Fig 1.
The IL-33 pathway promotes survival upon neuroinvasive flavivirus infection, independent of viral control.
(A) Survival analysis in Il33–/–, Il1rl1–/–, and B6/J mice following intracranial infection with 2e4 PFU WNV-E218A. (B) WNV-E218A Viral titers from indicated CNS components and tissues of mice 7 days post-infection via plaque assay on BHK cells. (C) Survival analysis in Il33–/–, Il1rl1–/–, and B6/J mice following intracranial infection of 1e6 PFU ZIKV-Dakar. (D) ZIKV-Dakar Viral titers from indicated CNS components and tissues of mice 7 days post-infection via plaque assay on Vero cells. (E) WNV-E218A Viral titers from indicated CNS components and tissues of mice 4 days post-infection via plaque assay on BHK cells. (F) WNV-E218A Viral titers from indicated CNS components and tissues of mice 14 days post-infection via plaque assay on BHK cells. Data are representative of 3 pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (A, C) (Gehan-Breslow-Wilcoxon test) (B, D, E) (2-way ANOVA with Holm-Sidak Multiple comparisons).
Fig 2.
Oligodendrocyte-derived IL-33 promotes survival to neurotropic flavivirus infection.
(A) Ratios of IL-33+ cells in indicated brain regions in adult B6/J mice with HBSS injection. (B) Representative immunofluorescent images of IL-33+ cell loss in B6/J Hippocampal CA3 7 days post intracranial infection with WNV-E218A (20x magnification, scale bars, 100 microns). Arrowheads indicate IL-33+Olig2+ and arrows IL-33+GFAP+ cells. (C) Quantification of IL-33 positivity in Olig2+ and GFAP+ in B6/J Hippocampal CA3 7 days post intracranial infection with WNV-E218A. (D) Representative images of MBP+IL-33+ cell loss in hippocampal CA3 10 days post intracranial infection with WNV-E218A (20x magnification, scale bars, 100 microns). (E) Quantification of MBP+IL-33+ cell number and MPB mean fluorescent intensity in Hippocampal CA3 10 days post intracranial infection with WNV-E218A. (F) Survival analysis of mice with oligodendrocyte-specific deletion of IL-33 following intracranial infection of 2e4 PFU WNV-E218A. (G) WNV-E218A Viral titers from indicated tissues of mice 7 days post-infection via plaque assay on BHK cells. (A-E) Data are representative of two independent experiments. (F) Data are representative of 3 pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. (C and E) two-tailed student’s T test (F) (Gehan-Breslow-Wilcoxon test).
Fig 3.
IL-33/ST2 signaling on adaptive immune cells does not impact host survival following intracranial flavivirus infection.
(A) WNV-E218A Viral titers from DCLNs of mice 4- and 14-days post-infection via plaque assay on BHK cells. (B) Cell number and CD44/CD69 gMFI of indicated adaptive immune cell types per ½ brain by FACs 7 days post intracranial infection with WNV-E218A. (C) Cell number and CD44/CD69 gMFI of indicated adaptive immune cell types per ½ brain by FACs 7 days post intracranial infection with ZIKV-Dakar. (D) Cell number and CD44/CD69 gMFI of indicated adaptive immune cell types per ½ brain by FACs 14 days post intracranial infection with WNV-E218A. (E) Percent ST2+ and ST2 gMFI of indicated immune cell types 7 days post vehicle injection. (F) Percent ST2+ and ST2 gMFI of immune cells 7 days post intracranial infection with WNV-E218A. (G) Survival analysis of mice with Treg deletion of ST2 following intracranial ZIKV-Dakar infection. Data are representative of 2 pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (A, B) (2-way ANOVA with Holm-Sidak Multiple comparisons) (C, D) (One-way ANOVA with Holm-Sidak Multiple comparisons). (E) (Gehan-Breslow-Wilcoxon test). Data are representative of 2 pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. (B, D, G, J, H, L) (2-way ANOVA with Holm-Sidak Multiple comparisons) (C, K) (Gehan-Breslow-Wilcoxon test) (I) (Student’s two-tailed t Test).
Fig 4.
IL-33/ST2 signaling is necessary for microglial activation and host defense following CNS flavivirus infection.
