Accepted Manuscript TRIF contributes to epileptogenesis in temporal lobe epilepsy during TLR4 activation Fa-Xiang Wang, Xiao-Lin Yang, Yuan-Shi Ma, Yu- Jia Wei, Mei-Hua Yang, Xin Chen, Bing Chen, Qian He, Qing-Wu Yang, Hui Yang, Shi-Yong Liu PII: DOI: Reference:

S0889-1591(17)30386-0 http://dx.doi.org/10.1016/j.bbi.2017.07.157 YBRBI 3198

To appear in:

Brain, Behavior, and Immunity

Received Date: Revised Date: Accepted Date:

22 March 2017 28 June 2017 26 July 2017

Please cite this article as: Wang, F-X., Yang, X-L., Ma, Y-S., Jia Wei, Y., Yang, M-H., Chen, X., Chen, B., He, Q., Yang, Q-W., Yang, H., Liu, S-Y., TRIF contributes to epileptogenesis in temporal lobe epilepsy during TLR4 activation, Brain, Behavior, and Immunity (2017), doi: http://dx.doi.org/10.1016/j.bbi.2017.07.157

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TRIF contributes to epileptogenesis in temporal lobe epilepsy during TLR4 activation Authors: *Fa-Xiang Wang1 , *Xiao-Lin Yang1, Yuan-Shi Ma1, Yu- Jia Wei1, Mei-Hua Yang1, Xin Chen3, Bing Chen4, Qian He2, Qing-Wu Yang2, Hui Yang1 and Shi-Yong Liu1 Affiliations: 1: Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China; 2: Department of Neurology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China; 3: Department of Neurosurgery, Chengdu Military General Hospital, Sichuan, 610083, China; 4: Department of Neurosurgery, Nanchong Central Hospital, Sichuan, 637900, China; * : These authors contributed equally to this work. Correspondence to: Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, 183 Xinqiao Main St, Shapingba District, Chongqing 400037, China. Shi-Yong Liu([email protected]); Hui Yang ([email protected])

Increasing evidence indicates that inflammatory processes play a crucial role in the etiopathology of epilepsy and seizure disorders. The Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) activated several transcriptions leading to the production of pro-inflammatory cytokines in the central nervous system, which suggests a potential role for TRIF in the epileptogenesis of epilepsy. In this study, we investigated the roles of TRIF in human and mice epileptogenic tissues.

Western blot and immunohistochemistry assays showed that the expression of TRIF was significantly upregulated in neurons and glial cells in both human epileptic tissues and mouse models, and positively correlated with seizure frequency. TRIF expression positively correlated with

high-mobility

group

box

1

(HMGB1)

expression.

In

TRIF-deficient

mice,

electroencephalograms displayed a significant decrease in seizure frequency and duration time, while KA induced seizures compared with wild-type(WT) mice. The number and duration time of spontaneous seizures were also decreased in the chronic KA-induced TRIF-deficient mouse models. In TLR4-deficient hippocampal neurons and mouse models, TRIF expression was lower compared with WT mice during HMGB1 and KA stimulation. Meanwhile, in KA-induced TRIF-deficient

mouse

models,

microglia

activation

was

significantly

suppressed;

pro-inflammatory factors including IL-1β, TNF-α, iNOS, HMGB1 and IFN-β were reduced; and the survival of the neurons in the hippocampus increased compared with WT mice. Our findings suggested that TRIF may be involved in the epileptogenesis of temporal lobe epilepsy, which would make it a potential therapeutic target for the treatment of epilepsy . Keywords:

Toll-like

receptors;

TRIF;

Epileptogenesis;

Temporal

lobe

epilepsy;

Neuroinflammation. Introduction Epilepsy is a chronic disorder characterized by episodes of disturbed brain activity that affect the patient’s attention and behavior, accounting for 1% of the global burden of disease (Engel, 2011). Temporal lobe epilepsy (TLE) represents the most common form of chronic and intractable epilepsy syndrome, and often progress refractory to anticonvulsant pharmacotherapy (Faber et al., 2013). Although a surgical resection is a promising option that renders approximately 70% of

