Article Figures & Data
Figures
- Figure 1.
Detection of Siglec-E on microglia. A, Detection of Siglece mRNA in primary microglia and the microglia line. Spleen tissue served as positive control. Siglece transcripts were detected in unstimulated microglia (unstim.) as well as in microglia stimulated with LPS, IFN-γ, IFN-α, and TNF-α. No Siglece transcripts were detected in primary neurons. Gapdh mRNA served as housekeeping standard. Representative data of three independent experiments are shown. Control, Water control. B, Flow cytometry analysis of the microglial line. Siglec-E was detected on unstimulated (unstim.) microglia at low levels. Treatment with interferons (IFN-γ, IFN-α) slightly increased expression of Siglec-E, whereas treatment with LPS or TNF-α had no effect. Representative data of three independent experiments are shown. Isotype, Isotype control antibody. C, Flow cytometry analysis of ex vivo and primary microglia. Low constitutive expression of Siglec-E on CD11b+ and CD45low cells was detected. Representative data of three independent experiments are shown. Isotype, Isotype control antibody. D, Microglia were transduced with lentiviral vectors expressing Siglece (SigE vector) or a control vector. Furthermore, lentiviral knockdown was performed by two lentiviral short-hairpin constructs targeting Siglece (shRNASigE1; shRNASigE2) or a corresponding nontargeting control vector (NTshRNA). qRT-PCR confirmed the successful modification of the microglial line by showing an increased (left graph) or decreased (right graph) Siglece cDNA, respectively. **p ≤ 0.01, ***p ≤ 0.001. E, Flow cytometry analysis confirmed overexpression (left graph) and reduced expression (right graph) of the Siglec-E protein. Representative data of three independent experiments are shown.
- Figure 2.
Siglec-E prevents phagocytosis and the associated reactive oxygen burst after challenge with neural debris. A, Uptake of red fluorescent-labeled neural debris into the microglial line was determined by confocal microscopy and 3D reconstruction. Microglial cells were transduced with the control vector, the Siglece overexpressing vector (SigE vector), the Siglece knockdown vectors (shRNASigE1, shRNASigE2) or the nontargeting vector (NTshRNA). Representative images of three independent experiments are shown. Scale bar, 20 μm. B, Phagocytosis of neural debris was quantified. Overexpression of Siglece mRNA reduced the uptake of neural material, whereas knockdown of Siglece increased the uptake of neural debris. ***p ≤ 0.001. C, Level of superoxide production as determined by DHE staining was quantified in the microglial line. After stimulation with neural debris, DHE intensity was increased after Siglece knockdown compared with the NTshRNA. ***p ≤ 0.001. D, Quantification of superoxide production as determined by DHE staining. After stimulation with neural debris in the presence of either 20 μg/ml SOD1 or 40 nm Trolox, increased DHE intensity after Siglece knockdown was antagonized. ***p ≤ 0.001. E, Quantification of superoxide production of microglial cells using the Amplex Red method. Knockdown of microglial Siglece via shRNASigE1 or shRNASigE2 increased the endogenous production of H2O2 equivalents after stimulation with neural debris compared with cells transduced with a control construct (NTshRNA). Arrow 1, Addition of cells; arrow 2, addition of 12,000 U/ml catalase. Representative data of three independent experiments are shown.
- Figure 3.
Siglec-E has anti-inflammatory effects and binds to neurons and astrocytes. A, qRT-PCR to detect Il-1β, Tnfsf2 (TNF-α), and Nos2 cDNA after 16 h incubation of microglia with neural debris. Microglia with knockdown of Siglece (shRNASigE1, shRNASigE2) showed a significant increase in gene transcription of Il-1β and Tnfsf2 (TNF-α) in the presence of neural debris compared with the control vector (NTshRNA). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n.s., not significant. B, C, Binding of Siglec-E to sialic acid residues of neural cells. Neurons/astrocytes were either untreated or treated with sialidase and then incubated with the Siglec-E:Fc fusion protein. Removal of sialic acids led to a decreased binding of Siglec-E:Fc to neurons (B) and astrocytes (C). Representative images of three independent experiments are shown. Scale bar, 30 μm.
- Figure 4.
Neurite protective effect of Siglec-E in neuron–microglia cocultures. A, Loss of neurites after knockdown of Siglece in microglia. Neurons were cocultured for 48 h with microglia. Siglece overexpression increased whereas knockdown of Siglece decreased the relative neurite length. Representative images without sialidase treatment of three independent experiments are shown. Scale bar, 30 μm. B, Relative neurite length and neuronal cell body density were quantified. Although the neuronal cell body density was unchanged (right graph), the relative neurite length was dependent on the expression level of Siglec-E on the microglial surface (left graph). After sialidase treatment, the relative neurite length was significantly reduced. **p ≤ 0.01, ***p ≤ 0.001. C, Neurons were cocultured with microglia in the presence of Trolox. Reduced neurite length after knockdown of Siglec-E was restored after treatment with 40 nm Trolox. ***p ≤ 0.001.
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