Fig. 4

A 16-fold reduction in initial viral inoculum results in 80% attenuation of the brain-infiltrating inflammatory monocyte response but maintenance of microglial activation. Mice were inoculated with 200,000 PFU or 12,500 PFU of TMEV and processed for BILs isolation (A–E, L–Q) or immunostaining (F–K) at 24 hpi. (A) Schematic of the two different cell isolation protocols employed in the study. Cells shown in B and analyzed in D, E were prepared with homogenization and a 1.100 g/mL Percoll gradient; cells shown in C were prepared with enzymatic digestion and a 30%:70% gradient. B BILs isolated at 24 hpi from mice inoculated with 200,000 PFU of TMEV are enriched in CD45^hiCD11b⁺⁺Gr1⁺1A8⁻ inflammatory monocytes (IM) and CD45^hiCD11b⁺⁺Gr1⁺1A8⁺ neutrophils (N) as compared to vehicle-inoculated mice. Both populations are substantially reduced in mice inoculated with 12,500 PFU of TMEV. CD45^midCD11b⁺Gr1⁻1A8⁻ microglia (Mi) isolated by the gradient are increased in both 200,000 PFU and 12,500 PFU inoculations relative to vehicle control mice. (C) The same pattern is observed using the alternative cell isolation method. D Inflammatory monocytes: F_(2,26) = 121.5718, P < 0.0001 by one-way ANOVA; Shapiro–Wilk P < 0.0001; Dunn's method pairwise analysis: 200,000 PFU vs 12,500 PFU: P = 0.0019; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P = 0.2616; d_s = 2.9; results from 4 separate experiments. E Microglia: F_(2,26) = 94.6556, P < 0.0001 by one-way ANOVA; Shapiro–Wilk P < 0.0001; Dunn's method pairwise analysis: 200,000 PFU vs 12,500 PFU: P = 0.8054; 200,000 PFU vs vehicle: P = 0.0009; 12,500 PFU vs vehicle: P = 0.0234; d_s = 2.6; results from 4 separate experiments. ** in panel (D) indicates P < 0.01; NS = not significant. F Schematic showing the location of immunostaining analysis in G–I (red box) and (J, K) (blue box). Iba-1⁺ activated microglia are shown in single channel (G–I) and in red with DAPI-labeled nuclei (blue) (J, K). Inoculation with 200,000 PFU of TMEV induced a large upregulation in Iba-1 expression in the stratum radiatum of CA1 (H) as compared to baseline levels in vehicle-inoculated mice (G). A similar increase in microglial activation is observed in mice inoculated with 12,500 PFU of TMEV (I). Analysis of LysGFP mice revealed strong Iba1⁺ microglia and GFP⁺ inflammatory monocytes in mice inoculated with 200,000 PFU TMEV (J) and equivalent Iba1⁺ microglia but absent GFP⁺ cells in mice inoculated with 12,500 PFU (K). Scale bar in I is 50 μm and refers to G–I. L Bone marrow chimerics were generated by reconstituting irradiated C57BL/6 recipient mice with bone marrow from LysGFP donor mice (B6:LysGFP) or LysGFP recipient mice with bone marrow from C57BL/6 donor mice (LysGFP:B6). B6:LysGFP mice exhibited GFP⁺ brain-infiltrating cell levels comparable to non-chimeric LysGFP mice, while LysGFP:B6 mice showed almost no GFP⁺ infiltrate. M Gating CD45⁺ cells in the cellular suspension from LysGFP mice indicated that GFP^negCD45⁺ cells (blue box and histogram) were Ly6C/G negative while GFP⁺CD45⁺ cells (red box and histogram) were Ly6C/G bright. N The specific association of Ly6C/G with inflammatory monocytes, not microglia, was confirmed by applying the same gates to the cellular suspension from B6 recipients receiving bone marrow from LysGFP donors. O CD45^mid cells are GFP^neg and TMEM119⁺ while CD45^hi cells are GFP⁺ and TMEM119^neg. P CD45^midLy6C/G^neg microglia exhibited increased levels of the CD44 and CD86 activation markers in mice inoculated with either 200,000 PFU or 12,500 PFU TMEV. CD11c levels are unchanged on this population. Q Two-dimensional expression analysis in CD45^midLy6C/G^neg microglia: expression level of first label shown along y-axis; expression level of second label shown by color coding; each dot represents a single cell. CD44^hiCD86^hi microglia are equivalently increased in mice inoculated with either 200,000 or 12,500 PFU TMEV; the CD44 brightest cells were CD11c low or negative in both groups. Data are from 3 mice per condition. P values were calculated using the Kolmogorov–Smirnov test comparing the cumulative frequency distributions