(A) Representative immunofluorescent images and quantification of microglial TMEM119 loss in hippocampal CA3 post WNV-E218A infection (20x magnification, scale bars, 100 microns). (B) Representative images and (C) Quantification of Iba1 MFI and Iba1+phospho-p38+CD68+ brain macrophage number post intracranial WNV-E218A infection. (D) Representative immunofluorescent images of Iba1+ brain macrophages in B6/J and Il1rl1–/–mice 7 days post-infection with WNV-E218A (20x magnification, scale bars, 100 microns). (E) Quantification of Iba1 by MFI in indicated brain regions of B6/J and Il1rl1–/–mice 7 days post-infection with WNV-E218A. (F) Survival analysis of mice with microglial-specific deletion of ST2 following intracranial infection with WNV-E218A. Data are representative of 2 and 3 (E) pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. (B, D) (2-way ANOVA with Holm-Sidak Multiple comparisons and Pearson’s Correlation) (E) (Gehan-Breslow-Wilcoxon test).
Fig 5.
Il33–/–hippocampal macrophages exhibit enhanced inflammatory and cell stress responses acutely during CNS WNV infection.
(A) Scheme of hippocampal macrophage isolation and RNAsequencing following intracranial WNV-E218A infection of B6/J and Il33–/–mice. Created using Biorender.com and exported via an academic subscription belonging to AO. (B) Principal Component Analysis of RNAseq samples. (C) Volcano plot showing differential gene expression in Il33–/–vs. B6/J hippocampal macrophages post-infection with WNV-E218A. (D) Top 20 Hallmark gene sets enriched in Il33–/–relative to B6/J hippocampal macrophages post-infection with WNV-E218A. (E) Microglial homeostatic signature genes in hippocampal microglia isolated post-vehicle and infection with WNV-E218A. ** p < 0.01 (E) 2-way ANOVA with Holm-Sidak Multiple comparisons.
Fig 6.
Disruption of IL-33 signaling during CNS WNV infection induces brain macrophage apoptosis and neuronal pathology.
(A) %ZombieNIR+ dying cell populations of indicated type by FACs analysis of brain homogenates 7 days post-infection with WNV-E218A in B6/J and Il1rl1–/–mice. (B) Representative images of CC3+ CA3 hippocampal brain macrophages 7 days post-infection with WNV-E218A in B6/J and Il33–/–mice (20x magnification, scale bars, 100 microns). Arrowheads indicate Iba1-CC3+ while arrows indicate Iba1+CC3+ cells. (C) Quantification of CC3+ CA3 hippocampal brain macrophages. (D) Quantification of monocyte count by FACs analysis of brain homogenates in B6/J and Il1rl1–/–mice 7 days post WNV-E218A infection. (E) Representative images of monocyte engraftment in hippocampal CA3 and cerebellum of B6/J and Il1rl1–/–mice 7 days post WNV-E218A infection (20x magnification, scale bars, 100 microns). Arrowheads indicate Iba1-CD68+ cells. (F) Quantification of CD68+Iba1- monocytes in hippocampal CA3 and cerebellum. (G) Representative images of pCJun+ cells and IgG deposition in hippocampal CA3 7 days post-infection with WNV-E218A in B6/J and Il33–/–mice (20x magnification, scale bars, 100 microns). Arrowheads indicated pCJun+ cells. (H) Quantification of pCJun+ cells and IgG MFI. (I) Representative images of pCJun+ projections and GFAP+ cells hippocampal CA3 7 days post-infection with WNV-E218A in B6/J and Il33–/–mice (20x magnification, scale bars, 100 microns). (J) Quantification of GFAP+ area and pCJun+ projection area. (K) Representative images of CC3+ neurons in hippocampal CA3 and cerebellum of B6/J and Il1rl1–/–mice 7 days post WNV-E218A infection (20x magnification, scale bars, 100 microns). Arrowheads indicate NeuN+CC3+ cells. (L) Quantification of CC3+ neuron number in CA3 and cerebellum. Data are representative of 2 (A-F) independent and 2 (K-L) pooled independent experiments (error bars, SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. (A, C, H) (two-tailed T test) (E, G, J, L) (Two-way ANOVA with Holm-Sidak multiple comparisons).