Enterovirus 71 (EV71) is a member of the family Picornaviridae
, which is composed of non-enveloped single-stranded RNA viruses. EV71 has been recognized for its ability to invade the central nervous system (CNS) and cause neurological symptoms. Data from clinical and animal studies suggest that after infection EV71 viral capsid proteins are present in neural cells in the brain, thus providing evidence that EV71 can directly infect neurons [1
]. In the brains of EV71-infected monkeys, EV71 antigen could be detected in the thalamus and motor cortex [1
]. Furthermore, EV71 RNA and protein expression could be detected in the brain neurons from deceased EV71-infected patients [2
]. However, the effects of EV71 infection in host neural cells remain to be identified.
Antiviral innate immune responses have been shown to play essential roles in defending cells against viral infections. Type I interferons (IFNs), the key regulators of innate immunity, are produced by cells in response to viral sensing. Once secreted, IFNs interact with their receptors resulting in the expression of interferon-stimulated genes (ISGs), which function to suppress viral replication and regulate the inflammatory process [3
]. For example, a previous study showed that IFNβ may have important functions in controlling the replication of enteroviruses [5
]. IFNβ has also been demonstrated to be effective in decreasing the virus yield in coxsackeivirus B3 (CVB3)-infected myocardial fibroblasts [6
]. In another study, mice treated with IFN inducers before EV71 infection showed increased survival rates with decreased viral loads in brain/muscle tissue [7
]. Nonetheless, conflicting results have been obtained when interferon was administered after EV71 infection, with mortality increasing in one study [8
]. Therefore, it has been suggested that interferon plays different roles during different phases of EV71 infection.
Viral pathogens can be detected through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), retinoid acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA-5). These receptors induce IFNβ expression by activating TIR domain-containing adaptor-inducing IFN-β (TRIF) and mitochondrial antiviral signaling protein (MAVS), which form a complex with TANK-binding kinase (TBK), IκB kinase ε (IKKε), and interferon regulatory factors (IRFs) [9
]. Subsequent activation of IRFs results in expression of type I IFNs and proinflammatory cytokines [10
]. However, not all infected cells can produce IFNs because certain viruses can subvert cellular IFN induction pathways [11
]. For example, some viral proteins, including influenza virus proteins PB1-F2 and PB2-S1, interact with the mitochondrial protein MAVS to inhibit the induction of IFN production [12
]; other viruses such as hepatitis C virus and dengue virus suppress IFN activation by cleaving MAVS and Stimulator of interferon genes (STING), respectively [14
Accumulating evidence indicates that the initiation of innate immune responses is regulated in a cell type-specific manner. Different cell types are equipped with specific PRR patterns to sense invading pathogens. Previous studies have demonstrated that some viruses such as dengue virus can induce IFNβ and ISG production in the brain; in contrast, IFNs are not detectable in infected dendritic cells, which suggests that IFN induction is disparately regulated in different cells [17
]. In the brain, TLR2 is expressed by astrocytes, whereas TLR1 and TLR9 expression is limited to infiltrating immune cells [19
]. RIG-I-like receptors (RLRs), including RIG-I, MDA-5, and laboratory of genomics and physiology 2 (LGP2), are widely expressed in most tissues. IFNs can also be generated by neurons [20
]; neurons express TLR3, and poly(I:C) alone can induce expression of IFNβ in NT2-differentiated neurons [21
]. In addition, RNA viruses such as rabies virus have been shown to evoke IFNβ expression in neuronal cells [21
]. Furthermore, a recent study showed that the brains of Theiler’s murine encephalomyelitis virus (TMEV) infected mice containing type I IFN mRNA during the acute phase of encephalitis [22
], indicating that enteroviruses might be able to activate IFN production.
