Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

To confirm that the suppression of prion infection by IFNs was associated with IFN signalling, we generated immortalized MEF cells from wild-type and IFNAR1-deficient (Ifnar1 À/À ) mice and performed prion infection ex vivo. Susceptibility to 22 L prion infection was remarkably higher in immortalized MEF cells from Ifnar1 À/À mice than in MEF cells from wildtype mice. Prions were dose-dependently propagated in both MEF cells. IFNAR1 À/À MEFs showed significantly higher sensitivity to prion infection with 1.5 Â 10 À1 % 22 L-brain homogenate than MEFs from wild-type (Fig. 3A) . Next, we generated Ifnar1-transduced Ifnar1 À/À MEF cells, which were rescued by delivering the Ifnar1 gene to Ifnar1 À/À MEFs with an MSCV retroviral vector, and investigated the efficiency of prion infection in an ex vivo model. The PrP C level was similar between Ifnar1 À/À MEFs and Ifnar1 À/À MEFs rescued by delivering the Ifnar1 gene with an MSCV retroviral vector, but the latter cells were significantly and continuously less susceptible to 22 L prion infection (Fig. 3B, Supplementary Fig. 3A and B) . To confirm whether the transduction restored IFN signalling, we performed ISRE-promoter activity analysis using the dual luciferase assay. The results indicated that the promoter activity in Ifnar1-transduced Ifnar1 À/À MEFs after poly I:C stimulation was significantly increased (3-fold) compared with non-stimulated cells, indicating that the transduced cells recovered cell signalling function via IFNAR1 whereas Ifnar1 À/À MEFs did not possess this signalling pathway (Supplementary Fig. 3C ). To elucidate the relationship between I-IFN dependent signalling pathways and prion infection in vivo, we inoculated 22 L prion into wild-type and Ifnar1 À/À mice and monitored prion pathogenesis. When mice were challenged intracerebrally with a 10 À1 dilution of 22 L prion brain homogenate, the survival periods of Ifnar1 À/À mice were significantly shorter (145 AE 5 days, n = 15; P = 0.0006, Log-rank test) than those of the wildtype mice (153 AE 8 days, n = 17) ( Fig. 4A and Table 1) . Furthermore, all groups of IFNAR1 À/À mice inoculated with 10 À2 dilution i.c., or 10 À2 and 10 À3 dilution i.p., also exhibited significantly shortened survival (Table 1) . To confirm pathogenesis in IFNAR1 À/À mice after prion inoculation, we performed immunoblotting to examine the PrP Sc levels in the brain and spleen at 100 dpi and at the terminal stage. The PrP Sc levels in the brain and spleen of Ifnar1 À/À mice were similar into those of wild-type mice ( Fig. 4B and Supplementary Fig. 3D ). However, vacuolation and gliosis with microglia (marker: IBA1) and astrocytes (marker: GFAP) were significantly more severe than in wild-type mice. In addition, accumulation of PrP Sc in some regions at 100 dpi was considerably more severe in Ifnar1 À/À mice (Fig. 4C, D and Supplementary Fig. 3E ). Taken together, these findings indicate that the I-IFN signalling pathway via I-IFN receptor, including IFNAR1, protects to some degree against prion infection. and NIH3T3 (C) cells after ex vivo prion infection with 2 Â 10 À3 or 2 Â 10 À2 % 22 L-brain homogenate (BH). Pre-infection of N2a-58 cells and normal brain homogenate treatment in 3T3 cells, white; 22 L-prion infection, black. Immunoblot shows PrP Sc levels in those cells after prion infection. (D) Irf3 and Ifnb levels in N2a-22 L cells for 48 h after PPS and 3S9 treatments. N2a-58 (white); -22 L (black). Immunoblot shows PrP Sc levels in the ce