Male C57BL/6 mice (8–12 week old, 20–24 g, from Scientific Research Center, Chengdu Medical College, Chengdu, China), and male adult Trif ^−/− mice (C57BL/6 J-AW046014 Lps2 /J; from Jackson Laboratory, Bar Harbor, ME, USA, gifts from Prof. Qingwu Yang, Xinqiao Hospital, Third Military Medical University, Chongqing, China), age 8–12 weeks (20–24 g). All animal-related procedures in this study were performed according to the Chengdu Medical College guidelines for the care and use of experimental animals. The Animal Ethics Committee of Chengdu Medical College approved all animal experimental procedures used in the present study, which are in accordance with the principles outlined in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. MPTP (Sigma-Aldrich, Shanghai, China) was freshly dissolved in 0.9% saline and administered to mice intraperitoneally (i.p. 20 mg/kg) four times within a 2 h interval.

Methyl-4-phenyl tetrahydropteridine (MPP+ iodide), MPTP and lipopolysaccharide (LPS) were purchased from Sigma (Shanghai, China). Recombinant Mouse IL-4 was purchased from R&D Systems (Shanghai, China). Poly(I:C) was purchased from InvivoGen (CA, USA).

The MN9D dopaminergic cell line and the immortalized murine BV2 microglial cell line were generous gifts by Prof. Qun-Yuan Xu, Capital Medical University, Beijing, China. These cells were cultured at 37°C plus 5% CO₂ in a high glucose (4500 mg/L) Dulbecco's Modified Eagle Medium (Gibco, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Shanghai, China) and 1% streptomycin and penicillin (Life Technologies, Shanghai, China). For all experiments, MN9D cells were differentiated for 4 days with 1.5 mM sodium butyrate (Sigma, Shanghai, China). All methods were carried out in line with relevant guidelines and regulations of Chengdu Medical College.

The BV2 cells were treated with LPS (100 ng/mL) or IL-4 (20 ng/mL) to induce polarity of BV2 cells followed by real-time RT PCR identification. Then, the cells were collected from the culture plates by using a rubber policeman and centrifuged at 300 × g for 5 min, washed with ice-cold PBS twice followed by TRIzol® (Life Technologies, Shanghai, China) lysis. RNA was collected by the TRIzol® lysis protocol according to the manufacturer's protocol.

After treatment total RNA was extracted from the BV2 cells with TRIzol® reagent (Life Technologies, Shanghai, China) according to the manufacturer's protocol. Total RNA was used for cDNA synthesis with the PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). SYBR™ Green quantitative PCR was performed with validated primers such as IFN-β, TNF-α, IL-6, CD86, inducible NO synthase (iNOS), IL-10, CD206, Arginase1, Ym1, and β-actin (Life Technologies, Shanghai, China) with SYBR™ premix Ex Taq™ II kit (Takara, Dalian, China) and monitored by a IQ5 Real-time PCR machine (Bio-Rad, CA, USA). The relative expression levels of each mRNA were calculated by using the 2^−ΔΔCt algorithm normalizing to β-actin and relative to the control samples.

MPTP treated WT and Trif ^−/− mice were sacrificed by CO₂ asphyxiation on the Day 7 after the last MPTP injection according to the method described previously (Liang et al., 2007). Briefly, the Str tissue of brains were dissected out on ice immediately. Dissected Str tissues were homogenized in 50 μl of 0.1 M perchloric acid. After centrifugation (15,000 × g, 10 min, 4°C) and filtration, 30 μl supernatant was injected onto a C18 reverse-phase HR-80 catecholamine column (ESA, Bedford, MA, USA). The concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (HE), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptamine (5-HT), and homovanillic acid (HVA) were quantified by HPLC-EC detection. The mobile phase (pH 2.9) consisted of 275 mg/l octane sulfonic acid in 90% 75 mM sodium phosphate and 10% methanol; the flow rate was 1 mL/min. Peaks were detected by an ESA Coulochem II with a model 5010 detector (E1 = 50 mV, E2 = 400 mV). Data were collected and processed by a Chromeleon™ computer system (Gynkotek, Gemering, Germany).

