Cell-type-specific transcriptional responses of leptomeninges from WT, Tlr4VEKO, and Tlr4MKO mice to neonatal E. coli infection.

(A) Schematic of the leptomeninges (pia, subarachnoid space, and arachnoid barrier) and adjacent structures. (B) Immunostaining of P6 mouse leptomeninges shown as a whole mount (left) or as a transverse section through a series of Z-planes (right). The vertical black bars indicate the extent of the leptomeninges. Scale bars, 20 µm. (C) UMAP plot of leptomeninges snRNA-seq from WT, Tlr4VEKO, and Tlr4MKO mice at P6, from uninfected and E. coli-infected mice, with cell clusters identified. N=126,235 nuclei. Fb, fibroblast. (D) UMAP comparison of P6 leptomeningeal snRNA-seq from control vs. 24-hour E. coli-infected mice of the indicated genotypes. (E) snRNA-seq from four leptomeninges cell clusters (Fibroblast-arachnoid, Dural border cells, Fibroblast-pia, and Arachnoid barrier cells) showing differentially expressed genes in control vs. E. coli-infected mice of the indicated genotypes at P6. For each cell cluster, DEGs were chosen based on a comparison of infected vs. uninfected WT cells. (F and G) As in panel (E) except for ECs (F) and myeloid cells (G). DEGs, differentially expressed genes. (H) As in panel (F) and (G) except DEGs were related to NF-κB and TNF-α signaling. (I) Summary of transcriptional responses of leptomeninges from WT, Tlr4VEKO, and Tlr4MKO mice to E. coli infection.

Non-myeloid TLR4 signaling drives ICAM-1 induction, increased vascular permeability, and myeloid cell activation during neonatal E. coli infection.

(A) Whole mount leptomeninges from P6 WT, Tlr4VEKO, and Tlr4MKO mice, under control or E. coli-infected conditions, immunostained for Cldn5 and ICAM-1 (upper panels) or immunostained for Cldn5 and incubated with fluorescent streptavidin (lower panels) to visualize the fate of intravascular sulfo-NHS-biotin. Scale bars, 20 µm. (B) Quantification of ICAM1 immunostaining relative to WT controls, as shown in (A). (C) Quantification of extravascular streptavidin binding relative to WT controls, as shown in (A). (D) Whole mount leptomeninges from P6 WT and Tlr4VEKO mice, control or E. coli-infected, immunostained for Cldn5 and either ASC or CD206 to visualize myeloid cells. Scale bars, 20 µm. (E) Quantification of ASC+ area (upper) and CD206+ area (lower), as shown in (D). Box plots show the median, interquartile range, and all individual data points. Each data point represents one image from a single mouse, with two locations imaged per mouse. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant (p > 0.05)

Claudin-5 redistribution in response to E.coli in leptomeningeal endothelial cells and in bEnd.3 cells.

(A) Whole mount leptomeninges from P6 WT, Tlr4VEKO, and Tlr4MKO mice, control or E. coli-infected, immunostained for Cldn5. Scale bars, 20 µm. (B) Quantification of Cldn5+ area in the leptomeninges, as shown in (A). (C) Control bEnd.3 cells and bEnd.3 cells exposed to live E. coli or live group B Streptococcus (GBS) for 1–6 hours were immunostained for Cldn5 and NF-κB. Scale bars, 20 µm. (D) Control bEnd.3 cells and bEnd.3 cells exposed to heat-killed E. coli or heat-killed group B Streptococcus (GBS) for 1–6 hours were immunostained for Cldn5 and NF-κB. Scale bars, 20 µm. (E) Quantification of experiments shown in (C) and (D). First plot, quantification of log10-transformed cytoplasmic-to-plasma membrane (Cyto/PM) intensity ratio for Cldn5 in bEnd.3 cells exposed to live E. coli for the indicated time in hours. Second plot, quantification of log10-transformed nuclear-to-cytoplasmic (Nuc/Cyto) intensity ratio for NF-κB in bEnd.3 cells exposed to live GBS for the indicated time in hours. The third and fourth plots are analogous to the first and second plots, except that bEnd.3 cells were exposed to heat-killed (HK) E. coli or GBS. Box plot in (B) shows median, interquartile range, and all individual data points. Each in vivo data point represents one image from a single mouse, with two locations imaged per mouse. For plots in (E), each data point represents a single bEnd.3 cell analyzed from representative images in three biological replicates. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant (p > 0.05).

