Fig 1: Identification of PRV UL13 as an inhibitor of cGAS-STING signaling.(A) Luciferase activities in UL13 PK-15 or control PK-15 cells cotransfected with IFN-β-luc reporter or ISRE-luc reporter. Twenty-four hours after transfection, cells were left untreated or stimulated with B-DNA (2 μg/ml) for 12 h, and cell lysates were analyzed for luciferase activity. (B and C) qPCR analysis of IFNB1 and downstream ISGs (MX1, ISG56) mRNA expression in UL13-PK-15 cells and control cells stimulated with B-DNA (1 μg/ml) (B) or cGAMP (1 μg/ml) (C) for the indicated times. (D and E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172)- and total TBK1 and IKKε, phosphorylated (Ser396)- and total IRF3 in whole-cell lysates of UL13-PK-15 cells and control cells stimulated for the various times (above lanes) with B-DNA (1 μg/ml) (D) or cGAMP (1 μg/ml) (E). Data are pooled from three independent experiments (A-C, mean ± SD) or representative of three independent experiments (D, E). *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).
Fig 2: Adoptive transfer of Tenc cells to Ifnb−/− mice causes elevated neuroinflammation associated with defective FoxA1+Treg cell generation.(a) Adoptive transfer of MBP89–101 Tenc cells to C57BL/10.RIII mice, EAE score from Ifnb+/+ and Ifnb−/− mice, n=24 mice per group. Non-parametric Mann–Whitney test was used, ***P<0.001. (b) Representative IF images in spinal cords from mice day 30 adoptive EAE. PDL1(green), FoxA1(red), TCR(purple) and DAPI(blue). Scale bars, 20 μm. (c) Quantification of number of spinal cord infiltrating T cells, (d) quantification of number of FoxA1+Treg cells. Graphs are mean±s.e.m., n=10 sections per group. Non-parametric Mann–Whitney test was used, *P<0.05 and **P<0.01. (e) IF images of T cells in close vicinity of neurons in spinal cords of a WT mouse, day 30 post-adaptive EAE. FoxA1 (red), TCR (purple) and DAPI (blue). Scale bars, 10 μm. (f) Representative IF images of neurons in cerebellums. PDL1 (green) and NF200 (purple) were stained and shown. Scale bars, 30 μm. (g) Closed up representative IF images of neurons in spinal cords. PDL1 (green), FoxA1 (red), TCR (purple) and DAPI (blue). (h) Quantification of number of NF200+FoxA1+PDL11+ neurons in spinal cords. Graphs are mean±s.e.m., n=7–8 sections per group. Non-parametric Mann–Whitney test was used, *P<0.05.
Fig 3: Generation of FoxA1+Treg cells requires neuronal FoxA1 and PDL1.(a) Percentage of FoxA1+Tregs upon co-culture of Tencs with CGNs, with or without transwell. Graphs are mean±s.e.m., n=3. One-way analysis of variance (ANOVA) test was used, ***P<0.001. (b) Fold change of Foxa1 mRNA and (c) Pdl1 mRNA in CGNs treated with or without rIFNβ (100 U ml−1). Graphs are mean±s.e.m., n=3, two-way ANOVA test was used, *P<0.05 and **P<0.01. (d) Foxa1 and (e) Pdl1 mRNA in CGNs (left) and quantification (right). Graphs are mean±s.e.m., n=3, Student's t-test was used, ***P<0.001. (f) Cytosolic (Cyto) and nucleus (Nu) fractions from CN (left). Graph shows normalized optical density (IOD) ratios between cytosolic/nucleus fractions of quantified FoxA1 protein bands (right). Graphs are mean±s.e.m., n=2–4. Non-parametric Mann–Whitney test was used for comparison of treated groups with control group. *P<0.05 and **P<0.01. (g) CGNs with or without treatment with rIFNβ. NF200 (green), FoxA1 (red) and DAPI (blue). Scale bars, 5 μm. (h) WB of FoxA1, PDL1 and vinculin in CNs extracts with or without treatment with rIFNβ. (i) Representative FACS histogram of neuronal FoxA1 and PDL1 after Foxa1 