patients as seizure-free，the remaining 30% of patients still suffer from seizures. Therefore, the mechanisms of the epileptic seizure of TLE need to be further elucidated. Over the past decade, clinical and experimental evidence has indicated that brain inflammation plays a prominent role in the mechanisms of seizure precipitation and recurrence. Several clinical studies have demonstrated that increased levels of inflammatory mediators, such as interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α and IL-1β, may play important roles in seizure disorders in the absence of infectious or immune-mediated etiology (He et al., 2013; Shu et al., 2010; Vezzani et al., 2013). Recent studies indicate that the Toll-like receptor (TLR) signaling pathways are activated in epilepsy (Bordon, 2010; Zurolo et al., 2011). TLR4, TLR2 and their endogenous ligand, the HMGB1, are overexpressed in the epileptogenic tissues of drug-resistant TLE, focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC) (Maroso et al., 2010; Zurolo et al., 2011). Additionally, animal experiments have shown that TLR4 knockout animal models are intrinsically less susceptible to experimental seizures, and HMGB1 increased NR2B phosphorylation, which promoted seizures through TLR4 signaling (Maroso et al., 2010). The above studies suggested that seizures are modulated via complex interactions between innate and adaptive immunity. These findings provide novel evidence for the intrinsic activation of these pro-inflammatory signaling pathways, which could contribute to the high epileptogenicity mediated by the TLR signaling pathways. Toll/IL-1R domain-containing adap tor-inducing IFN-β（TRIF), also called TCAM1, is an adapter molecule that contain TIR domain that interacts with TLRs via TIR:TIR domain and triggers downstream signaling (Brikos and O'Neill, 2008; Jenkins and Mansell, 2010; Ware et al., 2012). Recent studies suggested that the receptor-interacting protein homotypic interaction motif

(RHIM) of TRIF was associated with cell apoptosis (Hanafy, 2013; Kaiser and Offermann, 2005). Neuronal apoptosis can be the result of seizure activity in the hippocampus and promote further progression of TLE (Bengzon et al., 1997). TRIF mediates TLR4 signaling pathways involved in the activation of NF-κB and IFN-β production, as well as TLR3-dependent IFN-β production, which leads to the production of type-I IFN and pro-inflammatory cytokines, including IL1-β and TNF-α (Fitzgerald et al., 2003; Kawai and Akira, 2006; Matin et al., 2015; Siednienko et al., 2010). Although TLRs have somewhat similar signal transduction pathways, there is specificity with regard to their adaptor effects (Akira and Takeda, 2004; Siednienko et al., 2010). Toll-like receptor agonists can differentially induce the myeloid differentiation factor 88 (MyD88) and TRIF dependent pathways (Figueiredo et al., 2009). During TLR4 activation, the MyD88 and TRIF pathways conferred distinct single-cell signaling characteristics (Cheng et al., 2015). The MyD88 and TRIF signaling pathways play distinct roles in different diseases, such as endotoxic shock, polymicrobial sepsis, and acute cerebral infarction (Feng et al., 2011; Yang et al., 2008). Our previous study suggested that TLR4 may contribute to injury through the TRIF instead of MyD88 signaling pathway (Yang et al., 2008). The prior study further showed resveratrol, a phytoalexin with anti-inflammatory effects, decreased the KA-induced seizures and can specific inhibit TRIF signaling (Gupta et al., 2002; Wu et al., 2009; Youn et al., 2005).Those studies suggested that TLR4 may contribute to brain injury through the TRIF signaling pathway, TRIF may play a more important role in brain damage. Moreover, in a rat model, HMGB1 induced the TLR4-mediated signaling cascade in seizures following ischemic injury (Liang et al., 2014). Thus, we hypothesize that TRIF may be the key factor of epileptogenesis of TLE in the HMGB1-TLR4 signaling pathway. In this study, we examined the expression, distribution and function of TRIF in TLE

patients and experimental mouse models. Furthermore, whether the upstream and downstream factors of TRIF are involved in the TLE was also investigated. Materials and Methods Subjects All tissues were obtained after receiving informed consent, and the experimental procedures were performed with the approval of the Ethics Committee of the Third Military Medical University (Chongqing, China). The patients were diagnosed according to the International League Against Epilepsy (ILAE) Classification of Epilepsies and Epilepsy Syndromes Criteria (1989). Specimens from patients with intractable TLE (n = 20) were obtained between October 2009 and July 2014 and were comprised of 7 male (range 8–45, median age 24) and 13 female patients (range 6–47, median age 23). The clinical data were summarized in Supplementary Table 1. The five controls, temporal neocortical and/or hippocampal tissue samples without a history of epilepsy or other neurological diseases, were obtained from autopsies, with a maximum post-mortem delay of 24 hours (Supplementary Table 2). Animals WT mice, TRIF-deficient mice and TLR4-deficient mice in a C57BL/6 genetic background (n = 16 each group) were used at the age of 8-12 weeks (Hoebe et al., 2003). All experiments were approved by the Third Military Medical University Animal Studies Committee. We strictly adhered to the Guide for the Care and Use of Laboratory Animals (Guide) (NRC 2011). Cannulation and Infusion The mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate (400 mg/kg) and placed in the stereotaxic apparatus. A pair of stainless steel insulated bipolar depth electrodes

