Astrocytes evoke a robust IRF7-independent type I interferon response upon neurotropic viral infection

Fig. 1. IRF7 is dispensable for the survival of LGTV infection. WT, Mavs−/−, Irf3−/− and Irf7−/− mice were intraperitoneally infected with 104 FFU of LGTV (n = 10). A Survival and B body weight were monitored. Data represent mean with SEM of n ≥ 10 in each group per time point. Viral burden was determined by quantitative RT-PCR in the serum (C) and spleen (D). The dotted line represents the detection limit. IFN-I levels were determined by bioassay in serum (E) or in the supernatant of infected pDCs (F) or MØ (G). Asterisks indicate statistical significance calculated by Mann–Whitney test (A, C–G) or Student’s t test (B), *p<0.05, **p<0.01. Results are representative for at least two independent experiments

Fig. 2. IRF-7 limits viral replication in the periphery and the CNS. WT, Irf3−/− and Irf7−/− mice were intraperitoneally infected with 104 FFU of LGTV. Viral burden was determined by quantitative RT-PCR in the different brain regions (A) or by plaque assay in the olfactory bulb (B) (n = 10). The dotted line represents the detection limit. Asterisks indicate statistical significance calculated by Student’s t test, *p < 0.05. C Representative pictures of immunohistological analysis of LGTV-E protein in the olfactory bulb, cortex, hippocampus and cerebellum at 7 days post-infection (n = 5) [LGTV E protein (green), DAPI (blue)]. Magnification is represented by scale bar, 200 µm

Fig. 3. IRF7 deficiency increases cytokine response in the brain. WT, Irf3−/− and Irf7−/− mice (n ≥ 10) were intraperitoneal infected with 104 FFU of LGTV. A Level of IFNβ induction in LGTV-infected IFNβ+/Δluc mice. Shown is whole body imaging of Ifnβ+/Δluc WT, Irf3‑/‑ or Irf7‑/‑ reporter mice. Mice were imaged before treatment (0 h) and over time as indicated; one representative mouse is shown in dorsal position. The scale indicates the number of photons (p) measured per s per cm2 per steradian (sr). B Cytokines CCL2 and TNFα were determined by quantitative RT-PCR in the brain. The dotted line represents the detection limit. Asterisks indicate statistical significance calculated by Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4. IRF7 deficiency leads to inflammatory response and infiltration of immune cells into the brain. WT and Irf7−/− mice were infected intraperitoneally with 104 FFU LGTV (n = 6) and immune cells were isolated from brains for flow cytometric characterization on day 7 post-infection. Upon infection with LGTV, brain immune cells were manually gated based on their size and granularity in the forward scatter and side scatter light (FSC/SSC) and then separated from dead cells and doublets (not shown). Subsequently, infiltrating immune cells were distinguished from brain-resident microglial cells based on their high expression levels of CD45 and CD11b, respectively (red gate; A, B). Compared to WT littermates, infected Irf7−/−mice displayed high numbers of infiltrating immune cells in the brain parenchyma 7 dpi (C). Furthermore, the surface expression levels of CD45 (D), CD11c (E), and CD11b (F) by microglial cells were assessed. Representative histograms with dashed vertical lines show the frequency (with standard error of the mean) of microglial cells from WT (black tint) and Irf7−/− mice (red tint) expressing the respective surface marker in comparison to the corresponding FMO control…

Fig. 5. High diversity of infiltrating immune cells in brains of IRF7-deficient mice upon LGTV infection. WT and Irf7−/− mice were infected intraperitoneally with 104 FFU LGTV (n = 6) and immune cells were isolated from brains for flow cytometric characterization. A representative gating strategy (A-K). FSC and SSC gating of immune cells (A). Life cell selection (B) and separation from doublets (C). Neutrophil granulocytes Ly6G + CD11b + (D), differentiation of remaining mononuclear cells by CD45 and CD11b expression (E). Identification of NK1.1+ and NK1.1−CD3+ cells within CD45+CD11b− population (F). NK1.1−CD3+ cells were gated for CD4 and CD8 (G). Remaining NK1.1−CD3− cells from (F) were separated in B220+CD11c− B cells (H). B220+CD11c+ cells (H) which were further assessed for MHC class II (MHCII) and Ly6C expression show plasmacytoid dendritic cells (pDCs) (I). CD45+CD11b+ cells from (E) were gated for CD45 and CD206 separating brain-resident microglia from border-associated macrophages (BAMs) and infiltrating myeloid cells (J). MHCII and CD11c expression of myeloid cells to distinguish conventional dendritic cells (cDCs) and monocytes/macrophages (K). Monocytes and macrophages…

Fig. 6. IRF7 deficiency leads to differences in cellular tropism of LGTV. WT, and Irf7−/− mice were infected intraperitoneally with 104 FFU of LGTV. Brains were isolated for immunohistological analysis 7 days post-infection. A Representative pictures of IBA-1 (red), NeuN (red), GFAP (red) and, LGTV-E protein (green), and DAPI (blue). Scale bar is shown as 50 μm. B Quantification of LGTV-positive, NeuN+, GFAP+, and IBA-1+ cells in the glomerular layer of the olfactory bulb in infected mice, 7 days post-infection (n = 5). Asterisks indicate statistical significance calculated by Student’s t test, *p < 0.05, **p< 0.01, ***p < 0.001. C Proportion of microglia, neurons and astrocytes from infected cells

Fig. 7. Local antiviral role of IRF7 upon LGTV infection in the brain. WT, and Irf7−/− mice were infected intracranially with LGTV. A Kaplan–Meier curve of mice infected with 102 FFU (n = 5) or 10 FFU (n = 5). Data are cumulative from two independent experiments. Asterisks indicate statistical significance calculated by Mann–Whitney test, *p < 0.05. B IFN-β and IFN-α determination by quantitative RT-PCR in the brain of mice infected intracranial by 10 FFU with LGTV. Asterisks indicate statistical significance calculated by Mann–Whitney test (A) or Student’s t test (B), *p < 0.05, **p < 0.01. Determination of viral titers (B) and IFN-I production (C) from primary hippocampal neuron or whole-brain astrocytes cultures. D Primary astrocytes were transfected with increasing amounts of poly(I:C). Supernatants were harvested 24 h post-transfection and IFN-I was determined by a bioassay. E Survival analysis of LGTV-infected Ifnβ−/∆luc and Irf7−/− Ifnβ−/∆luc mice. Asterisks indicate statistical significance calculated by Mantel–Cox test (A, E) and 2-way ANOVA (B–D), *p < 0.05, ***p<0.001. Results are representative for at least two independent experiments

Fig. 8. Cell type-specific antiviral role of IRF7 upon LGTV infection in the brain. Determination of viral titers (A) and IFN-I production (B) from primary hippocampal neuron or whole-brain astrocytes cultures. C Primary astrocytes were transfected with increasing amounts of poly(I:C). Supernatants were harvested 24 h post-transfection and IFN-I was determined by a bioassay. Asterisks indicate statistical significance calculated by Student’s t test, ***p<0.001. Results are representative for at least two independent experiments

Fig. 9. IFNβ protects IRF7-deficient mice in LGTV infection. Ifnβ−/∆luc and Irf7−/− Ifnβ−/∆luc mice were intraperitoneally infected with 104 FFU of LGTV. A Kaplan–Meier curve. B Body weight during course of infection. Asterisks indicate statistical significance calculated by Mann–Whitney test, *p<0.05. Results are representative for at least two independent experiments