Gangliosides Trigger Inflammatory Responses via TLR4 in Brain Glia
2006; Elsevier BV; Volume: 168; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.050924
ISSN1525-2191
AutoresIlo Jou, Jee Hoon Lee, Soo Young Park, Hee Jung Yoon, Eun-hye Joe, Eun Jung Park,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoGangliosides participate in various cellular events of the central nervous system and have been closely implicated in many neuronal diseases. However, the precise molecular mechanisms underlying the pathological activity of gangliosides are poorly understood. Here we report that toll-like receptor 4 (TLR4) may mediate the ganglioside-triggered inflammation in glia, brain resident immune cells. Gangliosides rapidly altered the cell surface expression of TLR4 in microglia and astrocytes within 3 hours. Using TLR4-specific siRNA and a dominant-negative TLR4 gene, we clearly demonstrate the functional importance of TLR4 in ganglioside-triggered activation of glia. Inhibition of TLR4 expression by TLR4-siRNA suppressed nuclear factor (NF)-κB-binding activity, NF-κB-dependent luciferase activity, and transcription of inflammatory cytokines after exposure to gangliosides. Transient transfection of dominant-negative TLR4 also attenuated NF-κB-binding activity and interleukin-6 promoter activity. In contrast, these activities were slightly elevated in cells with wild-type TLR4. In addition, CD14 was required for ganglioside-triggered activation of glia, and lipid raft formation may be associated with ganglioside-stimulated signal propagation. Taken together, these results suggest that TLR4 may provide an explanation for the pathological ability of gangliosides to cause inflammatory conditions in the brain. Gangliosides participate in various cellular events of the central nervous system and have been closely implicated in many neuronal diseases. However, the precise molecular mechanisms underlying the pathological activity of gangliosides are poorly understood. Here we report that toll-like receptor 4 (TLR4) may mediate the ganglioside-triggered inflammation in glia, brain resident immune cells. Gangliosides rapidly altered the cell surface expression of TLR4 in microglia and astrocytes within 3 hours. Using TLR4-specific siRNA and a dominant-negative TLR4 gene, we clearly demonstrate the functional importance of TLR4 in ganglioside-triggered activation of glia. Inhibition of TLR4 expression by TLR4-siRNA suppressed nuclear factor (NF)-κB-binding activity, NF-κB-dependent luciferase activity, and transcription of inflammatory cytokines after exposure to gangliosides. Transient transfection of dominant-negative TLR4 also attenuated NF-κB-binding activity and interleukin-6 promoter activity. In contrast, these activities were slightly elevated in cells with wild-type TLR4. In addition, CD14 was required for ganglioside-triggered activation of glia, and lipid raft formation may be associated with ganglioside-stimulated signal propagation. Taken together, these results suggest that TLR4 may provide an explanation for the pathological ability of gangliosides to cause inflammatory conditions in the brain. Microglia and astrocytes are resident immunoeffector cells of the central nervous system. Although these cells are quiescent under normal conditions, they are rapidly activated in response to pathological stimuli. 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Gangliosides can alter the morphology of microglia and trigger the production of inflammatory mediators through activation of various inflammation-associated signaling molecules including NF-κB, Janus kinase (JAK), signal transducer and activator of transcription (STAT), and mitogen-activated protein kinase (MAPK).11Kim OS Park EJ Joe E Jou I JAK-STAT signaling mediates gangliosides-induced inflammatory responses in brain microglial cells.J Biol Chem. 2002; 277: 40594-40601Crossref PubMed Scopus (148) Google Scholar, 19Ryu JK Shin WH Kim J Joe E Lee YB