(30 mm OD) was implanted into the right dorsal hippocampus to record the hippocampal electroencephalogram. The coordinates for the injection in the CA1 region were as follows: nose bar, 0; anteroposterior, -1.5 mm; lateral, 1.2 mm; and dorsoventral, 1.5 mm from the bregma below the dura mater. The recording and reference electrodes were placed as follows: nose bar, 0; anteroposterior, -2.5 mm; lateral, 2.5 mm and dorsoventral 2.0 mm from the bregma below the dura mater. The cannula and electrodes were held in place with acrylic dental cement. After surgery, the animals were housed individually and recovered for at least 7 days. Aninjector needle (30 gauge) extending 200 µm from the tip of the guide cannula, which was connected to a 5 µl Hamilton microsyringe via PE20 tubing, was used to infuse either vehicle (PBS), KA (Sigma-Aldrich, USA) or HMGB1 at a rate of 0.5 µl/min, and the needle remained in situ for 5 min to block backflow. Acute model of seizures. For the acute mouse model of seizures, an intracerebral application of KA (20 ng in 2 µl, Sigma-Aldrich) and PBS (pH 7.4) for the control group [from bregma (mm): nose bar, 0; anteroposterior, -0.22;lateral, 1.1;and dorsoventral, 2.5 from bregma below the dura mater] was administered after the animals’ baseline EEG activity was recorded for 30 min. After the KA injection and during seizures, behavioral seizures, such as jumping, contralateral circling, sniffing and gnawing, and even the “Wet dog shakes”, were usually observed in mice. The behavioral seizures were observed after the KA injection and scored based on a modified Racine scale (Iori et al., 2013). Full-length and LPS-free recombinant HMGB1 protein(Maroso et al., 2010) (10 ug in 1 ul， sigma, USA ) had been dissolved in sterile 0.1 M PBS (pH 7.4). Drugs were

intracerebroventricularly injected 15 minutes before KA in acute mice into the septal pole of the hippocampus. Chronic model of spontaneous seizures. For the chronic mouse model of spontaneous seizures, the mice were injected with KA (200 ng dissolved in 1 µl of PBS for the chronic models of spontaneous seizures) after their baseline electroencephalogram activity was recorded for 30 min to serve as models of spontaneous seizures. Similar to the acute phase, behavioral seizures were observed and scored. Spontaneous seizures occur reproducibly at approximately 1 week and recur for up to 6 months (Maroso et al., 2010). During post-status epilepticus (SE) at 7-8 weeks, videos were recorded for 5 consecutive days (24 h per day). The electroencephalogram signals were recorded and analyzed using a CerePlex 32-channel data acquisition system (Blackrock Microsystems, Inc., USA). Seizure Assessment and Quantification The assessment and quantification of seizures has been extensively described using three parameters: the time of onset (i.e., the latent period from the KA injection until the occurrence of the seizure on the electroencephalogram), the total number of seizures (by summing every ictal episode during the electroencephalogram recording period), and the total duration of seizures (by summing the duration of every ictal episode) (Balosso et al., 2008; Maroso et al., 2010). The EEG recordings were terminated at 8 h after drug injection. Tissue processing Surgery samples from patients as well as brain tissue specimens from the KA-induced mice were instantly put in liquid nitrogen for storage, awaiting Western blotting at various times. For immunohistochemical detection, the specimens were isolated at the time of surgery and placed in a

