For single-nucleus RNA sequencing, leptomeninges were collected as previously described (Wang et al., 2023). In brief, P6 mice were euthanized 24 hours after infection, brains were rapidly removed, and the leptomeninges were dissected into ice-cold Hibernate-A medium. The leptomeninges were frozen at -80 °C. Single nucleus (sn)RNA-seq libraries were preparedusing the PIPseq T20 3' Single Cell RNA Kit v4.0 PLUS (Fluent BioSiences; Clark et al., 2023). Frozen tissue was suspended in homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH,pH 7.8) supplemented with 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, EDTA free protease inhibitor (Roche 11836 170 001), 0.5% IGEPAL-630, and 40 U/mL Protector RNase Inhibitor (Sigma, 03335402001). Samples were homogenized in a 2 mL Dounce homogenizer,  sing 15 strokes with a loose-fitting pestle followed by 30 strokes with a tight-fitting pestle. The sample was filtered through a 10 μm filter (CellTrix, Sysmex, 04-004-2324), and nuclei were pelleted in Low Retention 1.5 mL microcentrifuge tubes (Thermo Scientific, 3451) for 5 min at 500 x g at 4 °C. Nuclei were washed twice with Nuclei Suspension Buffer (supplied in the Fluent BioSciences kit) supplemented with 1% BSA and 40 U/mL Protector RNase Inhibitor. Nuclei were counted, and ~40,000 nuclei (for the T20 kit) or ~20,000 nuclei (for the T10 kit) were used for library production following the Fluent BioSiences protocol.  RNA-seq on WT and Tlr4KO bEnd.3 cells: libraries and sequencing bEnd.3 mouse brain endothelial cells were cultured in 10-cm tissue culture dishes and grown to confluency under standard conditions (37 C, 5% CO₂). Total RNA was isolated using the Zymo Research RNA purification kit according to the manufacturer's instructions. Briefly, culture medium was aspirated, cells were rinsed once with ice-cold PBS, and 600 μL of the provided lysis buffer with β-mercaptoethanol was added directly to the dish. The lysate was collected by scraping and transferred to a microcentrifuge tube, briefly mixed, and applied to the Zymo spin column. After binding, the column was washed with the supplied wash buffers, including an on-column DNase I treatment step to remove contaminating genomic DNA, and RNA was eluted in 50 μL of DNase/RNase-free water. RNA-seq library construction was performed with the Illumina mRNA stranded kit following the manufacturer's protocol, and sequencing was performed on an Illumina NovaSeqX