Cho KG Oh YJ Kim WU Jin BK Trisialoganglioside GT1b induces in vivo degeneration of nigral dopaminergic neurons: role of microglia.Glia. 2002; 38: 15-23Crossref PubMed Scopus (55) Google Scholar, 22Mclaurin J Franklin T Fraser PE Chakrabartty A Structural transitions associated with the interaction of Alzheimer β-amyloid peptides with gangliosides.J Biol Chem. 1998; 273: 4506-4515Crossref PubMed Scopus (164) Google Scholar, 34Yang MS Park EJ Sohn S Kwon HJ Shin WH Pyo HK Jin B Choi S Jou I Joe E Interleukin-13 and -4 induce death of activated microglia.Glia. 2002; 38: 273-280Crossref PubMed Scopus (94) Google Scholar In the present report, we show that TLR4 expression levels rapidly change after treatment with gangliosides in rat primary glia and demonstrate that TLR4 is essential for ganglioside-triggered inflammatory signaling pathways using TLR4-specific siRNA and a dominant-negative TLR4 gene. These results suggest that TLR4 can function as an upstream sensor for gangliosides and provoke intracellular inflammatory signaling in the brain. Our data provide interesting insights into the understanding of the signaling pathways through which gangliosides cause pathological conditions in the brain. Purified ganglioside mixture (Gmix), monosialo-ganglioside (GM) 1, disialo-ganglioside (GD) 1a, and trisialo-ganglioside (GT) 1b from bovine brain were purchased from Matreya (Pleasant Gap, PA) and Sigma (St. Louis, MO). Endotoxin-tested GM1 and asialo-GM1 were obtained from Sigma. Rat interferon (IFN)-γ was obtained from Calbiochem (La Jolla, CA). Salmonella typhimurium LPS, polymyxin B sulfate, filipin, and methyl β-cyclodextrin (MβCD) were purchased from Sigma. Minimal essential medium, Lipofectamine plus, oligofectamine, and G418 antibiotics were obtained from Life Technologies, Inc. (Gaithersburg, MD). Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Hyclone (Logan, UT). Primary microglia were cultured from the cerebral cortices of 1- to 3-day-old Sprague-Dawley rats. Briefly, cortices were triturated into single cells in minimal essential medium containing 10% fetal bovine serum and were plated in 75-cm2 T-flasks (0.5 hemisphere/flask) for 2 weeks. The microglia were detached from the flasks by mild shaking and applied to a nylon mesh to remove astrocytes and cell clumps. Cells were plated in six-well plates (5 × 105 cells/well), 60-mm2 dishes (8 × 105 cells/dish), or 100-mm2 dishes (2 × 106 cells/dish). One hour later, the cells were washed to remove unattached cells before being used in experiments. After removal of the microglia, primary astrocytes were prepared using trypsinization. Cells were demonstrated to be more than 95% authentic microglia and astrocytes because of their characteristic morphology and the presence of the astrocyte marker glial fibrillary acidic protein and the microglia marker CD11b. For blocking of CD14 or LPS-binding protein (LBP), cells were preincubated for 30 minutes with 10 μg/ml of anti-CD14 monoclonal antibody (mAb), anti-LBP mAb, or isotype control mAb (HyCult Biotechnology, Liden, The Netherlands), and then the cells were treated with LPS or gangliosides for indicated times. Total RNA was isolated using RNAzolB (Tel-Test Inc., Friendswood, TX), and cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (TaKaRa, Japan) according to the manufacturer's instructions. PCR was performed with 25 cycles of sequential reactions. Oligonucleotide primers were purchased from Bioneer (Seoul, Korea). The sequences for PCR primers were as follows: (forward) 5′-TCC CTC AAG ATT GTC AGC AA-3′ and (reverse) 5′-AGA TCC ACA ACG GAT ACA TT-3′ for GAPDH; (forward) 5′-TGA TGT TCC CAT TAG ACA GC-3′ and (reverse) 5′-GAG GTG CTG ATG TAC CAG TT-3′ for IL-1β; (forward) 5′-GTA GCC CAC GTC GTA GCA AA-3′ and (reverse) 5′-CCC TTC TCC AGC TGG GAG AC-3′ for tumor necrosis factor (TNF)-α; (forward) 5′-TTG AAG ACA AGG CAT GGC ATG G-3′ and (reverse) 5′-TCT C CCC AAG ATC AAC CGA TG-3′ for TLR4. Cells were treated