Figure 4. Lack of Usp18 enhances type I IFN gene expression in microglia due to prolonged Stat1 phosphorylation.

A Gene ontology enrichment network on differentially expressed genes in microglia from unstimulated Usp18^+/+ and Usp18^LacZ/LacZ (Usp18^−/−) microglia on the basis of an Affymetrix DNA microarray analysis. Diagram depicts results of GO clustering through GORilla. Only very highly significantly overrepresented GO terms are included with P-values ranging from P < 10⁻⁹ (yellow) to P < 10⁻²⁴ (red).

B Quantitative RT–PCR for Usp18 transcripts in primary microglia stimulated for the designated time points with 100 U/ml of IFN-β (left panel) or with 10, 100 or 1,000 U/ml of IFN-β and measured after 4 h (right panel). Bars represent means ± s.e.m with three to four samples in each group. Data are representative of two independently performed experiments.

C Fluorescence microscopy of the white matter (corpus callosum) of Mx1^Cre:R26-confettimice raised under specific pathogen-free conditions. Recombination of GFP, RFP, CFP or YFP (combined into one channel to XFP and displayed in red) was found in Iba-1⁺ microglia of the white matter. Scale bars: 20 μm (overview) and 10 μm (zoom).

D Flow cytometric quantification of Stat1 phosphorylation in BV-2 microglial cells transfected with control siRNA (siRNA co) or siRNA against Usp18. Representative dot blots of IFN-β-treated cells at indicated time points are shown that were obtained from two independent experiments. FSC: forward scatter.

E Immunoblot analysis of type I IFN signaling in microglia lacking Usp18. Upper panel: Absence of Usp18 protein leads to prolonged Stat1 activation upon IFN-β challenge (500 U/ml) of microglia from Usp18^+/+ and Usp18^LacZ/LacZ (Usp18^−/−) mice. Gapdh is shown as a loading control. Lower panel: Altered IFN signaling in the microglia cell line BV-2 transfected with control siRNA (siRNA co) or siRNA against Usp18. Quantification of band intensities is depicted next to the blots. Representative Western blots of three to four independently performed experiments are shown.

F Brain histology of the white matter reveals increased pStat1 and interferon-induced gene (ISG) 15 levels in white matter microglia in adult Cx3cr1^Cre:Usp18^fl/fl and Usp18^LacZ/LacZ (Usp18^−/−) mice but not in Usp18^+/+ individuals. Quantification of Isg15⁺ cells in the gray (GM) and white matter (WM) is presented next to the respective histological images. Scale bars: 200 μm (overview) and 10 μm (insert). Each symbol indicates the mean of one mouse. Error bars represent s.e.m. Significant differences are determined by an unpaired t-test and marked with an asterisk (*P < 0.05).

G Heat map (standardized and scaled to log₂ expression) of non-stimulated conditions (0 h) or after IFN-β (500 U/ml for 6 h and 24 h) treatment in primary microglia from Usp18^LacZ/LacZ (Usp18^−/−) and Usp18^+/+ mice or the microglia cell line BV-2 (transfected with control siRNA [siRNA co] or siRNA against Usp18). Expression profile of top 50 induced genes in Usp18^+/+ or siRNA co upon 6 and 24 h IFN-β is shown.