TLR4 signaling controls NF-κB activation, tight junction dynamics, and barrier integrity in bEnd.3 cells exposed to E. coli.

(A) WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 1 hour or 4 hours, were fixed and immunostained for Cldn5 and NF-κB and stained with DAPI. Scale bars, 20 µm. (B) WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 1 hour or 4 hours, were fixed and immunostained for Cldn5 and ZO-1 and stained with CellTrace. Scale bars: 20 µm. (C) Quantification of log10-transformed nuclear-to-cytoplasmic (Nuc/Cyto) NF-κB intensity ratio in WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli, as shown in (A). (D) Quantification of log10-transformed cytoplasmic-to-plasma membrane (Cyto/PM) Cldn5 intensity ratio in WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli, using ZO-1 localization to define the plasma membrane region, as shown in (B). (E) WT and Tlr4KO bEnd.3 cells were grown to confluence on biotinylated gelatin-coated coverslips, with or without E. coli exposure for 4 hours, were then incubated with fluorescent streptavidin, and were finally fixed and immunostained for ZO-1. Representative images are shown with merged (upper) and streptavidin-only (lower) channels. Scale bars, 20 µm. (F) Quantification of streptavidin+ area in WT and Tlr4KO bEnd.3 cells with or without exposure to E. coli, as shown in (E). Wells coated with gelatin alone or biotinylated gelatin (without cultured cells) served as negative and positive controls, respectively. Box plots show median, interquartile range, and all individual data points. Each data point represents a single cell (C and D) or one image field (F) from representative images in three biological replicates. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant (p > 0.05)

Comparisons of Cldn5 localization with junctional, plasma membrane, and trafficking markers in WT and Tlr4KO bEnd.3 cells with or without E. coli exposure.

(A-C) WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 4 hours, were fixed and immunostained for Cldn5 and the indicated markers. Scale bars, 20 µm. (D) Quantification of overlap between Cldn5 and β-catenin, GLUT1, PECAM1, and ZO-1, with each data point representing a 100 µm x 100 µm region of interest (ROI). (E) WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 4 hours, were fixed and immunostained for Cldn5 and the indicated markers. Scale bars, 20 µm. (F) Quantification of overlap between Cldn5 and EEA1, LAMP2, and Rab7, as in (D). (G) WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 4 hours, were fixed and immunostained for Cldn5 and the indicated markers. Scale bars, 20 µm. (H) Quantification of overlap between Cldn5 and Rab11, PDI, and RCAS1, as in (D). Box plots show median, interquartile range, and all individual data points. Each data point represents one image from representative images in three biological replicates. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant (p > 0.05)

In bEnd.3 cells, pharmacologic inhibition of NF-κB nuclear translocation and signaling does not impede rapid internalization of Cldn5 in response to E. coli exposure.

(A) bEnd.3 cells were pre-incubated for 30 minutes with the indicated fluorescent tracer, then either not exposed to E. coli (control) or exposed to E. coli for 4 hours and then fixed and immunostained for Cldn5. Scale bars, 20 µm. (B) Quantification of % overlap between tracer and Cldn5 from the experiment shown in (B). (C) Cldn5 internalization and recovery during 1 hour of E. coli exposure, followed by washout of E. coli, and then an additional 3 hours of incubation in medium without bacteria. Scale bars, 50 µm. (D) Quantification of the experiment shown in (D). The metric on the y-axis is (cytoplasmic signal – plasma membrane signal)/(cytoplasmic signal + plasma membrane signal). 100% cytoplasmic localization corresponds to 1.0. 100% plasma membrane localization corresponds to -1.0. 50% cytoplasmic localization and 50% plasma membrane localization corresponds to 0.0. (E) The subcellular localization of Lamp2 and Cldn5 were determined in bEnd3 cells in the presence or absence of 1 µM of the IKK inhibitor ACHP before E. coli exposure or after 1 hour of E. coli exposure. Scale bars, 20 µm. (F) Quantification of the experiment shown in (A). Each symbol represents a 100 µm x 100 µm region of interest (ROI). (G) NF-κB translocation from cytoplasm to nucleus was visualized in the presence or absence of 1 µM of ACHP before E. coli exposure or after 1 hour of E. coli exposure. Scale bars, 20 µm. (H) Quantification of the experiment shown in (C). Each symbol represents an individual cell. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant (p > 0.05).