siRNAKD in CGNs and (j) WB of FoxA1, PDL1 and vinculin after Foxa1 siRNAKD in CGNs. (k) Percentage of FoxA1+Tregs after co-culture of Tencs with CGNs after Foxa1 or control siRNAKD in CGNs. Graphs are mean±s.e.m., n=3, Student's t-test was used, ***P<0.001. (l) Neuronal PDL1 after Pdl1 or UNC (Universal Negative Control) siRNAKD in CGNs. (m) Percentage of FoxA1+Tregs upon co-culture of Tencs with CGNs after PDL1 or UNC siRNAKD in Ifnb+/+ CGNs. Graphs are mean±s.e.m., n=3, Student's t-test was used, ***P<0.001. (n) Percentage of FoxA1+Tregs upon co-culture of Tencs with CGNs after PDL1 was blocked in Ifnb+/+ neurons utilizing anti-PDL1 or PD1 was blocked in Tencs utilizing anti-PD1 antibodies. Graphs are mean±s.e.m., n=3, one-way ANOVA test was used, ***P<0.001. (o) Neuronal PDL1 after transfection of pIRES2-EGFP-PDL1 or pIRES2-EGFP in CGNs. (p) Percentage of FoxA1+Tregs upon co-culture of Tencs with overexpression of PDL1 in Ifnb−/− CGNs. Graphs are mean±s.e.m., n=3, one-way ANOVA test was used, ***P<0.001.
Fig 4: Neuronal ability to generate FoxA1+Tregs is IFNß-mediated PI3K-Akt-FoxA1 and PDL1 dependent.(a) Representative FACS plots of Tenc cells after co-culture with CGNs, with and without Akt siRNA KD or PI3K inhibitor (Wortmannin in DMSO, 200 nm). FoxA1+Treg cells were gated on CD4highPDL1high cells. (b) FoxA1 expression was gated in CD4highPDL1high cells (R1) and in CD4lowPDL1low cells (R2) and (c) quantified percentage of FoxA1+Tregs. Graphs are mean±s.e.m., n=3, one-way analysis of variance (ANOVA) test was used, ***P<0.001. (d) Schematic drawing of how neuronal endogenous IFNß signalling leads to PI3K and Akt phosphorylation. Total and phosphorylated (p)Akt binds to FoxA1, FoxA1-Akt complex translocates to nucleus and binds to Pdl1 promoter, consequently resulting in PDL1 expression. This signal is essential for neurons to convert Tenc cells to anti-inflammatory FoxA1+Treg cells. In contrary, lack of endogenous IFNß signalling in Ifnb-/- neurons leads to insufficient phosphorylated PI3K/Akt signalling, defective FoxA1 mediated PDL1 expression and thereby inability to generate FoxA1+Treg cells.
Fig 5: RNF5-deficiency potentiates PRV-induced antiviral immune responses.(A) Immunoblotting analysis of indicated proteins in immunoprecipitated samples and whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA (50 µM). Twenty-four hours after transfection, cells were further transfected with Flag-UL13, Myc-STING, and His-Ub for another 24 h before analysis. (B) Immunoblotting analysis of protein levels of RNF5, UL13, and STING in whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA for 24 h. Cells were then transfected with Flag-UL13 and Myc-STING for the indicated times (above lane). (C) Immunoblotting analysis of RNF5 protein expression in wild type (WT) and RNF5-deficient (RNF5 KO) MEFs. (D) qPCR analysis of transcription of Ifnb1 and downstream genes (Mx1, Isg56) in WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172) and total TBK1 and IKKe, phosphorylated (Ser396) and total IRF3, or RNF5 in whole-cell lysates of WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (F) qPCR analysis of mRNA expression of Ifnb1 and downstream genes (Mx1, Isg56) in RNF5 KO MEFs infected with PRV-WT or PRV-?UL13 (MOI = 1) for indicated times. Data are representative of two (A, C) or three (B, E) independent experiments, or are pooled from three independent experiments (D, F, mean ± SD). *p < 0.05, **p < 0.01 (Student’s t test).
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