The Journal of Neuroscience, December 13, 2006, ():
Suppression of Microglial Inflammatory Activity by Myelin Phagocytosis: Role of p47-PHOX-Mediated Generation of Reactive Oxygen Species
J. Neurosci. Liu et al.
26: 12904
Supplemental Data
Files in this Data Supplement:
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Table 1
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Table 2
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Supplemented Figure 1, Inhibition of phagocytosis reverses myelin-induced transcriptional suppression of TNF-α and IP-10 in primary cultured microglia.
IFN-γ-preactivated microglia were pre-treated with 5μM cytochalasin D for 1 hour and than incubated with myelin at 50 μg/ml in the presence of IFN-γ and cytochalasin D for 24 hours. Total RNA was isolated and transcripts of TNF-α (A) and IP-10 (B) were measured using real-time quantitative PCR. Myelin-induced transcriptional down-regulation of TNF-α and IP-10 are pronouncedly reversed by the treatment of cytochalasin D (independent samaples t-test, *: p <0.05, #: p>0.05, n= 3 per group).
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Supplemented Figure 2, Higher concentrations are needed for myelin pre-treatment to suppress lipopolysacchride-induced microglial activation.
Primary microglia were first treated with myelin at different concentrations (100 and 200 μg/ml) for 24 hours, then myelin was washed away and the culture medium was replaced with the fresh medium containing 10ng/ml lipopolysacchride for another 6 hours. We observed that myelin at a higher concentration (200μg/ml) was needed to suppress the TNF-α release (one-way ANOVA, vs. no myelin pre-treatment control, *: p <0.05, #: p>0.05, n= 3 per group).
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Supplemented Figure 3, Myelin phagocytosis does not affect viability of primary microglia.
We determined microglial viability following incubation of IFN-γ-activated microglia with varying concentrations of myelin for 24 hours. Viability assay was performed with the MTT assay with the Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannhein, Germany). Treatment with myelin (5~100μg/ml) did not cause a reduction of OD values, whereas 500μM and 1000μM H2O2 significantly reduced OD values (*:one-way ANOVA, p<0.05 vs. IFN-γ treatment only, n=3 per group) (A). Furthermore, a toxicity assay was performed by measurement of LDH release with CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega GmbH, Mannhein, Germany). Treatment with myelin (5~100μg/ml) did not increase release of LDH, whereas 500μM and 1000μM H2O2 significantly elevated LDH levels in the culture (*:one-way ANOVA, p<0.05 vs. IFN-γ treatment only, n=4 per group) (B).
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Supplemented Figure 4, Transfection of short interference RNA knocks down expression and function of p47-PHOX.
BV-2 cells were transfected with control siRNA (ct siRNA) or siRNA targeting mouse Ncf1 (Ncf1 siRNA), activated by IFN-γ for 24 hours and then incubated in the presence of IFN-γ with or without 10μg/ml myelin for a further 24 hours. Transcripts of Ncf1 were significantly knocked down by quantifying with real-time PCR (A); Expression of p47-PHOX in siRNA transfected BV-2 cells was measured by flow cytometry after intracellular immunofluorescent staining of p47-PHOX. Overlay of flow cytometry histograms of control siRNA (bold continuous line) and Ncf1 knock-down siRNA (thin continuous line) transfected BV-2 cells as well as isotype control staining (dotted line) demonstrated one typical experimental result that fluorescence intensity (shown in brackets) of p47-PHOX staining in control siRNA-transfected BV-2 cells was higher than that in Ncf1 knock-down siRNA-transfected cells after treatment of IFN-γ for total 48 hours (B); Production of ROS after treatment with myelin was significantly inhibited after Ncf1 knock-down siRNA transfection by measuring SOD-inhibitable reduction of WST-1 (C) (t-test between groups transfected with ct siRNA and Ncf1 siRNA, n=3 per group).
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Supplemented Figure 5, Inhibition of ERK1/2 and c-Jun MAPK signal pathways partially reverse myelin-induced suppression of TNF-α and IP-10 transcription and secretion in primary cultured microglia.
IFN-γ-preactivated microglia were treated with 10μg/ml myelin and IFN-γ in the presence or absence of mitogen-activated protein kinase (MAPK) inhibitors: 10μM SP600125 for Jnk (JNKi II); 20μM PD98059 for Erk (PD); and 10μM SB203580 (SB) for p38 for 24 hours. Transcription and secretion of TNF-α (A) and IP-10 (B) were measured using real-time quantitative PCR and ELISA, respectively. Myelin significantly reduced TNF-α (A) and IP-10 (B) transcription with or without co-treatments of MAPK inhibitors (independent samples t-test, vs. no myelin pre-treatment control, all p values <0.05). SP600125 could partially reverse myelin-induced both TNF-α and IP-10 transcriptional reduction (A and B) and PD98059 only reversed suppressed IP-10 transcription (B). Similarly, myelin decreased TNF-α and IP-10 secretion which was reversed by SP600125 (C and D). PD98059 only reversed myelin-induced reduction of IP-10 secretion (D) (independent samples t-test, *: p <0.05, #: p>0.05, n= 3 per group for TNF-α measurement and n=5 per group for IP-10 measurement).
