kinetics studies, infection with HSV-1 (F) was carried out for 1 h in a medium containing 10 % FBS, at a multiplicity of infection (moi) of 10 . Following infection, the virus was removed by washing, the media was replaced with a serum-free medium consisting of neurobasal medium (NB) supplemented with B27 and 0.5 mM l-glutamine, and the cells were further cultivated for 0, 0.5, 1, 2, 4, 8, 18 or 24 h post-infection (hpi). Infection of cultured astrocytes was corroborated analyzing the expression of viral genes HSV-1 gB and UL23 by semi-quantitative RT-PCR (Fig. S1).
In order to determine infectious HSV-1 particles present in the viral stock, a standard plaque assay was performed. Epithelial Vero cells (ATCC N°CCL-81) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and plated onto 12-well plates and grown to confluence. The growth medium was removed and an aliquot of 200 µl of the viral stock, at various dilutions, was incubated with the Vero cells for 1 h. Following incubation, the viral stock was removed and 1 % agarose-containing medium was placed on top of the cells. Plaque formation occurred within 3 days of the initial infection. In order to visualize the plaques, the agarose layer was removed and the cells were fixed and stained with 1 % (w/v) crystal violet in methanol. Lysis plaques were visualized under a light box and counted for each well. Finally, plaque-forming units per milliliter (PFU/ml) were calculated.
Cells were fixed in 4 % paraformaldehyde in PBS for 20 min, washed several times in fresh PBS for 10 min, permeabilized in 0.3 % Triton X-100 in PBS for 15 min, and incubated overnight at 4 °C with the following antibodies: anti-phospho-IRF3 (Cell Signaling Technology, Inc., MA, USA), anti-IRF7 (Santa Cruz Biotechnology, Inc., CA, USA), or anti-tubulin (Sigma-Aldrich Corporation, St Louis, MO, USA). After washing in PBS (three washes of 15 min each), cells were incubated with anti-rabbit or anti-mouse IgG-Alexa 488 conjugate (1:300; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and subsequently washed and mounted. Nuclei were stained with 1.7 µg/ml propidium iodide (Sigma-Aldrich Corporation, St Louis, MO, USA) for 15 min at room temperature. Fluorescent images were obtained using a Zeiss Axioscope II fluorescence microscope (Carl Zeiss, Göttingen, Germany). Cortical neurons and astrocytes were seeded onto 35-mm Petri dishes at a density of 1 × 106 cells/cm2. Data represent the mean ± SEM of three independent experiments.
Western blot analysis
Cultured cells were infected with HSV-1, for different time periods. Afterward, cells were homogenized in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 100 µg/ml PMSF, 2 µg/ml aprotinin, 2 µM leupeptin, and 1 µg/ml pepstatin) and the protein concentration was determined. Protein extracts were resolved by SDS-PAGE (30 µg per lane) in a 10 % polyacrylamide gel and transferred to Immobilon membrane (Millipore, Bedford, MA, USA). After blocking with 5 % (w/v) non-fat dry milk in PBS, the membranes were incubated overnight at 4 °C with primary antibodies in 1 % (w/v) BSA in PBS. Antibodies used were as follows: anti-TLR2, anti-TLR4 and anti-phospho-IRF3 (Cell Signaling Technology, Inc., MA, USA), anti-IRF7 (Santa Cruz Biotechnology, Inc., CA, USA), or anti-tubulin (Sigma-Aldrich Corporation, St Louis, MO, USA). After several washings, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 1 h at room temperature. The blots were developed by chemiluminescence (Amersham, Arlington Heights, IL, USA), and the signal was recorded on Kodak Biomax films. Finally, the films were scanned, and the resulting images were analyzed to determine the relative levels of each signal using Un-Scan-it gel 6.1 program.
