RIPK3 promotes ISG expression independent of CNS region in cultured microglia.

A-C) Transcriptional expression of LGTV (A) or indicated genes (B-C) in cultures of cerebral cortical (B) or cerebellar (C) microglia following 24-hour infection with 0.1 MOI LGTV TP21 (B). ns, not significant. *p (TIFF)</p

RIPK3 limits LGTV replication in cerebellar granule cell neurons.

(A-B) Ripk3 -/- (A) or Mlkl -/- (B) mice and littermate controls were infected subcutaneously with LGTV TP21. At 8 or 12 days post infection (dpi), viral loads in cerebral cortical and cerebellar tissues were determined by plaque assay. Data are pooled from 2–3 independent experiments. (C) Ripk3 -/- and littermate control mice were subcutaneously infected with LGTV TP21. BBB permeability was measured at 8 dpi by detection of sodium fluorescein accumulation in tissue homogenates derived from cerebral cortex or cerebellum. Data represent individual brain fluorescence values normalized to serum sodium fluorescein concentration. Individual mouse values were then normalized to the mean values for uninfected controls. (D-G) Multistep growth curve analysis following infection with 0.01 MOI LGTV TP21 in cortical neurons (D), cerebellar granule cell neurons (E), cortical astrocytes (F), and cerebellar astrocytes (G). n = 3 (cerebellar granule cell neurons) or 4 (astrocytes and cortical neurons) for growth curve experiments. ns, not significant. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

Isolated adult cerebellar granule cell neurons are highly enriched for neuronal genes and not glial genes.

Transcriptional expression of indicated genes in isolated GCNs derived from adult WT (C57BL/6J) mice. Values derived from a pool of GCNs isolated from 3 distinct animals. (TIFF)</p

RIPK3 limits LGTV pathogenesis independently of peripheral immunity.

(A-B) Survival analysis (A) and presentation of clinical signs of disease (B) in Ripk3-/- mice and littermate controls following subcutaneous inoculation with 3x104 PFU LGTV TP21. Data are pooled from two experiments. (C) Ripk3-/- and littermate control mice were infected subcutaneously with LGTV TP21. On indicated days following infection, splenic viral burden was measured via qRT-PCR. Data was normalized against a standard curve of known viral titers to generate plaque-forming unit (PFU) equivalents. Data for each day post infection are pooled from 2–3 experiments. LOD, limit of detection. (D-P) Ripk3-/- and littermate control mice were infected subcutaneously with LGTV TP21 for 8 days prior to harvesting splenocytes and profiling leukocytes by flow cytometry. (D) Representative flow cytometry plots showing CD8+ and CD4+ T cells among CD3+ leukocytes in the spleen. Numbers represent percentage of cells in each gate relative to total plotted cells. (E-F) Numbers of CD8+ T cells (E) and CD4+ T cells (F) among CD3+ leukocytes. (G-H) Percentage of CD44+ cells among CD8+ T cells (G) and CD4+ T cells (H). (I-N) Numbers of CD19+ B cells (I), CD11b+ NK1.1+ Natural Killer cells (J), CD11c+ MHC-II+ dendritic cells (K), CD45high CD11b+ F4/80+ macrophages (L), CD11b+ Ly6G+ neutrophils (M), and CD45high CD11b+ Ly6C+ monocytes (N) among total leukocytes in the spleen. (O-P) Percentage of CD80+ cells among CD11c+ MHC-II+ dendritic cells (O) and CD11b+ F4/80+ macrophages (P). ns, not significant. **p < 0.01, ***p < 0.001.</p

RIPK3 promotes expression of IFN receptors and IFN-dependent inflammatory genes in cerebellar granule cell neurons.

A-B) Transcriptional expression of indicated genes in wildtype (C57BL/6J) cultures of cerebellar granule cell neurons in the setting of 2-hour pretreatment with GSK 872 or vehicle followed by 1 hour treatment with 10ng/ml IFNβ (A) or 24-hour infection with 0.5 MOI LGTV TP21 (B). C) Expression of indicated genes in wildtype cerebellar granule cell neurons pretreated for 45 minutes with an anti-IFNAR1 neutralizing antibody or isotype control +/- cotreatment with GSK 872 or vehicle, followed by 24-hour infection with 0.5 MOI LGTV TP21. ns, not significant. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

MLKL signaling does not influence Langat virus pathogenesis.