with gangliosides or LPS in the presence of 5% serum for the indicated times. The cells were washed twice with phosphate-buffered saline containing 1% fetal bovine serum, collected, and stained with phycoerythrin-conjugated anti-mouse TLR4/MD2 antibody (eBioscience, CA) for 30 minutes at 4°C. After washing, the cells were analyzed with a FACS Vantage (BD Biosciences, CA), and the data were processed using the CellQuest program and WinMDI. The mean fluorescence intensity (MFI) was analyzed by CellQuest software, and the change in MFI of cells incubated with TLR4/MD2 antibody was calculated for LPS- or ganglioside-treated and untreated cells after subtraction of the MFI obtained with the isotype control antibody (eBioscience). Wild-type TLR4 [pDisplay-Tlr4(wt)] and a dominant-negative mutant of TLR4 [pDisplay-Tlr4(P712H)], were gifts from Dr. Lynn Hajjar and C. Wilson (University of Washington, Seattle, WA). The 5×NF-κB-luciferase reporter construct and IL-6-luciferase reporter construct were from Dr. Seong Ho Jeon (Hanllym University, Korea). Chemically synthesized, double-stranded small interfering RNAs (siRNAs), with 19-nucleotide duplex RNA and 2-nucleotide 3′ dTdT overhangs, were purchased from Dharmacon Research (Lafayette, CO) in a deprotected and desalted form. To design TLR4-specific siRNA duplexes, the mRNA sequence for TLR4 was screened for unique 21-nucleotide sequences in the National Center for Biotechnology Information database using the BLAST search algorithm. The siRNA sequence targeting TLR4 in this study is 5′-ACG CUG UUC UGC UCA GGA GdTdT-3′. Forty to fifty percent of confluent cells were transfected with siRNA oligonucleotides using oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. All assays were performed at least 24 hours after RNA transfection. Transient transfections were performed in triplicate on 35-mm dishes using Lipofectamine plus reagents as instructed by the manufacturer (Life Technologies, Inc.). To normalize the variations in cell number and transfection efficiency, all cells were co-transfected with pCMV-β-GAL for 24 hours. Luciferase assays were performed according to the manufacturer's instructions (Promega, Madison, WI). Luciferase activity was measured using 20 μl of cell extract in 100 μl of assay buffer. Light intensity was measured for 30 seconds on a luminometer (Berthold Lumat LB9501). Luciferase activity was normalized by measuring β-galactosidase activity (in OD420). All plasmid DNAs were prepared using endotoxin-free DNA isolation kit (Qiagen, Valencia, CA). Cells were stimulated in the presence of 2.5% serum, and then the cell extracts were suspended in 9× packaged cell volume of a hypotonic solution (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 0.5% Nonidet P-40) and centrifuged at 5000 rpm for 10 minutes at 4°C. The pellet (nuclear fraction) was resuspended in 20 mmol/L HEPES (pH 7.9), 20% glycerol, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethyl sulfonyl fluoride, incubated on ice for 60 minutes with occasional gentle shaking, and centrifuged at 13,000 rpm for 20 minutes. The crude nuclear proteins in the supernatant were collected and stored at −70°C. Electrophoretic mobility shift assay was performed for 30 minutes on ice in a volume of 20 μl containing 2 μg of nuclear protein extract in a reaction buffer containing 8.5 mmol/L EDTA, 8.5 mmol/L EGTA, 8% glycerol, 0.1 mmol/L ZnSO4, 50 μg/ml poly (dI-dC), 1 mmol/L dithiothreitol, 0.3 mg/ml bovine serum albumin, 6 mmol/L MgCl2, and γ-32P-radiolabeled oligonucleotide probe (3 × 104 cpm), with or without a 20- to 50-fold excess of unlabeled probe. DNA-protein complexes were separated on 6% polyacrylamide gels in Tris/glycine buffer, and the dried gels were exposed to X-ray film. The following double-stranded oligonucleotides were used in these studies: NF-κB gel shift oligonucleotides, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, 25 bp (sc-2505; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). 