solution of 10% methanal in 0.1 M PBS (pH 7.4) for 24 hours. The mice were anesthetized and intracardially perfused with a solution of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were excised and post-fixed in the same fixative for 24 hours; they were then embedded in paraffin, cut into 8mm coronal sections, and mounted on coverslips coated with poly-L-lysine. For fluorescence staining, the human tissues and mouse brains were post-fixed overnight, followed by cryoprotection in 30% sucrose. RNA isolation and qPCR Using our previously described method, the total RNA samples (n=8 for each subunit) were isolated from the hippocampus using an RNA extraction kit (Invitrogen, USA). The quality and quantity of the final RNA samples were detected with a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and verified by agarose gel electrophoresis. The total RNA was reverse transcribed using the Transcriptor RevertAidTM First Stand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., USA) and the following program: 65°C for 5 min, 25°C for 5 min, 60°C for 42 min, 70°C for 5 min, and 4°C for 5 min. The cDNA was then amplified in a final volume of 25 µl by PCR. The optimized sense and antisense primer sequences used for amplification are listed in Supplementary Table 3. PCR was performed in accordance with the instructions of the Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Canada). The samples were denatured at 94°C for 30 s followed by 40 cycles of 95°C for 1 min, 60°C for 30 s and 72°C for 40 s, with a primer annealing temperature of 61°C. The experimental data were analyzed using the ∆∆Ct method. All independent experiments were repeated three times, and the data were plotted as the mean ±SD. The results were normalized to the endogenous control, GAPDH.

Western blotting The protein extracts were diluted with loading buffer and separated on 12% SDS-polyacrylamide gels by electrophoresis before being transferred to nitrocellulose membranes. The membranes were blocked in blocking buffer (LI-COR Bioscience, Lincoln, NE) at room temperature for 2 h and then incubated with anti-TRIF (1:1000; Abcam, USA) or anti-GAPDH (1:1000; Cell Signaling Technology, Beverly, MA) primary antibodies overnight at 4°C. The membranes were washed and incubated with goat peroxidase-conjugated secondary antibodies (1:5,000; Zhongshan Biotechnology, China) for 1 hour at room temperature followed by enhanced chemiluminescence (ECL) substrate. The blots were analyzed using a digital scanner. The target proteins were quantified by normalization with the GAPDH levels in the same lysates. The band intensity was quantified using the ImageJ 1.43 µ program (National Institutes of Health, USA). Immunohistochemistry Following our previous methods, the tissues were de-waxed in xylene, rehydrated in alcohol, and then immersed in 3% hydrogen peroxide in water/methanol solutions for 10 min to block the endogenous peroxidase activity. Antigen retrieval was performed by heating the sections at 100°C for 20 min in 0.01 mol/l sodium citrate buffer. After washing three times with PBS, the sections were incubated with the following primary antibodies overnight at 4°C: anti-TRIF (1:50, Abcam, USA), rabbit anti-glial fibrillary acidic protein (GFAP; 1:100; Sigma, USA), and anti-Iba-1(1:100; Sigma, USA), respectively. After 3 consecutive washes with PBS for 15 min, the sections were incubated with a goat polyclonal secondary antibody to mouse IgG (Boster, China) for 30 min at 37°C. After extensive washes with PBS, the color reaction was developed with diaminobenzidine and counterstained with Meyer’s hematoxylin. Then, all of the stained sections were dehydrated

and mounted. Non-immunized serum that was pre-absorbed with a ten-fold excess of specific blocking antigen or PBS was substituted for the primary antibodies as the negative control. Six contiguous slices per mouse were analyzed, and three mice per group were studied. We used a light microscope (BX51, Olympus, Japan) at 200× magnification to count the cell number. The coordinates according to Paxinos and Franklin (2001) were -1.94 mm caudal to bregma, 1.5 mm right lateral to midline, and 1.3 mm ventral to the surface of brain.The groups were blinded to three researchers who obtained the data, and this value was used for the statistical analysis. Fluorescence staining The tissues were fixed in 4% paraformaldehyde for 24 hours, dehydrated with 30% sucrose/PBS, and frozen in compound. The sections (20-µm-thick) were blocked with 0.1% Triton X-100 and 5% normal donkey serum in PBS for 20 min, and they were then incubated with the following primary antibodies overnight at 4°C: anti-TRIF (rabbit polyclonal, 1:100; Abcam, USA); anti-NeuN (mouse monoclonal, 1:200; Millipore, USA); anti-GFAP (mouse monoclonal, 1:500; CST, USA); anti-Iba-1 (mouse monoclonal, 1:200; Abcam, USA); anti-CD11b (rat monoclonal, 1:200; Millipore, USA); and anti-HLA-DP, DQ, and DR (mouse monoclonal, 1:100, Dako, Denmark). The sections were then incubated with a mixture of AlexaFluor 647-conjugated donkey anti-rabbit IgG and AlexaFluor 488-conjugated donkey anti-mouse IgG or AlexaFluor 594-conjugated donkey anti-rat IgG (1:500; Life Technologies) antibodies for 30 min at 37°C. Next, 4′,6-diamidino-2-phenylindole (DAPI, 1:3000) was applied for 5 min, and the samples were rinsed 3 times in PBS for 15 min. The sections were mounted on SuperFrost slides, and all images were captured using a confocal fluorescence microscope (TCS-TIV; Leica, Nussloch, Germany). Nissl and Fluoro-Jade B (FJB) staining