Conditioned Medium (CM) was collected at desired time points after cell treatment, filtered through 0.45 μm filters (Millipore, Shanghai, China) and quickly frozen at −80°C for further MN9D cell culture. MN9D cells grown on poly-D lysine (Sigma, Shanghai, China)-coated slides were pretreated with MPP+ (300μM) for 12 h after which time the medium was removed, and cells were exposed to conditioned medium from BV2 cells for 24 h.

BV2 cells were plated in 6-well-tissue culture plates at about 80% confluence. The TRIF siRNA (5′-GGGUUACCACACGAAAUUAtt-3′, 5′-GCCUCUCAUUAUUCACCAUtt-3′) and scrambled negative control siRNA (Cat. 4390843, Life Technologies, Shanghai, China) were obtained from Life Technologies. Transfections were performed using Lipofectamine™ 2000 (Life Technologies, Shanghai, China) according to the manufacturer's instructions. Four to six hours after transfection, media were removed and replaced with fresh media. Cells were treated with poly(I:C) (25 μg/mL) and vehicle for further experiments on the following day.

BV2 cells are placed on the upper layer of a cell permeable membrane and MN9D cells were placed on the bottom of the cell culture plate. Following a culture period of 24 h, the BV2 cells that have migrated through the membrane are quantified and stained by cresyl violet.

Total protein from each group was digested in radioimmunoprecipitation (RIPA) assay buffer supplemented with a protease inhibitor cocktail (Roche, Shanghai, China). Supernatant was collected after homogenate centrifugation at 12,000 g for 10 min at 4°C. After protein denaturation, the proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the resolved proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, CA, USA). Each membrane was blocked and then incubated with primary antibody at 4°C overnight followed by incubation with HRP-conjugated secondary antibodies for 1 h at 25°C. The intensity of the protein signal of three duplicates from each sample was calculated using ImageJ software (NIH, USA). The antibodies used were anti-TLR3 (1:1,000, ab62566, Abcam, Cambridge, UK), anti-TRIF (1:1,000, ab13810, Abcam, Cambridge, UK), anti-IRF3 (1:1,000, sc-9082, Santa Cruz Biotechnology, CA, USA), anti-phospho-IRF3 (1:1,000, 4,947, Cell Signaling Technology, MA, USA), anti-TH (1:2,000, AB152, Millipore, MA, USA), and anti-DAT (1:1,000, ab111468, Abcam, Cambridge, UK). Anti-mouse HRP (1:10,000, ZB2305, Zhongshan Goldenbridge, Beijing, China) and anti-rabbit HRP (1:10,000, ZB-2301, Zhongshan Goldenbridge, Beijing, China) were used as secondary antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5,000, KC-5G4, KANGCHEN) served as a loading control.

The CM-treated cells were washed with icecold PBS twice followed by 4% paraformaldehyde fixation (PFA, Sigma, Shanghai, China) for 20 min. TUNEL (Shanghai, China) staining was performed according to manufacturer's instructions. Briefly, the cells were permeabilized with 0.1% Triton™ X-100 and 0.1% sodium citrate for 2 min on ice followed by a PBS rinse. The TUNEL mixture was prepared freshly and added to each sample for incubation at 37°C for 1 h in the dark. The samples were rinsed twice with PBS, then blocked with 5% bovine serum albumin (0.05% Tween® 20) in PBS for 45 min at room temperature. Cells were then incubated with primary antibody anti-βIII tubulin (1:400, ab78078, Abcam, Cambridge, UK) at 4°C overnight. Appropriate secondary antibodies (Alexa Fluor® 568, Life Technologies; A11031, 1:400) were used for incubation at 25°C for 1 h. After washing with PBS, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) for 5 min. Cells were mounted with an inverted fluorescent mounting medium (DAKO, Glostrup, Denmark), and images were captured with a digital camera (Sterling Heights, MI).