5′-End-labeled probes were prepared with 40 μCi of [γ-32P]-ATP using T4 polynucleotide kinase (Promega) and purified on Sephadex G-25 quick spin columns (Roche Molecular Biochemicals, Indianapolis, IN). All ganglioside preparations were tested for bacterial contamination using the chromogenic Limulus amebocyte lysate assay according to the manufacturer's specifications (WinKQCL, Bio-Whittaker). The purity of ganglioside mixture by thin layer chromatography was more than 98% according to the manufacturers' reports (Matreya and Sigma), and the purity of individual ganglioside was 93 to 98%. Media nitrite concentration was measured as an indication of NO release. After the indicated cell incu-bations, 50 μl of culture medium was removed and mixed with an equal volume of Griess reagent (0.1% naphthylethylene diamine, 1% sulfanilamide, 2.5% H3PO4), and absorbance of the mixture at 540 nm was measured. All data are expressed as the mean ± SD. Statistical analysis was performed using Student's t-test. In an effort to address how gangliosides initiate inflammatory conditions in the brain, we examined whether TLR4 could function as an upstream sensor for gangliosides in rat primary microglia. First, we tested the engagement of TLR4 in ganglioside-induced inflammation by its expression level because TLR4 expression has been shown to be strictly regulated in a stimulus-dependent manner.35Rehli M Of mice and men: species variations of Toll-like receptor-expression.Trends Immunol. 2002; 23: 375-378Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 36Weiss DS Raupach B Takeda K Akira S Zychlinsky A Toll-like receptors are temporally involved in host defense.J Immunol. 2004; 172: 4463-4469PubMed Google Scholar Rat primary microglia were treated with 50 μg/ml of brain ganglioside mixture (Gmix) for 18 hours, and TLR4 cell surface expression levels were analyzed by flow cytometric analysis using phycoerythrin-conjugated TLR4/MD2 (MTS510) antibody. Interestingly, we found that cell surface expression of TLR4 was markedly low in ganglioside-treated primary glia and that the reduced level of TLR4 in ganglioside-treated cells was similar to that in cells treated with 100 ng/ml of LPS, a representative TLR4 ligand (Figure 1A). We also observed that gangliosides significantly reduced TLR4 cell surface expression in BV2 mouse microglia (Figure 1A). To evaluate further the effect of gangliosides on TLR4 surface expression, we examined the time- and dose-dependent effects of gangliosides in primary microglia. Cell surface expression of TLR4 was rapidly reduced within 3 hours of exposure to Gmix (Figure 1B). Moreover, TLR4 cell surface expression was slightly changed even in 10 μg/ml ganglioside-treated cells, and the increasing concentrations of gangliosides further diminished TLR4 expression (Figure 1C). These results indicate that gangliosides rapidly modulate TLR4 expression in primary microglia, suggesting the possibility that gangliosides may trigger inflammation through TLR4 in the brain. The major types of gangliosides in the brain are GM1, GD1a, GD1b, GT1b, and GQ1b, each of which have a different number and position of carbohydrate-linked sialic acid residues.37Dreyfus H Guerold B Freysz L Hicks D Successive isolation and separation of the major lipid fractions including gangliosides from single biological samples.Anal Biochem. 