Fig 1.
Ifit2-/- mice showed increased disease severity and increased infectious viral load upon RSA59 infection.
: 4–5 weeks old WT (n = 28) and Ifit2-/- mice (n = 30) were infected intracranially with 2000 PFU of RSA59 and monitored for development of clinical disease (A) and viral titers in brains (B). Clinical scores were assigned by an arbitrary scale of 0–4 as described in Materials and Methods. Viral burden in brain tissue homogenates was analysed by plaque assay and shown as PFU/ brain (approx. 300 mg) from individual mice (n = 5-6/timepoint). The solid line represents the mean viral titer. Asterix (*) indicate statistical significance by Two-Way ANOVA analysis for clinical score and unpaired t-test for viral titers. (**P<0.01, ***P<0.001, ****P<0.0001).
Fig 2.
Ifit2 deficiency increases RSA59 viral spread throughout the brain parenchyma.
5–10 μm thin cryosections were prepared from brains of 4–5 weeks old WT and Ifit2-/- infected mice at day 5 p.i. Representative EGFP fluorescence in whole brain section is shown from WT (A; n = 2) and Ifit2-/- (B; n = 4) mice. Similar anatomic locations from the WT and Ifit2-/- mice brain stem regions were magnified for both WT (C) and Ifit2-/- (D) infected mice. Scale bar for Panels A and B = 1mm and for Panel C and D = 50 μm.
Fig 3.
Ifit2 deficiency results in decreased neuroinflammation upon RSA59 infection.
5 μm thin paraffin embedded serial sections of RSA59 infected WT and Ifit2-/- brain tissues processed for H & E (Panels A-D and H-K) and Iba1 staining (Panels E-G and L-N) at 5 days p.i. as indicated. Representative scanned images of whole brain are shown for WT (A) and Ifit2-/- (H) mice with selected meningeal infiltration indicated by Red box, perivascular cuffing by blue box and microglial nodule formation by green box. Selected boxed areas are enlarged for WT and Ifit2-/- brains in Panels B, C, D and Panels I, J, K, respectively. Representative Iba1 staining in meninges, perivascular cuffs and microglial nodules is shown for WT and IFIT2-/- brains in Panels E, F,G and L,M,N, respectively. Ifit2-/- mice showed reduced overall inflammation, reduced or no perivascular cuffing and only scattered Iba-1+ cells without apparent nodule formation. Red arrow depicts menegitis, Dark blue arrow depicts encephalitis (perivascular cuffing), green arrow shows encephalitis (microglial nodules). The experiment was repeated three times and total n = 8. The scale bar for Panel A and H is 2 mm and for Panel B-G and Panel I-N is 75 μm.
Fig 4.
Ifit2 deficiency results in increased viral infection, impaired microglial activation but induce no changes in astrocyte activation upon RSA59 infection.
5 μm thin serial sections from brain tissues used in Fig 3 were processed for Viral N protein (Panels A,B), Iba1 (Panels D,E) and GFAP (Panels G,H) immunostaining as indicated. Quantification of Viral N protein, Iba1 and GFAP expression are graphically represented in Panel C, F, and I respectively. The experiment was repeated three times and total n = 8.The scale bar for the whole brain section is 2.8 mm and for the insets is 80 μm. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (**P<0.01, ****P<0.0001).
Fig 5.
Differential mRNA expression levels of selected proinflammatory cytokines and chemokines in brains of infected Ifit2-/- and WT mice.
RNA extracted from individual brain tissues of RSA59 infected WT and Ifit2-/- mice at day 3, 5, and 7 p.i. was analysed for mRNA levels of the indicated cytokines and chemokines by Real time PCR. Solid lines represent the mean levels of mRNA expression (n = 4–11). Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis (Mann-Whitney Test) (*P<0.05, **P<0.01, ***P<0.001).
Fig 6.
Ifit2 deficiency impairs CD45+ leukocyte migration into the CNS at day 5 and 7 post infection.
Brains from WT and Ifit2-/- mice infected with RSA59 were harvested at days 3, 5, and 7 p.i. for flowcytometric analysis. Purple colour denotes WT and green represents Ifit2-/- mice. Panel A shows representative flow cytometry plots at day 5 and 7 p.i., indicating percentages of CD45hiand CD45lo cells in brains after gating on live cells. Absolute numbers of CD45hi cells are graphically represented across timepoints (Panel B) and in between WT and Ifit2-/- groups for day 5 p.i., and day 7 p.i. (Panel C) to better represent respective statistics. Graphs represent n = 14–20 mice from 3 separate experiments. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (*P<0.05, **P<0.01).
Fig 7.
Ifit2 deficiency does not alter myeloid cell populations in the brain.