Naïve, MPTP, MPTP+poly(I:C) and poly(I:C) treated mice were anesthetized by chloral hydrate (4%) and were fixed by 2% PFA perfusion through the heart. After 10, 20, and 30% sucrose dehydration, the fixed brains were then sectioned in a cryostat to get the SNc sections (30 μm). After PBS rinsing three times (5 min each), the sections were treated with 0.3% H₂O₂ for 25 min at room temperature (r.t.). After PBS rinsing, the sections were blocked in goat serum (5%) for 1 h at r.t., and primary antibody (TH, 1:2,000; Millipore, CA, USA) was added for incubation overnight at 4°C. On Day 2, following PBS rinsing, biotinylated goat anti-rabbit secondary antibody was added onto the slides which were incubated for 1 h, followed by incubation with Streptavidin-HRP for 1 h at r.t. and visualization by reaction with nickel-intensified, 3-diaminobenzidine tetrahydrochloride (DAB: 0.25% nickel ammonium sulfate/0.05% DAB as a chromagen and 0.003% hydrogen peroxide) for 5 min. Sections were mounted on gelatin-coated slides, dehydrated through graded ethanol, and cleared in xylene and finally followed by coverslipping using Permount. The images were taken by an Olympus microscope (Japan).

Based on our previous quantification method and protocol (Liang et al., 2006, 2007, 2008), density of TH-immunopositive cells was counted on both hemispheres by serial section analysis of the total number of neurons. Briefly, according to the atlas of mouse brain (The Mouse Brain in Stereotaxic Coordinates, Academic Press, New York, 2001), every sixth brain section throughout the entire extent of the SNc was numbered from the rostral to the caudal plane by blinded investigators (Crocker et al., 2003). Adjacent SNpc tissue sections from each animal were also stained with methylene blue (Nissl's staining) to validate immunohistochemical determination of nigral neuron survival. TH positive cells were quantified in SNpc tissue sections from B-2.8 to B-3.52 according to our previous method (Liang et al., 2006). Estimate of total TH positive neuron populations was calculated using Abercrombie's correction (Abercrombie, 1946). The images of the SNc region were taken by a BX63 microscope (Olympus, Japan). The data output is shown as number of TH positive cells.

The occurrence of apoptosis was determined by the fluorescein isothiocyanate (FITC) annexin V Apoptosis Detection Kit (Keygen, Nanjing, China) after cell treatment with MPP+ (300 μM) and BV2-conditioned medium for 24 h using a C6 flow cytometer (BD Biosciences, NJ, USA).

All data shown are mean ± S.E.M of triplicate values from three separate experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 were indicated as compared with the control group. Independent Student t-test or one-way ANOVA was used to compare the continuous variables between the two groups or more than two groups. Statistical analysis was carried out with statistical analysis software program SPSS13.0 software (IBM) and Prism® 5.0 software (GraphPad).

DA and its metabolites DOPAC, HE, 5-HIAA, 5-HT, and HVA were detected at Day 1, 3, 7, and 14 after MPTP injection in WT and Trif ^−/− mice (Figure 1). MPTP significantly reduced DA levels, DOPAC levels and 5-HT levels in WT and Trif ^−/− mice from Day D1 to D14 post-MPTP treatment, respectively. Interestingly, the levels of DA (D1, D3, and D7), DOPAC (D3 and D7), 5-HIAA (D3 and D7), 5-HT (D1 and D3), and HVA (D7) were dramatically decreased in Trif ^−/− mice compared to respective levels in WT mice (Figure 1, p < 0.05, n = 6). However, no effect on the genotype was found with respect to the NE level.

To confirm the effect of an MPTP-induced decrease of DA and related metabolites, we then observed dopamine neuron loss by tyrosine hydroxylase (TH) staining in SNpc by quantification in serial sections from level (from Bregma, −2.8 to 3.52 mm) B2.8 to B3.52. A decreased number of these cells were clearly observed in the MPTP treatment group in WT and Trif ^−/− mice on D7 from B2.8 to B3.52 levels. However, less TH positive cells were found in MPTP treated Trif ^−/− mice (Figures 2F,I) vs. the WT group in B2.8, B2.92, B3.08, B3.16, and B3.28 levels (Figures 2B,I, p < 0.05, n = 6), which suggests that TRIF may play a protecting role in the neuronal MPTP-induced DA neuron loss. To verify the function of the TRIF signaling pathway in DA neuron loss, we used poly(I:C), an agonist of the TLR3-TRIF signaling pathway, to rescue the neuronal loss phenotype in MPTP-treated WT and Trif ^−/− mice at a concentration of 1.25 μg/g dose via i.p. injection. By poly(I:C) treatment, more TH-positive cells survived in the WT group (Figure 2C) compared with Trif ^−/− mice (Figure 2G) in B2.92, B3.08, and B3.16 levels (Figure 2I, P < 0.05, n = 6), which suggests that the TLR3-TRIF signaling pathway may contribute to DA neuron protection from MPTP-induced neuron loss. No difference in the number of DA neurons was observed between WT and Trif ^−/− mice in the poly(I:C) treatment group (Figures 2D,H, p > 0.05, n = 6) as well as in the vehicle group (Figures 2A,E, p > 0.05, n = 6).