RNA isolation and RT-PCR analyses
For total RNA extraction from cultured cells, the media was removed and cells were immediately lysed by the addition of 1 ml of RNAwiz reagent (Ambion Inc.) for 4 × 106 cells according to the manufacturer’s instructions. Extracted RNA was treated with amplification grade DNase (20 kU/μL) for 15 min at 37 °C to eliminate genomic DNA contamination. RNA was quantified by absorbance at 260 nm and used immediately or stored precipitated in ethanol at −70 °C until use. To evaluate integrity, total RNA (30 μg per sample) was incubated in 1× MOPS buffer, 2.2 mM formaldehyde, and 50 % deionized formamide for 10 min at 65 °C; mixed with formaldehyde loading buffer [50 % (v/v) glycerol, 1 mM EDTA pH 8.0, 0.25 % (w/v) bromophenol blue, and 0.25 % (w/v) xylene cyanol]; and separated by electrophoresis in 1 % (w/v) agarose gel containing 6 % (v/v) formaldehyde. Standard RT-PCR analyses were performed using 1 µg of total RNA for reverse transcription, followed by PCR amplification with Superscript III One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) using the specific primers and conditions described in supplementary Table 1. To verify the complete elimination of DNA contamination, negative controls omitting reverse transcriptase were performed in parallel. GADPH was used to normalize the relative expression of the different transcripts. Agarose gels were scanned with a BIO-RAD Molecular Imager FX Pro System, and the pixel intensity of the bands (same area) was measured using the Quantity One program for Windows. Total counts (arbitrary units) from each band were obtained after subtracting the background.
All the results are representative of at least 3 independent experiments. Results were analyzed by one-way ANOVA, and the data were expressed as means ± standard error of the mean (SEM). The p values were reported in each case; p < 0.05 was considered significant.
HSV-1 in vitro infection induces the expression of Toll-like receptors 2 and 4 in astrocytes
HSV-1 activates Toll-like receptor-dependent signaling pathways
HSV-1 replication is required for a strong induction of TLR expression
IL6 and SAA3 are up-regulated during productive HSV-1 infection
Brain inflammation due to infection, aging, and other deleterious processes is associated with activation of the local innate immune system. This could be crucial, if not a causal mechanism leading to the neuronal damage seen in various CNS diseases. In fact, increasing evidence indicates that TLRs play a major role in several inflammatory CNS pathologies . In this context, the neurotropic persistent pathogen HSV-1 constitutes an important candidate to be included among the risk factors for the development of neuropathies.
Previous studies have suggested that activation of TLR4-dependent pathways in microglia contribute to neuronal death and play a key role in the pathogenesis of cerebral injuries, such as those caused by ischemia [22, 23]. In this type of injury, TLR4 was shown to be up-regulated in activated microglia of wild-type mice, while much less neuronal damage and activated microglial cells were observed in the ischemic area of the brains of TLR4 KO mice . Interestingly, these neuroprotective effects were not observed in TLR3 and TLR9 KO mice.
Also, using primary cultures of microglial cells from wild-type and TLR2 KO mice challenged with HSV-1, Aravalli et al.  showed that TLR2 signaling is critical for robust production of multiple major proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, in response to HSV-1 infection. Also, Wang et al.  showed recently that TLR2 KO mice had a significantly increased survival rate following intracranial inoculation of HSV-1, compared to wild-type and TLR9 KO mice.
Here, we show that productive infection of HSV-1 on astrocytes cultures induces strong TLR2 and TLR4 expression and activation, probably through IRF3- and IRF7-dependent pathways, suggesting a possible mechanism by means recurrent reactivations of the virus in the CNS could lead to neuronal damage.
The signaling pathways activated by TLRs are broadly classified into myeloid differentiation factor 88 (MyD88)-dependent and independent pathways, as MyD88 is the universal adapter protein recruited by TLRs . TLR3 and TLR4 are capable of signaling through MyD88-independent pathways and are unique among TLRs, with respect to their capacity to activate IRF3 . Our results suggest that in astrocytes infected with HSV-1, TLR4 up-regulation and activation would be responsible for IRF3-dependent signaling, since no important up-regulation of TLR3 was observed. However, TLR4-independent activation of IRF3 cannot be ruled out since a previous study that characterized the innate antiviral response in human fibroblasts showed that infection with HSV-1-based amplicon vectors triggered an IRF3 and IRF7-dependent, TLR-independent antiviral response, rendering the cells resistant to vesicular stomatitis virus infection and inducing significant changes in the pattern of cellular gene expression, including the nuclear translocation and subsequent degradation of IRF3 .