1997; 249: 67-78Crossref PubMed Scopus (46) Google Scholar We therefore investigated the role of the sialic acid residues in the regulatory effect of gangliosides on TLR4 expression. We first compared the effects of GM1, which has one sialic acid residue, with Gmix on TLR4 expression. As shown in Figure 2A, no significant differences in the surface expression level of TLR4 were observed between GM1- and Gmix-treated primary microglia. Reduced TLR4 surface expression was observed in cells treated with either GD1a, which has two sialic acid residues, or GT1b, which has three sialic acid residues; this decrease was also similar to that caused by Gmix (Figure 2, A and B). These results indicate that the number of sialic acid residues per ganglioside molecule has little effect on modulation of TLR4 expression at the cell surface expression level. To further examine the role of sialic acid residue in ganglioside-induced changes of TLR4 expression, we compared the effects of Gmix and asialo-GM1, which has no sialic acid residue, on TLR4 expression. Compared to untreated control cells, cell surface expression of TLR4 was decreased in microglia treated with 25 μg/ml of Gmix, whereas transcription of IL-1β was markedly increased. In contrast, cell surface expression of TLR4 as well as IL-1β transcription was unchanged in cells treated with asilao-GM1 (Figure 2C). Taken together, these results suggest that the sialic acid residue is necessary for ganglioside-induced modulation of TLR4 expression, although the number of sialic residues per ganglioside molecule is not relevant. To investigate whether ganglioside-dependent modulation of TLR4 could be attributable to LPS contamination in preparation of gangliosides, we carefully tested all ganglioside preparations using chromogenic Limulus amebocyte lysate assay (WinKQCL, Bio-Whittaker). The average endotoxin level of Gmix was ∼0.007 EU/ml (±0.002), which was not sufficient to induce LPS-induced inflammatory effects including modulation of TLR4 expression in glia. Endotoxin levels for GT1b, GD1a, GM1, and asialo-GM1 used for this study were 0.008 (±0.002), 0.009 (±0.002), 0.005 (±0.003), 0.009 (±0.002) EU/ml, respectively. To further eliminate the possibility for misinterpretation of our results by endotoxin contamination of gangliosides, we examined the effect of polymyxin B, a well-known pharmacological LPS scavenger, on the ganglioside-dependent change of TLR4 expression. As expected, pretreatment of polymyxin B did not affect ganglioside-induced modulation of TLR4 expression as well as ganglioside-triggered transcription of inflammatory cytokines (Figure 3). These results suggest that TLR4 may specifically recognize gangliosides in microglia. Astrocytes, which are another glial cell type vital to brain immune responses, express various cell surface receptors and produce inflammatory mediators such as cytokines. To define better the association of TLR4 with brain inflammatory responses, we investigated whether TLR4 expression levels were altered in ganglioside-treated astrocytes. Primary astrocytes were treated with 50 μg/ml of Gmix or 100 ng/ml of LPS for 18 hours, and the TLR4 surface expression level was analyzed by flow cytometry. Consistent with the results in microglia, cell surface expression of TLR4 was significantly reduced on primary astrocytes treated with Gmix as well as LPS (Figure 4). Similar patterns of reductions in TLR4 expression were observed in astrocytes treated with GM1, GD1a, or GT1b (Figure 4). These results indicate that ganglioside treatment can alter TLR4 expression in both astrocytes and microglia, suggesting that TLR4 may play important roles in ganglioside-triggered inflammatory responses. Because our results suggest a possible involvement of TLR4 in ganglioside-induced inflammatory events, we examined whether TLR4 could indeed contribute to ganglioside-triggered inflammatory signaling using TLR4-specific short interfering RNA (siRNA). To do this, we designed the synthetic siRNA that targets the TLR4 and chemically synthesized the small RNA duplexes corresponding to TLR4 (Dharmacon). After validating our siRNA as an inhibitory reagent capable of depleting TLR4 levels in rat primary astrocytes (Figure 5A), we tested whether siRNA-mediated repression of TLR4 could specifically inhibit the ganglioside-stimulated transcription of inflammatory cytokines. Rat primary astrocytes were transfected with either TLR4-siRNA or nonsilencing control siRNA, and the cells were t
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