IRF-3 is one of the downstream molecules of TRIF (Liu et al., 2015), which can be activated by phosphorylation by the inhibitory kappa B kinase (IKK) and/or TANK-binding kinase 1 (TBK1) in response to stimulation (Bruni et al., 2013; Liu et al., 2015). Poly(I:C) is the classic agonist of TLR3, which stimulates TLR3 via a TRIF-dependent pathway which is a unique adaptor in TLR3 and TLR4 signaling pathways contributing to interferon (IFN)-beta production (Yamamoto et al., 2003). To investigate the role of the TLR3-TRIF-IRF3 signaling pathway in BV2 cell stimulation, we treated BV2 cells with siTRIF for 24 h at a concentration of 25 μg/ml and found that the expression of TRIF decreased about 60% compared with the siNC group (Figure 3B, p < 0.01) as well as poly(I:C) stimulation group decreased about 70% compared with the siNC group (Figure 3A). Moreover, p-IRF3 which reflects the activation status of the IRF3 signaling pathway also decreased about 50% compared with siNC+poly(I:C) group (Figure 3B, p < 0.01) which suggests that the TRIF-IRF3 signaling pathway can be inhibited significantly by siTRIF. The results can be used to set up an inhibition model that will be useful for subsequent experiments.

TRIF, but neither MyD88 nor TIR domain-containing adaptor protein (TIRAP), is able to activate the IFN-β promoter (Yamamoto et al., 2002). The outcome of activation is usually followed by the activation of IFN-inducible genes, such as interferon-inducible protein-10 (IP-10) and glucocorticoid attenuated response gene 16 (GARG16), which were induced in response to LPS in MyD88 knock out cells (Doyle et al., 2002). IFN-β is the typical factor that can be released when the TRIF-IRF3 axis is stimulated (Sharma et al., 2003). We used Real-Time-PCR to identify the change of IFN-β mRNA after siTRIF treatment in the BV2 cell line. The results showed that the gradual upregulation of IFN-β mRNA depends on the time course of the siTRIF treatment. There are significant differences of IFN-β mRNA fold-change between the 12 and 24 h treatment groups, in which the siNC group reached the highest level of mRNA expression at 24 h (Figure 3C, p < 0.01). The present results indicate the function of siTRIF on BV2 cells depends on the time course of the siTRIF treatment.

Continuous microglial cell activation and migration are important for the pathogenic processes of Parkinson's disease (Kim et al., 2013; Wang et al., 2015). To investigate the role of TRIF in the microglial migration in response to MPP+ (200 μM) treated MN9D cells, we co-cultured BV2 and MN9D cells in a transwell co-culture system to mimic the pathological process between dopamine neurons and resident microglia that exists in Parkinson's disease. By quantifying the number of BV2 cells that located on the other side of the transwell membrane, we found that the number of migrated BV2 cells decreased about 56.4% when compared with the siNC group. There is no significant difference between siTRIF+ (MPP+ treated MN9D cells) and siTRIF+poly(I:C)+(MPP+ treated MN9D cells) groups, i.e., columns 3 and 4 (Figure 4, n = 6, p < 0.01).

Lipopolysaccharide (LPS) is known as a classical M1 microglial cell polarization inducer. M1 microglia express pro-inflammatory molecules such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and nitric oxide (NO). The typical cell surface markers like CD86 and CD68 are also expressed by M1 microglial cells (Liao et al., 2012). On the other hand, IL-4 is known to induce M2 microglial polarization. M2 microglial cells express different molecules, such as IL-4, arignase1, Ym1, CD206, and IL-10, which are beneficial to neuroprotection (Ponomarev et al., 2007; David and Kroner, 2011; Liao et al., 2012). To explore the role of TRIF in the M1/M2 regulation, we added LPS (100 ng/ml) and IL-4 (20 ng/ml) to treat BV2 for 24 h. As indicated by Real-Time PCR results, mRNA expressions of TNF-α, IL-6, CD86, iNOS were upregulated after 24 h LPS treatment, while IL-10, CD206, YM1, and Arg1 were upregulated significantly different from control and LPS treated groups (Figure 5A, n = 3, p < 0.01). The siTRIF treatment of the different groups resulted in significantly different expressions of TNF-α, IL-6, CD86, CD206, and Arg1 mRNA compared with the control groups (Figures 5B–I, n = 3, p < 0.01). The results indicated the essential role of TRIF in microglial M1/M2 marker polarization.