(A-B) Survival analysis (A) and presentation of clinical signs of disease (B) in Mlkl-/- mice and littermate controls following subcutaneous inoculation with 3X104 PFU LGTV TP21. Data are pooled from two experiments. (C) Mlkl-/- and littermate control mice were infected subcutaneously with LGTV TP21. On indicated days following infection, splenic viral burden was measured via qRT-PCR. Data was normalized against a standard curve of known viral titers to generate plaque-forming unit (PFU) equivalents. Data for each day post infection are pooled from 2–3 experiments. LOD, limit of detection. (D-P) Mlkl-/- and littermate control mice were infected subcutaneously with LGTV TP21 for 8 days prior to harvesting splenocytes and profiling leukocytes by flow cytometry. (D) Representative flow cytometry plots showing CD8+ and CD4+ T cells among CD3+ leukocytes in the spleen. Numbers represent percentage of cells in each gate relative to total plotted cells. (E-F) Numbers of CD8+ T cells (E) and CD4+ T cells (F) among CD3+ leukocytes. (G-H) Percentage of CD44+ cells among CD8+ T cells (G) and CD4+ T cells (H). (I-N) Numbers of CD19+ B cells (I), CD11b+ NK1.1+ Natural Killer cells (J), CD11c+ MHC-II+ dendritic cells (K), CD45high CD11b+ F4/80+ macrophages (L), CD11b+ Ly6G+ neutrophils (M), and CD45high CD11b+ Ly6C+ monocytes (N) among total leukocytes in the spleen. (O-P) Percentage of CD80+ cells among CD11c+ MHC-II+ dendritic cells (O) and CD11b+ F4/80+ macrophages (P). ns, not significant.</p

Neither RIPK3 nor MLKL is required for restriction of LGTV replication in bone marrow-derived macrophages and dendritic cells.

(A-B) Multistep growth curve analysis following infection with 0.01 MOI LGTV TP21 in primary macrophages (BMDMs) (A) and dendritic cells (BMDCs) (B) cultured from bone marrow of C57BL/6J (WT), Ripk3-/-, or Mlkl-/- mice. (n = 4) No comparisons are statistically significant. (TIFF)</p

RIPK3, but not MLKL, restricts Langat virus pathogenesis following intracranial infection.

Survival and body weight analysis from Ripk3-/- (A-B) and Mlkl-/- (C-D) mice and their respective littermate controls following intracranial inoculation with 50 PFU LGTV TP21. Data are pooled from two (A-B) or three (C-D) experiments. ns, not significant. **p < 0.01, ***p < 0.001.</p

RIPK3 promotes protein expression of CXCL10 in the cerebellum and cultured GCNs.

A-B) ELISA analysis of CXCL10 abundance in homogenates of cortex (A) or cerebellum (B) derived from mice of indicated genotypes at 8 dpi (footpad). C) CXCL10 ELISA analysis in culture supernatants of GCNs at 24 hpi. ns, not significant. ****p (TIFF)</p

RIPK3 does not impact IFN-mediated responses to LGTV in cortical neurons.

A-B) Transcriptional expression of indicated genes in wildtype (C57BL/6J) cultures of cerebral cortical neurons in the setting of 2-hour pretreatment with GSK 872 or vehicle followed by 1 hour treatment with 10ng/ml IFNβ (A) or 24-hour infection with 0.5 MOI LGTV TP21 (B). C) Expression of indicated genes in wildtype cerebral cortical neurons pretreated for 45 minutes with an anti-IFNAR1 neutralizing antibody or isotype control +/- cotreatment with GSK 872 or vehicle, followed by 24-hour infection with 0.5 MOI LGTV TP21. ns, not significant. *p (TIFF)</p