In bEnd.3 cells, TLR4 signaling plays a central role in the transcriptional response to E. coli exposure.

(A) Volcano plot of differential transcript abundances in WT bEnd.3 cells, with or without exposure to E. coli for 3 hours. The plot shows transcript-level log2 fold change (LFC) on the horizontal axis versus −log10(false discovery rate; FDR) on the vertical axis. Red points mark significantly changed transcripts (FDR < 0.05 and |LFC| ≥ 1); grey points are not significant. (B) Volcano plot of differential transcript abundances in Tlr4KO bEnd.3 cells, with or without exposure to E. coli for 3 hours, plotted as in (A). Note the change of scale of the vertical and horizontal axes between (A) and (B). (C) Pathway effect map from ssGSEA Hallmark scores. Each symbol is a pathway. The horizontal axis is the effect of E. coli exposure for 3 hours on WT bEnd.3 cells (ΔssGSEA = E. coli exposed – not exposed) and the vertical axis is the effect of E. coli exposure for 3 hours on Tlr4KO bEnd.3 cells. Symbol size encodes the within-genotype significance [max(−log10 FDR) across both genotypes], and the color code indicates the genotype x E. coli exposure interaction FDR. The 45-degree diagonal line represents equal effects for the two genotypes. (D–F) ssGSEA pathway activity based on genotype and E. coli exposure for three Hallmark immune response pathways: TNF-α signaling via NF-κB (D), Inflammatory response (E), and IL6–JAK–STAT signaling (F). Symbols represent ssGSEA scores for individual samples. The vertical axis shows the interaction FDR score from a linear model for genotype x E. coli exposure. (G) Transcript abundance changes for 12 immune system genes in WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli for 3 hours. (H) Heatmap of transcript abundance changes in WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli for 3 hours, for TNF-α signaling via NF-κB. Rows correspond to transcripts with the greatest changes; values are variance-stabilized transformation (VST) z-scores derived from DESeq2-normalized counts. Columns are arranged by genotype and condition. (I) Scatterplot showing transcript abundance changes in WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli for 3 hours for TNF-α signaling via the NF-κB pathway. Each symbol represents one gene. Red points indicate genes significant for either genotype (FDR < 0.05). The top 15 genes by effect size are labeled. Note the different scales for the horizontal and vertical axes. The diagonal line represents equal effects for the two genotypes.

Specificity of Cdh5-CreER in leptomeninges.

(A and B) Cdh5Cre-mediated recombination of the loxP-stop-loxP (LSL) reporters Rosa26-LSL-SUN1-sfGFP (A) and Rosa26-LSL-tdT-2A-nlsGFP (B). Immunostaining for markers ERG, CD206, and PU.1 was performed on coronal sections of P6 brains following 4HT administration at P2. Nuclear-localized GFP is observed in nearly all ECs, as visualized with ERG immunostaining, but not in myeloid cells, as visualized with PU.1 and CD206 immunostaining. The region corresponding to the leptomeninges is labeled on the right. Scale bars, 100 µm. (C) WT uninfected leptomeninges UMAPs showing the expression of the genes encoding the markers used in (A) and (B): Erg (ECs), Mrc1 (CD206; myeloid cells); Spi1 (PU.1; myeloid cells). (D) Quantification of the co-localization of nuclear GFP from Rosa26-LSL-tdT-2A-nlsGFP with cell-type-specific markers in the leptomeninges. The left set of three data points show the percent of marker+ cells that express GFP. The right set of three data points show the percent of GFP+ cells that express each of the three cell-type specific markers. Approximately 75% of GFP+ cells are ECs and approximately 0% are myeloid cells. Therefore, the remaining 25% of GFP+ cells correspond to some combination of other leptomeningeal cell types. Each data point represents one mouse. A mean of 158 cells was scored per datapoint.