Recent studies have demonstrated that EV71 can actively replicate in neural lineage ranging from neuroblastoma cells to primary neurons [23
]. Regardless, it is not completely clear whether EV71 evokes innate immune responses in neural cells. Although EV71 has been demonstrated to inhibit IFNβ induction by affecting the pathways mediated by RIG-I, TLR3, and MDA-5 via viral proteases [25
], animal experiments have indicated that IFNβ expression is enhanced in the brain tissue of EV71-infected mice [27
]. Therefore, we hypothesized that EV71 infection may trigger activation of IFN expression in the CNS. Our results also demonstrate that EV71 can induce IFNβ expression in neural cells and that this increased IFNβ expression is associated with TLR3, TLR8, and MDA5. Furthermore, ISGs are upregulated in EV71-infected neural cells, indicating that these cells can respond to secreted IFN.
2. Materials and Methods
2.1. Cells and Viruses
EV71 strain TW-2231 (EV71 2231) (subgenotype C2) and EV71 strain BrCr (subgenotype A) were used in this study. Unless otherwise stated, cells were infected with EV71 TW-2231. RD (human rhabdomyosarcoma) cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% non-essential amino acid, 1% L-Glutamine, and 1% penicillin/streptomycin (all from Thermo-Fisher Scientific, Waltham, MA, USA). SF268, SH-SY5Y, and IMR32 cells were obtained from Bioresource Collection and Research Center, Taiwan. SF268 cells are defined as human malignant glioblastoma cells, while IMR32 and SH-SY5Y cells were derived from human neuroblastoma. These cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% L-Glutamine (all from Thermo-Fisher Scientific, Waltham, MA, USA). Human neural stem cells (hNSCs) were obtained from commercial sources (HUXNF-01001, Cyagen Biosciences or U7800-100, Thermo-Fisher Scientific, Waltham, MA, USA). The human neural stem cells were cultivated in a neural stem cell medium containing neural stem cell supplements. For neuronal differentiation, hNSCs were plated in the CELLStart coated plate and incubated for 2 days. The culture medium was then changed to knockout DMEM/F12 with 2% StemPro Neural Supplement and 1% Glutamax, and incubated for 5–7 days at 37 °C and 5% CO2. The differentiation was confirmed by detecting the expression of neuron-specific markers including MAP-2 and Neuron-specific class III β-tubulin. All cells were maintained in a 37 °C humidified incubator equilibrated with 5% CO2.
2.2. Viral Infection
Cells were seeded on 12-well plates at the concentration of 2 × 105 cells per well. The cells were washed by PBS once after overnight seeding. Virus was then added at specified multiplicity of infection (MOI) with serum-free DMEM. After one hour of adsorption, the virus-containing medium was decanted and DMEM containing 2% FBS was then added. To inactivate EV71, virus stock was kept on ice and exposed to UV light in UV crosslinker (Spectronics corporation, NY, USA) at 2 × 105 μJ/cm2 for 20 min.
Poly(I:C) (Sigma-Aldrich, St. Louis, MO, USA) was prepared using PBS. Motolimod (Selleckchem, Houston, TX, USA) were prepared at 1 mM in DMSO. Poly(I:C)_HMW/LyoVecTM (InvivoGen, San Diego, CA, USA) was prepared at 0.125 μg/mL in endotoxin-free water. Poly(A:U) (InvivoGen, San Diego, CA, USA) was prepared at 1 μg/mL in sterile physiologic water. The cells were seeded in 12-well plates and incubated overnight. Lipofectamine 2000 (Thermo-Fisher Scientific, Waltham, MA, USA) was used for transfection and reagents A and B were prepared according to the manufacturer’s protocol. Reagent A contained 100 μL opti-MEM with EV71 RNA, 1 μg poly(I:C), 1 μg poly(I:C)_HMW, 1 μg poly(A:U), or 10 μM motolimod. Reagent B contained 100 μL opti-MEM with 2 μL Lipofectamine 2000. Reagents A and B were mixed and incubated at room temperature for 20 min. The mixtures were then added into tested cells for transfection.
