Fig 1: Brain endothelial cells upregulate inflammatory signatures and downregulate BBB marker expression following GAS infections.a, Experimental workflow for single-cell RNA sequencing (scRNAseq) and MERFISH experiments. b-c, UMAP visualization of olfactory bulb (OB) scRNAseq from PBS controls (gray) and GAS-infected mice (blue), with enrichment of CD31+ brain endothelial cells (BECs) and CD11b+ (myeloid cells). d, Quantification of transcriptional shifts across OB cell types between PBS and GAS conditions using L∞ distance (cross-entropy test). e, Gene set enrichment analysis (GSEA) of differentially expressed genes in BECs after GAS infection. Inflammatory and interferon-related pathways are enriched, whereas blood-brain barrier (BBB) gene sets are depleted. Bars show significance of enrichment (−log2FDR) and blue dots indicate the number of significant genes per set. f-i, Heatmaps of interferon response, antigen presentation, endothelial LPS response and extracellular matrix in BECs from PBS and GAS conditions [log(z-score)]. The red colors mean a high z-score and the blue colors mean a low z-score. Significant genes are in balck (adjusted p < 0.05) and non-significant ones are in gray (adjusted p > 0.05). j-m, Downregulation of BBB-associated transcripts (Itm2a, Itih5 and Mfsd2a) in BECs measured by scRNAseq (left; Wilcoxon Rank Sum test, *adjusted p < 0.05). Representative images (center) and quantification (right) of changes in the BBB transcripts by fluorescence in situ RNA hybridization (FISH) for Itm2a, Itih5 mRNAs (red) combined with immunofluorescence for BEC marker Glut1 (j-l; cyan) and immunofluorescence staining for Mfsd2a (red) and BEC marker CD31 (m; red). Scale bars = 25 μm. Comparisons were performed with unpaired t test with Welch’s correction (* p < 0.05; n = 7-9 per condition). Data are mean +/− SEM.