Specificity of Lyz2Cre in leptomeninges.

(A) Lyz2Cre-mediated recombination of the loxP-stop-loxP (LSL) reporter Rosa26-LSL-tdT-2A-nlsGFP, with the tdTomato reporter (false-colored green) shown in the left set of nine panels and the nuclear-localized GFP reporter (green) shown in the right set of nine panels. Immunostaining for markers CD206, PU.1, and ASC was performed on P6 leptomeninges+cortex whole mounts, as described for Figure 1B. The leptomeninges region is shown. Scale bar, 200 µm. (B) WT uninfected leptomeninges UMAPs showing the expression of the genes encoding the markers used in (A): Mrc1 (CD206; myeloid cells), Pycard (ASC; myeloid plus arachnoid barrier cells), and Spi1 (PU.1; myeloid cells). (C) Quantification of co-localization data, as shown in (A). Each data point represents one mouse. A mean of 136 cells was scored per datapoint. The Cre reporters, tdTomato and GFP, are expressed in 90-100% of CD206+ myeloid cells, in 40-70% of ASC+ cells, and in 50-80% of PU.1+ cells.

snRNA-seq cell cluster assignments and analysis of differentially expressed genes related to NK-κB and TNF-α responses.

(A) UMAP plot of leptomeninges snRNA-seq data from WT, Tlr4VEKO, and Tlr4MKO mice at P6, either uninfected control or with E. coli infection, with cell clusters identified. N=126,235 nuclei. Fb, fibroblast. (B) snRNA-seq transcript abundances among the six major leptomeninges cell clusters for a set of 33 genes, for which the transcript abundances distinguish these clusters. We note that the dural border cell cluster was originally referred to as “fibroblasts dura3” in Wang et al. (2023). (C) UMAP plots of leptomeninges snRNA-seq showing the abundances of transcripts that are highly enriched in each of the six major leptomeningeal cell clusters. (D) snRNA-seq from four leptomeningeal cell clusters showing differentially expressed genes related to NK-κB and TNF-α responses in control vs. E. coli-infected mice of the indicated genotypes at P6. The corresponding dot plots for ECs and myeloid cells are shown in Figure 1H.

Comparison of WT P6 leptomeninges snRNA-seq using the PIP-seq and 10X Genomics platforms.

(A) WT P6 leptomeninges snRNA-seq determined with the PIP-seq platform. Expression of 24 genes is illustrated to highlight: Cdh5 (upper left), EC and immune cell genes (left column), and, the four principal cell clusters (right two columns; from left to right within each UMAP these are Dural border cells (DB), Arachnoid barrier cells (AB), Fibroblast arachnoid (Fb-ar.), and Fibroblast-pia (Fb-pia). (B) As for (A), except that the WT P6 leptomeninges snRNA-seq was determined with the 10X Genomics platform. Note that the yields of immune cells and ECs differ between the two datasets.

Principal component analysis of snRNA-seq-derived transcriptomes for each of the six major leptomeningeal cell clusters divided by genotype and infection status.

The two experimental conditions (control vs. infected) and the three genotypes (WT, Tlr4VEKO, and Tlr4MKO) are indicated by the colors and symbols in the upper right. The datapoints represent the same leptomeningeal snRNA-seq datasets analyzed in Figure 1C-H, from control vs. E. coli-infected mice at P6.

Transcriptome changes by cell cluster for the JAK-STAT and IFN-ɣ pathways.

(A and B) snRNA-seq from each of the major leptomeninges cell clusters showing differentially expressed genes that are related to the JAK-STAT pathway (A) or the IFN-ɣ pathway (B). Dot plots are based on the same leptomeningeal snRNA-seq datasets analyzed in Figure 1C-H, from control vs. E. coli-infected mice at P6.

Genes regulated by E. coli infection in leptomeningeal endothelial cells and myeloid cells analyzed at the level of individual mice and individual genes.

(A) Genes regulated by E. coli infection in leptomeningeal ECs. Left panel, most differentially regulated genes based on adjusted p-value. Right two panels, most differentially regulated genes based on fold change. (B) Genes regulated by E. coli infection in leptomeningeal myeloid cells. Left panel, most differentially regulated genes based on adjusted p-value. Right two panels, most differentially regulated genes based on fold change. The data are derived from the same leptomeningeal snRNA-seq datasets analyzed in Figure 1C-H, from control vs. E. coli-infected mice at P6.