Hematoxylin and eosin (H&E) staining and Nissl staining were performed according to our previous methods. For the FJB staining, the tissues were cryoprotected with gradually increasing concentrations of sucrose, frozen and sectioned at a thickness of 25 µm (The sections were obtained at 1.7, 2.0 and 2.3 mm posterior to the bregma). The slides were firstly immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 10 minutes. This was followed by 2 minutes in 70% alcohol and 5 minutes in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate for 10 minutes in a dark room, preferably on a shaker table to ensure consist coverage. Subsequently, the slides were rinsed in distilled water for 2 minutes and then incubated in the FJB staining solution that was prepared from a 0.01% stock solution of Fluoro-Jade® B (catalog number: AG325, Chemicon International, USA)that was prepared by adding 10 mg of the dye powder to 100 ml of distilled water. After 20 minutes in the staining solution, the slides were rinsed. The dry slides were cleared by immersion in xylene for 2 minutes before adding a coverslip with DPX (Fluka, Milwaukee WI, or Sigma Chemical Co., St. Louis, MO), which is a non-aqueous non-fluorescent plastic mounting media. We further examined the tissues using a fluorescence microscope with a green (450-490 nm) excitation light. (Olympus Microscope System BX51; Olympus, Tokyo, Japan). To reduce counting bias, the number of cells was counted by two independent investigators who were blinded to the groups. The results were expressed as the average number of cells within each frame per section.The number of cells in each standardized microscopic field was evaluated using the NIH ImageJ program (version 1.46J). Cell culture and treatment

The primary hippocampal neuron cultures were prepared from neonatal mice as previously described, with minor modifications (Wang et al., 2014; Zhang et al., 2010). Briefly, the hippocampal tissue was dissected from neonatal WT mice or TRIF-deficient mice and incubated in 1 ml of 0.25% trypsin-EDTA (Life Technologies, Birmingham, MI); the discrete hippocampi were dissociated in dissociation medium (10% fetal calf serum, DMEM/high glucose). To precipitate the necrotic cells and other unnecessary tissues, the cells were incubated in ice water for 2 min. Subsequently, the cells were plated onto 6-well plates (500 µl) or coverslips (200 µl) that had been pre-coated with poly-L-lysine at a density of 1.1×105 cells/cm2. After 1 hour, the starter medium (Neurobasal medium with B27, 200 U/ml penicillin + streptomycin, 0.5 mM glutamine, and 25 µM glutamate; Life Technologies) was added. On the third day, half of the medium was replaced, and the culture was maintained at 37°C with 5% CO2. Both of these culture conditions allowed the growth of differentiated neuronal cultures with > 90% homogeneity, as assessed by immunocytochemistry for MAP2 and GFAP. After 7 days, the cells were treated with PBS, KA (100 µM), or HMGB1 (500 ng/ml) (Yang et al., 2011). After 3 h of treatment, the cells were fixed or the protein was extracted to measure the expression of TRIF. Statistical analyses The data are expressed as means ±SEM. The differences were analyzed using the Student’s t-test for comparisons between two groups, whereas one-way ANOVA tests followed by Fisher’s protected least significant differences were used for the comparisons between multiple groups. Spearman’s rank correlation tests were used for the correlations between the TRIF protein levels and the different clinical variables. In each of the statistical tests, p values of less than 0.05 were

considered statistically significant. The statistical data were obtained using the SPSS software package (version 13.0; SPSS, Chicago, IL, USA).

Results Expression and distribution of TRIF in human TLE tissues To determine the role of TRIF in epileptogenesis, we investigated the expression of TRIF in epileptic tissues from human drug-resistant TLE. Western blot analyses showed a significant up-regulation of TRIF in the temporal cortex and the hippocampal tissues of TLE patients compared with controls (** P