Fig. 1

Immunoproteasome inhibition reduces IFNγ-dependent complement gene activation. Wild-type or β5i knockout BV-2 cells were treated with IFNγ in the absence and presence of immunoproteasome inhibitor, ONX-0914 and levels of C1q genes were analyzed. (A) Gene expression analysis of C1qa revealed a significant difference between treatment groups using one-way ANOVA ([F(5, 20) = 9.21], p < .001, n = 5). Tukey’s HSD post hoc test revealed that IFNγ resulted in a significant increase of C1qa compared to all other groups (control, p < .001; ONX-0914, p = .001; ONX + IFNg, p < .01; KO control, p < .001; K.O. IFNγ, p < .001). C1qa levels were not increased by IFNγ in b5i KO cells (p = .996, n = 3, t-test comparing two groups). (B) Gene expression analysis of C1qb revealed a significant difference between treatment groups using one-way ANOVA ([F(5, 20) = 10.56], p < .001, n = 5). Tukey’s HSD post hoc test revealed that IFNγ resulted in a significant increase of C1qb (control, p < .001; KO control, p < .001; KO IFNγ, p < .05). C1qb levels were not increased by IFNγ in b5i KO cells (p = .974, n = 3, t-test comparing two groups). (C) Gene expression analysis of C1qc revealed a significant difference between treatment groups using one-way ANOVA ([F(5, 24) = 10.56], p < .001, n = 6). Tukey’s HSD post hoc test revealed that IFNγ resulted in a significant increase of C1qc (control, p < .001; ONX + IFNγ, p = .012; KO control, p < .001; K.O. IFNγ, p < .01). C1qc levels were not increased by IFNγ in b5i KO cells (p > .999, n = 3, t-test comparing two groups) (D) Western blot analysis confirms that IFNγ treatment increases C1q protein levels in WT BV2 cells but not in β5i KO BV-2 cells. (E) Analysis of C3 gene expression revealed a significant difference between groups using one-way ANOVA (F(3,12) = 15.76, p < .001, n = 4). Tukey’s HSD post hoc test revealed that ONX-0914 treatment reduced C3 levels compared to control and IFNγ treatments (p < .01 and p ≤ .001, respectively). Further, ONX-0914 co-treatment with IFNγ reduced C3 levels compared to IFNγ alone (p = .002). (F) Gene expression analysis of C1qa, C1qb and C1qc in iPSC-derived microglia were determined by qRT-PRC (control n = 4, SMA n = 4; *p < .05, **p = .01, ***p < .001, ****P < .0001). Heat Map of Cytokine Levels. BV-2 cells were treated for 24 h then submitted to cytokine analysis. Values are mean pixel density of 4 independent experiments. * indicates a significant difference between control and IFNγ groups. ** (p < .05) indicates that ONX-0914 treatment reversed the IFNγ−dependent increase.