Infection responses in the leptomeninges and adjacent cerebral cortex in WT and Tlr4VEKO mice.

(A) UMAP plots of leptomeninges snRNA-seq data from WT and Tlr4VEKO mice at P6, either uninfected control or with E. coli infection, showing Icam1 transcripts. (B) Whole mount cortical surface, with attached leptomeninges, from P6 WT and Tlr4VEKO mice, control or E. coli-infected, immunostained for Cldn5 and ICAM1. Upper panels, stacked Z-planes at the level of the leptomeninges; lower panels, stacked Z-planes at the level of the adjacent cerebral cortex. Scale bars, 20 µm. (C) Quantification of ICAM1 immunostaining in the leptomeninges and adjacent cerebral cortex, as shown in (B). Note the different scales for the vertical axis in the two plots. (D) As in (B), except immunostained for Cldn5 and ASC. These are the same four leptomeninges+cortex flat mounts as shown in Figure 2D: the upper row of four images shown here reproduces the upper row of four images shown in Figure 2D, and the lower row of four images shown here correspond to the regions in the top row but were captured at a greater depth to visualize the underlying cortex. (E) Quantification of ASC immunostaining in the cerebral cortex, as shown in the lower row of images in (D). (F) As in (B), except immunostained for Cldn5 and CD206. These are the same four leptomeninges+cortex flat mounts as shown in Figure 2D: the upper row of four images shown here reproduces the lower row of four images shown in Figure 2D, and the lower row of four images shown here correspond to the regions in the top row but were captured at greater depth to visualize the underlying cortex. These data are not quantified because CD206 cells were extremely rare in the cortex in all samples.

Cleaved Caspase-3+ cells accumulate in the cortex in E. coli-infected WT and Tlr4MKO mice but not in Tlr4VEKO P6 mice.

(A) Flat mount images showing the meninges and adjacent cortex for the indicated genotypes and conditions. Scale bar, 50 µm. (B) Quantification of cleaved Caspase-3+ cells from flat mount images like the ones shown in (A). The region of cortex analyzed is adjacent to the leptomeninges. (C) Coronal sections showing meninges (upper) and cortex (lower). For quantification, two regions were imaged per hemisphere, as shown in the schematic on the right and labeled ‘a’ and ‘b’. Examples of ‘a’ and ‘b’ images are shown at left. Scale bar, 200 µm. (D) The number of cleaved Caspase-3+ cells in leptomeninges and cortex were quantified from 1 mm x 1 mm images like those shown in (C). The different planes of sectioning and the different cortical depths in the images quantified in (B) and (D) account for the different numbers of cleaved caspase3+ cells. (E) Correlation between cleaved Caspase-3+ cells in the leptomeninges and cortex for individual coronal images as shown in (C). (F) Cleaved Caspase-3+ cells in cortex are NeuN-, GFAP-, Cldn5-, and PU.1+, implying a myeloid identity. Scale bar, 100 µm.

An increase in cortical Iba1+ immunostaining with E. coli infection.

(A) Coronal sections of P6 cortex and leptomeninges showing Iba1 immunostaining with or without E. coli infection in P6 WT and Tlr4VEKO mice. Scale bars, 50 µm. (B) Quantification of the relative area occupied by Iba1+ pixels in the cortex of WT and Tlr4VEKO mice with or without E. coli infection at P6, as shown in (A).

Assessment of clinical progression at 24 hours in WT and Tlr4VEKO mice following E. coli infection.

(A) Representative maximum intensity projection images of RFP-expressing E. coli (red) and DAPI (blue) in lung, liver, and brain from control (uninfected) and E. coli-infected WT and Tlr4VEKO mice at 24 hours post-infection. Boxed regions (marked 1 and 2) indicate areas of bacterial accumulation. All scale bars, 100 µm. (B) Quantification of bacterial burden in the brain normalized to tissue area, expressed as the ratio of RFP-positive area to DAPI-positive area for each image. Each point represents an individual field of view. Boxes indicate median and interquartile range (IQR); whiskers represent 1.5 x IQR. (C) Bacterial titers in blood measured as colony-forming units (CFUs) per 20 µL of blood at P6 (log₁₀ scale). Each point represents an individual animal. Plates with colony numbers exceeding the reliable counting range were assigned a value of 2,000 CFUs. (D) Body weight measurements at P5 (pre-infection) and P6 (24 hours post-infection). Each point represents an individual animal. For (B-D), statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test. ns, not significant.

