(A–C) Coronal sections of P6 brain with leptomeninges, 1 day after a subcutaneous injection of 1.2×105 RFP-expressing E. coli K1. Regions within the rectangles labeled (B) and (C) are enlarged below. The Tie2-GFP transgene is expressed in ECs. (D) Leptomeninges flatmount showing scattered E. coli (RFP; false colored magenta). (E) Leptomeningeal macrophages, visualized with nuclear immunostaining for transcription factor PU.1 and cytoplasmic staining for CD206, show cytoplasmic enlargement upon infection (left), but only a small increase in cell number (right). (F) Infection leads to a selective reduction in LYVE1 immunostaining, and little or no change in CD206 immunostaining, in macrophages in the leptomeninges and dura. (G) Infection leads to little or no change in CD180 and CD206 immunostaining in macrophages in the leptomeninges. (H) IL6 increases in dural fibroblasts in the central sinus. (I) Quantification of images in F-H, in arbitrary units. Each point in this and other quantifications of immunofluorescent data represents the analysis of a single Z-stacked confocal image that encompasses the full depth of the tissue (leptomeninges or dura), unless noted otherwise. (J and K) Increase in cells immunostained for S100A8 in the leptomeninges and dura of infected mice. All infected tissue and data in this and other figures are from P6 mice that had been infected 22 hr earlier. Scale bars: A and A’, 500 µm; B-I, 100 µm. In this and all other figures showing quantification: (1) unless stated otherwise, each symbol in the immunofluorescent quantifications represent a single confocal image, (2) the bars show mean ± SD; (3) the number of mice used for each sample are listed in Supplementary file 4; (4) the Wilcoxon rank sum test was used to measure statistical significance, except for Figure 5D and G, in which the sample size is too small and the student t-test was used instead; and (5) abbreviations are: n.s., not significant (i.e. p>0.05); *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.