Abstract
The blood–brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2,3,4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7,8,9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP–CD14 LPS transfer and internalization pathway10,11,12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.
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Data availability
The scRNA-seq dataset generated in this study is available under the GEO accession number GSE211099. The public datasets used in this study include the following: Vanlandewijck et al.26 (GEO accession number: GSE98816), Duan et al.31 (GEO accession number: GSE112436), Winkler et al.43 (https://adult-brain-vasc.cells.ucsc.edu), and Yang et al.44 (GEO accession number: GSE163577). All other data supporting the findings of this study are included in this manuscript. Source data are provided with this paper.
Code availability
The codes for the analysis of the dataset are deposited at https://github.com/RuiyuRayWang/Gsdmd_BBB.
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Acknowledgements
The authors thank F. Han and Y. