Fig. 5

IDO1 regulates pain sensitivity through IFN signaling pathway in chronic migraine. (A) PCA plot indicating distinct clustering of gene expression profiles in the ACC of WT and IDO1 KO mice treated with saline or NTG (n = 4). (B-D) Volcano plotsillustrating differentially expressed genes (DEGs) in the ACC: (B) WT + Control vs. WT + Model, (C) KO + Control vs. KO + Model, (D) WT + Model vs. KO + Model (n = 4). (E) Venn diagrams depicting the overlap of DEGs between WT + Control vs. WT + Model and KO + Control vs. KO + Model groups, identifying 430 DEGs unique to NTG-treated WT mice but not altered in NTG-treated KO mice. (F) Heatmap representing the expression levels of the 430 unique DEGs in the ACC across all experimental groups, indicating distinct expression patterns. (G-H) GO and KEGG were used for functional enrichment analysis of the 430 unique DEGs. (I) GSEA plot for the IFN signaling pathway. (J) Enrichment plots for the top 10 pathways by GSEA. (K) Increased IFN-γ protein expression in the ACC of NTG- treated WT mice. (L) Quantitative analysis of the data in (K). The IFN-γ band density was normalized to the loading control GAPDH (n = 4). (M) No changes of IFN-β between NTG- or saline-treated mice. (N) Quantitative analysis of the data in (M). The IFN-β band density was normalized to the loading control GAPDH (n = 4). (O) Experimental timeline for treatment with Tilorone, an IFN agonist, in combination with NTG to assess its effects on pain sensitivity (red triangle) in WT mice injected with AAVs. (P-Q) Basal and acute mechanical paw withdrawal thresholds measured by von Frey filament in response to mechanical stimuli in mice treated with saline + mCherry, NTG + mCherry, NTG + shRNA, and NTG + shRNA + Tilorone (n = 12–14). The data are shown as mean ± SEM, ^*p < 0.05, ^**p < 0.01, ^**** or ^####p < 0.0001; one-way ANOVA in (I, N), two-way ANOVA in (P, Q)