2.4. Immunofluorescence Staining
Cells were fixed with ice-cold 4% paraformaldehyde for 15 min at room temperature. After being washed by 1× PBS three times, the fixed cells were then permeabilized by addition of 0.5% triton X-100 for another 5 min. After being washed by PBS three times, the cells were then blocked by PBS containing 2% FBS for 1 hour at room temperature. After blocking, the cells were incubated with primary antibodies: mouse anti-EV71 3D (1:500, Genetex, Irvine, CA, USA), rabbit anti-MAP2 (1:200, Millipore, Burlington, MA, USA), TUJ1 (mouse anti-neuron-specific class III β-tubulin)(1:200, Millipore, Burlington, MA, USA), and rabbit anti-phosphorylated IRF3-Ser396 (1:200, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The cells were then washed three times with 1× PBS and incubated with Dylight 594 conjugated donkey anti-mouse secondary antibody or Dylight 488 conjugated goat anti-rabbit secondary antibody (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The cells were washed three times with 1× PBS, and cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO, USA). The images were collected by a fluorescence microscope (Olympus BX51, Olympus, Tokyo, Japan).
2.5. Protein Isolation and Western Blot
Total protein was extracted from mock- and EV71-infected cells. Protein samples were separated by 8% or 12% SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (PVDF) (GE Healthcare Life Sciences, Boston, MA, USA). The protein-containing membranes were blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST, 20 mmol/mL Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0.1% Tween-20) at room temperature for 1 h. The membrane was then incubated with primary antibodies: mouse anti-EV71 3D (1:2000, Genetex, Irvine, CA, USA), mouse anti-EV71 3C (1:500, a generous gift from Dr. Shin-Ru Shih, Chang Gung University), mouse anti-EV71 VP (1:2000, Millipore, Burlington, MA, USA), anti-IRF3 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phosphorylated IRF3-Ser396 (1:1000, cell signaling, Danvers, MA, USA), rabbit anti-TRIF (1:1000, cell signaling, Danvers, MA, USA), mouse anti-MAVS (1:1000, Santa Cruz, Dallas, TX, USA), mouse anti-TLR3 (1:1000, Abcam, CAMB, UK), mouse anti-TLR8 (1:1000), rabbit anti-TLR7 (1:1000, both from Thermo-Fisher Scientific, Waltham, MA, USA), rabbit anti-MDA5 Ab (1:2000, Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-RIG-I (1:1000, Pro-Sci, San Diego, CA, USA), and mouse anti-β-actin (1:20,000, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the membrane was probed with anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:5000, Jackson ImmunoResearch Laboratories, St. Louis, USA). The protein was detected with a chemiluminescence reagent (PerkinElmer, Waltham, MA, USA) and a ChemiTM imaging system (Bio-rad, Hercules, CA, USA).
2.6. RNA Isolation and RT-PCR
The total RNA was collected by TRI reagentTM
solution (Thermo-Fisher Scientific, Waltham, MA, USA) at various times. The cells were homogenized by TRI reagent solution and mixed with chloroform. The homogenate was incubated for 5 min at room temperature and centrifuged at 12,000× g
for 15 min at 4 °C. The aqueous phase was transferred to fresh tubes. The aqueous phase containing RNA was added with an equal amount of isopropanol and incubated at room temperature for 10 minutes. The mixture was centrifuged at 12,000× g
for 10 min at 4 °C and the supernatant was removed. The RNA pellet was washed by 1 ml 75% ethanol at 7000× g
for 5 min at 4 °C. The 75% ethanol was removed and the RNA pellet was air dried at room temperature. The RNA pellet was then dissolved by sterile water. One microgram of total RNA was used for cDNA synthesis. The synthesis of cDNA was performed with using RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific, Waltham, MA, USA). One μL of cDNA sample with 5 μM primers was performed for the qPCR and SYBR green (KAPA Biosystems, Wilmington, MA, USA) was used as the quantifying expression. qPCR assay was carried out in a 384-well plate and analyzed by Roche Lightcycle 480 (Roche, Basel, SW). Each sample was assayed in triplicates and 18S rRNA was used as a reference gene. The relative quantification of each gene was analyzed by 2−∆∆CT
method. The primers were designed according to the gene sequence published in NCBI (Table 1
2.7. siRNA Knockdown
The cells were seeded in 12-well plates and incubated overnight. The siRNAs specific for TLR3, TLR7, TLR8, RIG-I, and MDA-5, as well as scrabble siRNA, were used in this study (all from Sigma-Aldrich, St. Louis, MO, USA). The stock of siRNA was prepared in 100 μM with RNase-free distilled water and the working concentration was 100 nM. Lipofectamine 2000 RNAiMAX (Thermo-Fisher Scientific, Waltham, MA, USA) was used for siRNA transfection, and reagent A and B were prepared. Reagent A contained siRNA diluted in 100 μL opti-MEM and reagent B contained 4 μL lipofectamine 2000 RNAiMAX diluted in 100 μL opti-MEM. Reagent A was added to reagent B and incubated for 5 min at room temperature. The cells were washed once with PBS and fresh culture medium was added to the cells. The mixture of reagent A and B was added to the cells and incubated at 37 °C with 5% CO2 for 72 h.