To investigate the stimulating effect of MN9D (MPP+) cells on BV2 cells, which were co-cultured in a transwell system, Real-Time PCR was utilized to quantify the mRNAs of M1/M2 differentiation markers such as IL-1β, IFN-γ, NO, CD86, and CD68. The expressions of M1 markers increased gradually as the co-culture progressed. From 4 to 36 h of co-culture, mRNAs of TNF-α, IL-6, CD86, iNOS, IL-10, CD206, and Arg1 were upregulated gradually. TNF-α and IL-10 reached their highest levels at 24 h, while IL-6, CD86, and CD206 reached their highest levels at 2 h. iNOS and Arg1 reached their highest levels at 36 h. (Figure 6, p < 0.01). The present results suggest that the typical microglial M1/M2 markers can be changed by MPP+-treated MN9D cells and the paradigm regulation depends on injured MN9D cell stimulation. Moreover, the inhibition of TRIF had a different effect on the inhibition of mRNA expression levels of typical M1/M2 markers, such as the expression of TNF-α upregulated in 24 and 36 h, IL-6 upregulated from 12 and 24 h, CD86 upregulated from 4 to 24 h, iNOS upregulated in 36 h, IL-10 upregulated from 12 to 36 h, CD206 upregulted from 4 to 36 h and Arg1 upregulted from 4 to 36 h, except for YM1 which has no difference of mRNA fold change from 4 to 36 h (Figures 6A–H).

As we have found the M1/M2 paradigm of microglia was triggered by MPP+-injured MN9D cells, the microglial cells can be polarized by the release of different typical pro/anti-inflammatory factors. However, we did not know how MN9D cell apoptosis was affected by microglial polarization. In this section, the co-cultured MN9D cells and BV2 cells were studied especially when siTRIF was applied. As a result, by βIII-tubulin and TUNEL dual labeling, the number of apoptotic MN9D cells in the MPP+ group accounts for 24 ± 5% of the total number of MN9D cells, however, the number increased drastically to 35 ± 3% when co-cultured with BV2 cells. And the number of apoptotic MN9D cells reached over 48 ± 4% with siTRIF treatment (Figures 7A–D, n = 3, p < 0.05). The exact number of apoptotic cells was quantified and analyzed by flow cytometry with Annexin-V-FITC /PI double staining (Figure 8, n = 3, p < 0.05). The siTRIF/MPP+ group showed the highest number of apoptotic MN9D cells. Pro-inflammatory stimuli and stress conditions increase the DA neuron apoptosis in vivo (Glass et al., 2010). In this section, we have found that the siTRIF treatment of BV2 cells may aggravate the apoptosis of MPP+-treated MN9D cells. Here, TRIF may play a protective role in MN9D cell apoptosis.

MPP+ is the conversion product of the neurotoxin MPTP in the brain, which is a high-affinity substrate for DAT as well as for norepinephrine and serotonin transporters (Javitch et al., 1985). DAT is the required step for MPTP neurotoxicity as evidenced by the fact that antagonist inhibition or genetic ablation of DAT prevents MPTP-induced dopaminergic neurodegeneration (Uhl et al., 1985). As TH functioned as a rate-limiting step in catalyzing the formation of L-DOPA, PD can be also considered as a TH-deficiency syndrome (Haavik and Toska, 1998). In this study, DAT and TH were detected by Western blot to verify the TRIF function in regulating MN9D cell dopaminergic function. As a result, it was indicated that the siTRIF treated BV2 cells affect the DAT and TH expression by MPP+-treated MN9D cells (Figure 9, n = 3, p < 0.05). There is a significant difference between the siNC-CM group and the siTRIF-CM group, which suggests TRIF suppression may aggravate MN9D cell dopaminergic functions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.