Cell suspensions from brains of infected WT and Ifit2-/- mice were analysed as described in Fig 6 by flow cytometry, following staining for CD45 and the myeloid marker CD11b. Panel A shows representative flow cytometry plots, indicating percentages of CD11b+CD45hi and CD11b+CD45lo cells at days 5 and day 7 p.i. Absolute numbers of CD11b+CD45hi cells are graphically represented across timepoints (Panel B) and in between WT and Ifit2-/- groups for day 5 p.i., and day 7 p.i. (Panel C) to better represent respective statistics. Similarly, absolute numbers of CD11b+CD45lo cells are graphically represented for same group at each timepoint (Panel D) and in between WT ad Ifit2-/- groups at two timepoints (Panel E). Graphs represent n = 4–7 mice and the experiment were repeated 2–3 times. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (*P<0.05).
Fig 8.
Ifit2 deficiency does not alter neutrophil migration into the CNS.
Cell suspensions from brains of infected WT and Ifit2-/- mice were analysed as described in Fig 6 by flow cytometry following staining for CD45 and the neutrophil marker Ly6G. Panel A shows representative flow cytometry plots at days 3,5,and 7 p.i. Absolute numbers of Ly6G+CD45hi cells are graphically represented across timepoints for each group (Panel B) and between WT and Ifit2-/- groups for day 3 p.i., 5 p.i., and day 7 p.i. (Panel C) to better represent respective statistics. The experiment is repeated twice and representative graphs are presented with n = 5–9. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (**P<0.01, ****P<0.0001).
Fig 9.
Ifit2 deficiency significantly impairs migration of NK1.1, CD4 and CD8 T cells among the CD45hi population at acute stage of RSA59 infection.
Dot Plot representation of the percentage of NK1.1, CD4 and CD8 expressing cells were gated from CD45hi populations in the brain lysates, which was gated on live cells for both day 5 and 7 p.i. (Panel A). Purple colour denotes WT and green colour denotes Ifit2-/- mice. Absolute number of NK1.1 expressing cells gated from CD45hi population for both the WT and Ifit2-/- total brain lysate is comparatively represented across timepoints (day 5 and day 7) (Panel B) and also between WT and Ifit2-/- mice for day 5 p.i., and day 7 p.i. (Panel C). Absolute number of CD4 expressing cells gated from CD45hi population for both the WT and Ifit2-/- total brain lysate is comparatively represented across timepoints (day 5 and day 7) (Panel D) and also between WT and Ifit2-/- mice for day 5 p.i., and day 7 p.i. (Panel E). Absolute number of CD8 expressing cells gated from CD45hi population for both the WT and Ifit2-/- total brain lysate is comparatively represented across timepoints (day 5 and day 7) (Panel F) and also between WT and Ifit2-/- mice for day 5 p.i., and day 7 p.i. (Panel G). The data were pooled from two independent experiments with n = 5–10. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (*P<0.05, **P<0.01).
Fig 10.
Ifit2 deficiency decreases CX3CR1 expression in microglia following infection.
Brain derived cells from WT and Ifit2-/- mice either mock infected (Panel A) or infected with RSA59 (Panel B) were stained for CD45, CD11b and CX3CR1 expression at 5 days p.i. Dot plots show gating and percentages of CD45hi CD11b+ and CD45lo CD11b+ populations as indicated. Histograms show CX3CR1 expression on the respective gated myeloid cell populations as indicated by arrows. Panels C and D show Median Fluorescent Intensity of CX3CR1 expression by CD45lo CD11b+ microglia and CD45hi CD11b+ infiltrates of WT (Purple) and Ifit2-/- (Green) mice, respectively.The experiment is repeated 2–3 times with n = 5–6. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (***P<0.001).
Fig 11.
Ifit2 deficiency resulted in downregulation of CX3CR1 upon RSA59 infection.
Brain derived cells from WT and Ifit2-/- mice infected with RSA59 were stained for CD45 and CX3CR1 expression at 5 days p.i. Dot plots show gating and percentages of CD45hi and CD45lo populations as indicated. Dot Plot representation of the percentage of CX3CR1 expressing cells were gated from CD45hi and CD45lo populations in brain lysates at day 5 p.i. (Panel A; gated on live cells). Purple colour denotes WT and green colour denotes Ifit2-/- mice. Panels B and C show absolute numbers of CX3CR1 expressing CD45lo cells and CD45hi cells as gated in Panel A from WT and Ifit2-/- mice, respectively. Panels D and E show Median Fluorescent Intensity of CX3CR1 expressing CD45lo and CD45hi cells, respectively. The experiment is repeated 2–3 times with n = 5 for absolute number of cells and n = 10–11 for median fluorescent intensity. Asterix (*) represents differences that are statistically significant by Student’s unpaired t-test analysis. (*P<0.05, ****P<0.0001).
Table 1.
List of Primers.