Table 1.
Antibodies used for flow cytometry analyses.
Fig 1.
Murine NCC induces a strong eosinophil response in the brain.
Immuno-fluorescent staining shows SiglecF+ cells in brain sections of mock-infected (A, B) and 2 wks p.i (C, D) in WT (A, C) and ΔdblGATA mice (B, D); red staining. Cytocentrifuged brain infiltrates confirm strong eosinophil responses in WT mice (E, F) and absent in 2 wk-infected ΔdblGATA mice (G).
Fig 2.
Kinetics of eosinophil response during NCC.
Representative flow cytometry analysis of brain infiltrates shows gating strategy based on SSC-A/FSC-A (A), single cells (B) and CD11b+ myeloid cells (C). Myeloid cells were further analyzed based on SiglecF and Ly6G expression (D) to differentiate eosinophils from neutrophils. Eosinophils, gated as CD11b+SiglecF+ cells, were abundant in WT brains (E) and diminished in ΔdblGATA mice (F). Total cellularity was compared between infected WT and ΔdblGATA mice (G) and flow cytometry analysis shows that eosinophils accounted for about 80% of the myeloid population in WT mice (H) by 3 wks p.i and absent in ΔdblGATA mice (I).
Fig 3.
Increased frequency of mast cells / basophils and neutrophils in eosinophil deficient mice.
Brain infiltrates from WT (A) and ΔdblGATA mice (B) mice at 2 wks p.i were cytospun followed by Diff Quick staining to identify and quantify mast cells/basophils and IF staining using anti-Ly6G for neutrophils (C, F). Brain tissues were stained with anti-Ly6G antibodies (D, E; red staining). Representative images of flow cytometry analyses gating singlets for Ly6G and CD11b (G, H) confirms the increased percentage of neutrophils in ΔdblGATA mice although the overall neutrophil number did not appear different between WT and ΔdblGATA mice 2 wks p.i (I, J).
Fig 4.
Decreased expression of myeloid specific Arg1 in ΔdblGATA mice.
Brain infiltrates were analyzed by flow cytometry in WT (A-F) and ΔdblGATA mice (G-L). Gating strategy based on side and forward scatter (A, G), followed by singlets discrimination (B, H), then gating on hematogenous derived infiltrating cells defined as CD45hi (C,I), and myeloid cells CD11b+ (D, J), were further analyzed to gate eosinophils, and neutrophils based on SiglecF and Ly6G expression (E, K) and the negative population (Gate 1), was analyzed for Ly6C expression to identify macrophages (F, L). Total Leukocyte infiltration (M), macrophages (N), and Arg1 levels (O) were compared between WT and ΔdblGATA mice. Arg1 expression was confirmed in brain infiltrates subjected to Imaging Flow Cytometry in WT (P, left panel) and ΔdblGATA mice (P, right panel), Arg1 MFI for the total CD45hiCD11b+ myeloid population is shown in (Q).
Fig 5.
Increased T cell infiltration in ΔdblGATA mice.
Brain tissues from WT (A) and ΔdblGATA mice (B) at 2 wks p.i were stained with anti-TCRβ antibodies. Percent of total infiltrates (C) and total cell numbers (D) assessment of αβ T cells based on IF staining of cytospun brain infiltrates revealed an increased T cell infiltration in ΔdblGATA mice brains. Flow cytometry analysis of brain infiltrates of WT (E) and ΔdblGATA mice (F) also revealed an increased frequency and overall numbers of T cells (G, H) in eosinophil-deficient mice. Further assessment of CD4 and CD8 subpopulation (I, J) by flow cytometry showed an increased frequency and number of CD8 T cell in ΔdblGATA mice (filled bars) when compared to WT mice (open bars).
Fig 6.
Eosinophil-deficient mice have reduced Th2 response during murine NCC.
Immunofluorescent staining for IL-4 shows negative signals in mock-infected WT (A) and ΔdblGATA brains (B). However, IL-4 is upregulated after infection and abundant in inflammatory infiltrates of WT mice (C) and to a lesser extent in ΔdblGATA mice (D, E). Total RNA isolated from enriched brain T cells was subjected to reverse transcription and transcript levels of IL-4 and Gata3 assessed by qRT-PCR and results shown as fold change over values from ΔdblGATA mice (F) revealed an enrichment of both transcripts in WT T cells. CD4 T cells were isolated from infected brains and incubated with PMA/Ionomycin and Brefeldin A/Monensin followed by intracellular IL-4 staining (G, H). The proportion of CD4+IL-4+ T cells (I) was increased in T cells isolated from infected WT mice.
Fig 7.
Eosinophil-deficient mice are less vulnerable to infection.
Total parasites in brain tissues were counted in HE stained sections from WT (A) and ΔdblGATA mice (B), and parasite numbers compared between groups at 2wks (C) and 4 wks pi (D). Overall assessment of cell death was assessed by TUNEL staining in mock infected WT (E) and ΔdblGATA mice (F) brain tissues, and 4 wks p.i infected WT (G) and ΔdblGATA mice (H) brain tissues. TUNEL+ cells were more abundant in WT tissues (I). Disease susceptibility to infection in both WT and ΔdblGATA mice is shown in (J).