Fig 1.
ASC-dependent inflammasomes contribute to the pathogenesis of HSV-1 encephalitis.
(A) Survival curve of WT (n = 17), NLRP3-/- (n = 14), AIM2-/- (n = 11), and ASC-/- (n = 15) mice infected intracranially with 3x104 PFU HSV-1 KOS. Results are combined from seven inoculations. (B) Whole brain titers of infected WT, NLRP3-/-, and ASC-/- mice at days 1, 3, and 4 post-infection (n = 7–12 per group, 8 inoculations). (C) Whole brain IL-1β and (D) IL-18 from WT, NLRP3-/-, and ASC-/- mice either before infection or at days 1 and 3 post-infection (n = 4–5 per group). Values are expressed as means ± SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 2.
Microglial ASC-dependent inflammasomes are activated during HSE.
Cell-specific caspase-1 activity was analyzed by flow cytometry and FLICA assay at day 3 post-infection following intracranial inoculation of 3 x 106 PFU HSV-1 KOS. (A) Representative flow cytometry histograms of CD45midCD11b+ microglia from mock-infected or HSV-1-infected WT and ASC-/- mice stained with FLICA reagent to quantify active caspase-1. Percentages of each cell type that are FLICA+ (B) or FAM-FLICA mean fluorescence intensity (C) in astrocytes, microglia, and CD11b+ infiltrating leukocytes (n = 3–4 mice per group). After excluding multiplets and dead cells, cells were gated using the following strategy: astrocytes CD45lo/ACSA-2+, microglia CD45mid/CD11b+, infiltrating leukocytes CD45hi/CD11b+. Values are expressed as means ± SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001). Results are representative of two independent experiments.
Fig 3.
IL-1β production is microglia-dependent in an organotypic brain slice culture model of HSV encephalitis.
Microglia were depleted from organotypic brain slice cultures by incubation with clodronate-filled liposomes and IL-1β production was quantified after HSV-1 infection. (A) Diagram of experimental set-up. Slices were allowed to settle for 24 hours in culture before incubation with either clodronate-filled or empty liposomes. Slices were then infected with 106 PFU HSV-1 KOS and culture media was collected at 48 hour post-infection. (B) Microglia quantification by flow cytometry after 6 days of clodronate or empty liposome treatment (n = 4 slices per group). (C) Protein levels of IL-1β as quantified by ELISA (n = 3 slices per group). Values are expressed as means ± SEM (*P < 0.05, **P < 0.01).
Fig 4.
Glial inflammasome activation drives expression of the monocyte chemokine CCL6 during HSV-1 infection.
Glial co-cultures were infected at an MOI of 5 and cellular RNA was isolated at 12 hours post-infection. Relative gene expression of (A) Ccl6 and (B) Il1rn in glial co-cultures (n = 3–4 per group). (C) CCL6 protein levels from the brains of either uninfected or infected mice at days 1 and 3 post-infection (n = 4–5 per group). Values for gene expression are expressed as means ± SD and values for CCL6 levels are expressed as means ± SEM (*P < 0.05, **P < 0.01).
Fig 5.
ASC-dependent inflammasomes promote leukocyte migration into the CNS during HSV-1 encephalitis.
Infiltrating leukocytes in the brain were analyzed by flow cytometry in WT, NLRP3-/-, and ASC-/- mice at day 3 post-infection after intracranial inoculation with 3 x 106 PFU HSV-1 KOS. Total CD45hi cells (A), dendritic cells (B), neutrophils (C), CD4+ and CD8+ T cells (D). Representative FACS plots of CD11c and CD11b expression within CD45hi and Ly6G- cells. Macrophages are gated as CD11b+ and CD11c- (E). Absolute numbers of macrophages (F) calculated from numbers of total cells at day 3 post-infection (n = 5, 3 replicates for myeloid cells; n = 3–4, 2 replicates for T cells). After excluding multiplets, cell types were gated within the CD45hi population using the following gating strategy: macrophages/monocytes Ly6G-/CD11c-/CD11b+, dendritic cells Ly6G-/CD11c+, neutrophils Ly6G+/CD11b+/CD11c-, T cells CD3+/CD4+ or CD3+/CD8+. No significant decrease was observed between WT and ASC-/- mice for total CD45hi, DCs, neutrophils, or T cells. Values are expressed as means ± SEM (**P < 0.01).