ZO-1 in leptomeningeal endothelial cells with or without E. coli infection.

(A) Whole mount leptomeninges from P6 WT and Tlr4VEKO mice, control or E. coli-infected, immunostained for Cldn5 and ZO-1. Scale bars, 20 µm. (B) Quantification of ZO-1 immunostaining in the leptomeninges, as shown in (A). (C) Enlarged images of whole mount leptomeningeal vasculature immunostained for Cldn5. Visual inspection reveals a modest elevation in non-junctional Cldn5 in E. coli-infected (right) compared to control (left). Scale bar, 50 µm.

CRISPR knockout of Tlr4 in bEnd.3 cells

(A) Functional testing of three bEnd.3 clones following CRISPR KO of Tlr4. For each clone, the TLR4-mediated NF-κB response to E. coli exposure (migration of NF-κB from cytoplasm to nucleus) was quantified by immunostaining, as shown in Figure 3C-E. (B) Sanger sequencing from two plasmid subclones carrying genomic PCR products encompassing the region targeted by the Tlr4 guide RNA from bEnd.3-Tlr4KO-m1 cells. Each sequencing run shows a distinctive frameshift mutation. The sequences show the WT sequence on the left and the mutant sequence on the right (with the altered nucleotides in red). Left, a single nucleotide insertion. Right, a two-nucleotide deletion. (C) Compendium of ten plasmid subclone sequences from each of the three bEnd.3 clones shown in (A). Each clone shows three distinct alleles, all of which differ from WT and all of which produce a frameshift. No WT sequences were observed in any subclone. These data imply the Tlr4 gene is present at three copies in bEnd.3 cells, and that each subclone is likely null for Tlr4 function.

The streptavidin leak assay requires a confluent monolayer of bEnd.3 cells.

(A) Among bEnd.3 cells, junctional localization of Cldn5 requires cell-cell contact as shown by immunostaining of cells grown at different densities. Scale bars, 20 µm. (B) bEnd.3 cells were grown at either low or high density on biotinylated gelatin-coated coverslips, incubated with fluorescent streptavidin, and then fixed and immunostained for ZO-1. In regions where the bEnd.3 cells had not formed a contiguous network of tight junctions, the streptavidin gained access to the underlying biotinylated gelatin. Scale bars, 20 µm.

Cldn5 dynamics in bEnd.3 cells following exposure to E. coli.

The overlap between Cldn5 and β-catenin, GLUT1, PECAM1, and ZO-1, in WT and Tlr4KO bEnd.3 cells, either not exposed to E. coli (control) or exposed to E. coli for 4 hours. Shown here is a more granular quantification of the experiment shown in Figure 5A-D, with each datapoint representing a 100 µm x 100 µm region of interest (ROI). Box plots show median, interquartile range, and all individual data points. Each data point represents one image from representative images in three biological replicates. Statistical comparisons (p-values) were calculated with the Wilcoxon rank-sum test.

Transcriptome analysis of WT and Tlr4KO bEnd.3 cells, with or without exposure to E. coli.

(A) Principal component analysis (PCA) showing the relatedness of the 14 bEnd.3 RNAseq samples. Two of the four orange symbols are nearly superimposed. (B) Pearson correlations for the 14 bEnd.3 RNAseq samples. (C) Pathway-level differences between WT and Tlr4KO bEnd.3 cells under control and E. coli-exposed conditions. Red, false discovery rate (FDR) less than 0.01. Orange, FDR between 0.01 and 0.05. Green, FDR between 0.05 and 0.25. Grey, FDR greater than 0.25. (D-F) Individual transcript abundances in the 14 bEnd.3 RNAseq samples for three GSEA categories. The transcriptome responses to infection are minimal in Tlr4KO bEnd.3 cells.