2.8. In Vitro Proteinase Cleavage Assay
The protein extracts of SF268 and 293T cells were prepared upon treatment with CA630 lysis buffer (1% CA630, 50 mM Tris-base, 150 mM NaCl, pH8.0, without protease inhibitor) for 30 min on ice. The cells were harvested and centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were collected. 30 μg of SF268 or 293T protein extract was incubated with 15 μg of viral proteinase EV71 3C or EV71 3CC147S (kindly provided by Dr. Shin-Ru Shih, Chang Gung University, Taiwan) and cleavage buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM DTT and 1 mM EDTA, pH 7.5) at a total volume of 15 μL. The mixture was incubated for 4 h at 37 °C and the signal of proteolytic cleavage was analyzed by immunoblotting.
2.9. IFN-β Antibody Blocking Assay
SF268 was seeded on 12-well plates at the concentration of 2.5 × 105 cells/well. After incubation overnight, the cells were infected with EV71 at an MOI of 40 and then IFN-β antibody (Thermo-Fisher Scientific, Waltham, MA, USA) was added in the fresh DMEM containing 2% FBS after virus adsorption. The total RNA was harvested at 24 h post infection and RT-qPCR was performed to detect viral replication.
2.10. Plaque Assay
The viruses were harvested at different time points and then quantified by plaque assay. RD cells were expanded in DMEM/10% FBS and seeded on 6-well plates at the concentration of 5 × 105 cells/well. After incubation overnight, the cells were infected by serially diluted virus solution. After one hour of adsorption, the virus suspension was decanted and replaced by DMEM (supplemented with 2% FBS and 0.3% agarose). After 96 h, the medium was removed and the cells were stained by crystal violet solution.
2.11. Statistical Analysis
Results were expressed as the mean ± standard deviation. Statistical significance was determined by Student’s two-tailed t-test. Statistical significances are indicated as follows: *, p < 0.05, **, p < 0.01, ***, p < 0.001.
Accumulating evidence demonstrates that neuronal cells can produce IFNβ in response to viral infections. For example, Theiler’s virus and La Crosse virus induce IFN production in the brain neurons of infected animals [20
]. Moreover, differentiated NT2-N cells secrete IFNβ in response to rabies virus infection [21
]. A recent study demonstrated that Sabin attenuated type 1 poliovirus-induced IFNβ expression in SK-N-SH cells [31
]. However, knowledge regarding the abilities of other non-polio neurotropic enteroviruses to induce type I IFN production in human neuronal cells is limited. To the best of our knowledge, this is the first paper to show that EV71 can induce IFNβ expression in neural lineage cells.
Several members of the Picornaviridae
family have evolved strategies to inhibit IFN production by interfering with the cascades involved in the induction of type I IFN expression [32
], and thorough studies have been performed to investigate the inhibitory effect of EV71 on innate immune response regulation. It has recently been shown that EV71 3Cpro
can cleave TRIF and thus suppress the transcription of IFNβ initiated by RIG-I recognition [25
]. Furthermore, 2Apro
targets MAVS and thus reduces IFNβ expression [34
]. A previous study also demonstrated that EV71 3Cpro
inhibits the TLR3-mediated innate immune response by blocking TRIF [26
]. The results of these studies suggest that EV71 viral proteins can efficiently suppress IFNβ induction in host cells. However, because we can observe enhanced expression of IFNβ transcripts in neural cells, we assumed that this phenomenon might be attributed to postponed expression of viral proteins in infected neural cells and efficient simulation of IFNβ transcription by vRNA. Nevertheless, other factors may also contribute to EV71-induced IFNβ upregulation in these specific host cells.
