Fig 2: Surviving aged mice show less microglial activation than young adult mice. 16 week and 85 week mice were infected with 100 PFU WNV-NY via footpad inoculation. At 30 dpi, mice that survived the infection were euthanized, and leukocytes were isolated from the cortex and analyzed by flow cytometry. (a) Representative flow cytometry plots of CD45+-gated cells analyzed by CD11b vs CD45 expression. (b) Quantification of number of CD11b+CD45+ cells in each group. (c) Representative flow cytometry plots of CD11b+-gated cells analyzed by CD45 vs P2RY12 expression with gating for P2RY12+ cells. (d) Quantification of number of P2RY12+CD11b+ cells in each group. (e) Representative flow cytometry plots of P2RY12+-gated cells analyzed by P2RY12 vs CD68 expression. (f) Quantification of number of CD68+P2RY12+ cells in each group. (g) Representative flow cytometry plots of P2RY12+-gated cells analyzed by P2RY12 vs MHCII expression. (h) Quantification of number of MHCII+P2RY12+ cells in each group. These data are the representative of two independent experiments with 7–15 mice per group. Statistical significance was calculated using one-way ANOVA with Welch's correction and Dunnett's multiple comparisons test. Exact p values noted; *p < 0.05; **p < 0.01

Fig 3: Uninfected aged mice exhibit myeloid cells that lack typical microglial markers. Adult (16 week) and aged (85 week) mice were infected with 100 PFU WNV-NY via footpad inoculation. At 9 dpi, a time point just prior to when mice begin succumbing to viral infection, leukocytes were isolated from the cortex and analyzed by flow cytometry. (a) Representative flow cytometry plots of CD45+-gated cells analyzed by CD11b vs CD45 expression. (b) Quantification of percent of CD11b+CD45+ cells in 16 week mock, 85 week mock, 16 week infected, and 85 week infected cortex. (c) Representative flow cytometry plots of CD11b+-gated cells analyzed by CD45 vs P2RY12 expression. (d) Quantification of percent CD45hi, P2RY12+CD45lo, and P2RY12negCD45lo cells in each group. (e) Representative flow cytometry plots of P2RY12+CD45lo-gated cells analyzed by P2RY12 vs MHCII expression. (f) Quantification of percent MHCII+ of P2RY12+CD45lo-gated cells in each group. (g) Representative flow cytometry plots of P2RY12negCD45lo-gated cells analyzed by P2RY12 vs MHCII expression. (h) Quantification of percent MHCII+ of P2RY12negCD45lo-gated cells in each group. These data are representative of two experiments with 6–10 mice per group. Statistical significance was calculated using one-way ANOVA with Welch's correction and Dunnett's multiple comparisons test. (i) Representative images of 16 week (top row) and 85 week (bottom row) cortical tissue sections collected from mock- (left) or WNV-infected mice at 9 dpi (middle), or 30 dpi (right). Sections were immunostained with antibodies to Iba1 (green) and CD68 (red), and counterstained with DAPI (blue). Arrowheads point to colocalization of Iba1 and CD68. Asterisks denote regions magnified in inset panels. (j–l) Quantification of (j) percent area covered by Iba1 staining, (k) percent area covered by CD68 staining, and (l) Manders coefficient of colocalization between CD68 and Iba1 and calculated by ImageJ. Statistical significance was calculated by two-way ANOVA with Sidak's multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001