Activation of interferon and interferon-stimulated genes (ISG) expression has been associated with hyperphosphorylation, dimerization, and nuclear translocation of IRF3; however, several data indicate that depending on the virus and cell type, virus entry is enough to trigger innate antiviral response mediated by IRF3, and that subsequent virus replication is required for hyperphosphorylation, dimerization, and nuclear translocation of IRF3 [29, 30]. In fact, Noyce et al.  failed to detect IRF3 dimerization or hyperphosphorylation upon entry of replication-deficient Newcastle disease virus, despite robust IGS and antiviral state induction. In addition, two independent groups have shown that UV inactivation of HSV-1 fails to fully activate the type I IFN pathway in murine embryo fibroblasts (MEFs) and in vivo [31, 32]. Accordingly, our results suggest that productive infection is required for the establishment of a TLR-dependent antiviral state in astrocytes.
Among the endogenous ligands that have been shown to activate TLR are members of the family of acute phase protein SAA. In fact, SAA can stimulate cells via TLR2 to elicit a robust signaling cascade in human monocytes  and mouse macrophages . In addition, a previous study showed that macrophage activation by SAA is TLR4-dependent, and the downstream pathways involved in SAA-induced NO production includes ERK1/2 and p38 activation but not the MyD88 pathway . Moreover, a recent study reported that SAA is the first natural proinflammatory mediator that can provide signals for the production of pro–IL-1β and for the activation of the NLRP3 inflammasome, resulting in the secretion of mature IL-1β . The expression of isoforms SAA1 and 2 occurs mainly in the liver under the stimuli of proinflammatory cytokines such as IL-6, IL-1, and TNF-α and is subsequently released into the blood during the acute phase response, where they associate with HDL particles. In contrast, SAA3 in mice has been shown to be expressed in a wide variety of cells and tissues, including leukocytes and epithelium, and has never been identified bound to HDL , but rather appears to have autocrine or paracrine effects [35, 36]. In fact, it has been demonstrated that following exposure to different stimuli, such as oropharyngeal administration of LPS, and airway epithelial-specific NF-κB activation, lungs of C57BL/6 mice exhibit a preferential mRNA induction of SAA3 over SAA1 or SAA2 . Accordingly, in the current study, we found that HSV-1 strongly induces SAA3 expression in infected astrocytes, while SAA1 transcript was hardly detectable, confirming that SAA3 would be the isoform involved in local immune response. These results are in agreement with the induced expression of SAA3 transcript observed during cerebral ischemia in mice, which is explained by the up-regulation of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) that binds to the SAA3 promoter, activating its transcription . C/EBPβ also regulates the expression of key genes in inflammation such as tumor necrosis factor α, IL-6, and IL-1β, among many others, and has been shown to be up-regulated in activated astrocytes and microglia . Also, the induction of SAA3 transcript in HSV-1-infected astrocytes paralleled the increased expression of TLR2 and TLR4 receptors, suggesting that TLR activation could not only be triggered by the virus but also amplified by this locally produced danger signal.
The results presented herein suggest that repeated reactivation of HSV-1 at CNS would likely promote neuroinflammation, in part through TLR2 and TLR4 activation directly triggered by the virus components or indirectly through endogenous danger signals, such as SAA proteins synthesized locally by glial cells. Accordingly, preventive and therapeutic measures against HSV-1 or other neurotropic pathogens would be recommendable, with the aim of reducing the incidence of both sporadic and chronic neurodegenerative diseases.
The study was supported by Fondecyt Project 11080067 and DID-UACH S-2009-40.
Conflict of interest
The authors have no conflict of interest to declare.