The translation efficiency of enteroviruses is affected by both viral and cellular factors. It has been demonstrated that the lower translational efficiency of Sabin-PV correlates with its superior ability to induce type I IFN production in neuronal cells when compared to that of wild type PV [31
]. Thus, IFNβ induction may not be obvious in RD cells because viral proteins are expressed in large amounts at the early stage of infection. In contrast, EV71 viral protein expression levels are significantly lower than those observed in RD cells. Accordingly, the low translation activity of viral proteins may be in part attributed to the significant IFNβ induction in EV71-infected SF268 cells.
TLR3 has been demonstrated to cause an antiviral response in bronchial epithelial cells infected with rhinovirus [35
]. In addition, TLR3-mediated type I interferon signaling is important in limiting the replication of CVB3 in cells [36
]. A recent study also demonstrated that silencing TLR3 impairs IFNβ expression in EV71-infected immune cells [37
]. Except TLR3, EV71 infection has been shown to increase expression of TLR7 and -8 in brain tissues from fatal EV71 cases [38
]. Additionally, TLR8 is associated with the cardiac inflammatory responses induced by infection with Coxsackie B viruses [39
]. These findings indicate that TLR3 and TLR8 play roles in enterovirus infection. As our results reveal that TLR3 and TLR8 play essential roles in mediating IFN upregulation in SF268 cells, TLR3 and -8 may play essential roles in mediating EV71-induced IFNβ induction.
In addition to TLRs, neurons are equipped with functional RLRs such as RIG-I and MDA-5 [40
]. Recent studies have shown that JEV infection activates neural production of proinflammatory cytokines including IL-6, IL-12p70, MCP-1, IP-10, and TNF-α via RIG-I-dependent pathways, and that ablation of RIG-I in neurons results in increased viral load [41
]. Furthermore, Co et al. demonstrated that simian immunodeficiency virus (SIV) infection enhances expression of RIG-I and MDA-5 in the brains of infected monkeys [42
]. Our knockdown experiments revealed that IFNβ mRNA expression is dependent on MDA-5, in accordance with the results obtained by Kuo et al. [43
Differentiated neural cells are known to express more TLR3 than undifferentiated neural progenitors and thus evoke more potent immune responses [39
]. Therefore, we hypothesized that enhanced IFNβ expression in neural cells may involve with IFNβ induction upon PAMP stimulation. Furthermore, our vRNA transfection experiments showed that when similar amounts of vRNA were detected in SF268 and RD cells, many more IFNβ transcripts were present in SF268 cells. Poly(A:U), motolimod, and poly(I:C)_HMW, agonists for TLR3, TLR8, and MDA-5, respectively, stimulate SF268 cells to produce more IFNβ transcripts in a dose-dependent manner. We also demonstrated that TLR3 and MDA-5 protein expression levels were significantly higher in SF268 cells than in RD cells. The discrepant PRR expression patterns between these two cell types may be associated with their different IFNβ induction capacities. Interestingly, our results showed that in addition to SF268 cells, other neural lineage cells are also capable of producing IFNβ when infected by EV71. Hence, these neural cells may also have superior ability to upregulate IFN expression. However, more experiments need to be performed to support this conclusion.
In summary, our results demonstrate that EV71 infection is able to upregulate expression of IFNβ in neural cells via TLR3, TLR8, and MDA-5, which may be associated with inefficient translation of EV71 RNA in neuronal cells and the superior ability of neural cells to produce IFNβ transcripts upon recognizing the virus. Additionally, IFNβ secreted by infected SF268 cells can interact with cells to limit viral growth, which is in accordance with previous observations [3
]. Interestingly, MxA is the ISG that is able to regulate cell cycles [44
]. A recent study showed the evidence that replication of EV71 could be affected by cell cycle arrest [45
]. However, more experiments have to be performed to link cell duplication and anti-viral activities of IFNβ. The difference in IFNβ-inducing abilities in neural and RD cells reveals the unique properties of neural cells in restricting viral replication.