Fig 1.
RIPK3 contributes to the pathogenesis of HSV-1 encephalitis, independent of its pro-necroptotic kinase activity.
Corneas of 6-8-week-old WT, Ripk3−/− and Ripk3K51A/K51A mice were scarified and inoculated with 5 × 105 pfu of HSV1 strain McKrae on the left eye. (A) Survival plot showing percent survival of WT (n = 7), Ripk3−/− (n = 6), and Ripk3K51A/K51A(n = 9) mice after HSV1 infection. Tear film swab (B), trigeminal ganglion (TG) (C), brainstem (D), and (E) cerebrum and cerebellum tissues were collected at 5 dpi for determining viral titers (n = 4–5 per group). Values are expressed as means ± SEM (*P<0.05, **P<0.01, ***P<0.001).
Fig 2.
Caspase 8 contributes to RIPK3 protection within CNS against HSV1.
Mice were infected as above described. (A) Plot showing percent survival of WT (n = 8), Ripk3-/-Casp8-/- (n = 9) and Ripk3K51A/K51ACasp8 -/- (n = 7) mice after HSV1 infection. Viral titers at 5 dpi in (B) tear film, (C) trigeminal ganglion (TG), (D) brainstem, and (E) combined cerebrum and cerebellum tissues were tested (n = 4–5 per group). Values are expressed as means ± SEM (*P<0.05, **P<0.01, ***P<0.001).
Fig 3.
H&E sections of brainstem at 7 dpi.
(A) Images represent examples of pathologic findings indicated on each panel. (B) Pathological scores of sections. (C) Number of glial nodules or perivascular cuffing within the parenchymal of the brainstems. (D) Measurement of the average thickness of the meningeal tissue at the ventral surface of the brainstem. Each dot represents different section. Distribution of meningeal thickness in micrometers. Values are expressed as means ± SEM (*P<0.05, **P<0.01, ***P<0.001).
Fig 4.
Impact of RIPK3 on tissue cytokine levels.
(A) Heat map comparing mRNA patterns of immune-related genes in brain from WT and Ripk3−/− mice (n = 3 for each group) at 5 dpi, where mean fold-change of each transcript was compared to normalized expression of all WT immune-related transcripts. Blue, white and red indicate down-regulated, unchanged and up-regulated expression, respectively. (B and C) mRNA levels of IFNs, cxcl10, cxcl11 were measured by qRT-PCR in brain homogenates of WT and Ripk3-/- mice. (D and E) Levels of IFNγ and TNF were measured by ELISA in combined cerebrum and cerebellum or brainstem homogenates as well as serum of WT and Ripk3-/- mice. (F and G) Levels of IFNγ and TNF were measured by ELISA in cerebrum and cerebellum, brainstem homogenates as well as serum of Ripk3K51A/K51A and Ripk3K51A/K51ACasp8 -/-mice (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5.
RIPK3 collaborates with Casp8 on NK and T cells recruiting to CNS during HSV1 infection.
Numbers of NK, CD4 and CD8 T cells of total brain leukocytes (A) or spleen (B) from WT, Ripk3-/-, Ripk3 K51A/K51A and Ripk3K51A/K51ACasp8 -/-mice. Total IFNγ+ and granzyme B+ NK or T cell numbers by intracellular cytokine staining were also evaluated (n = 5–7 per group). All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.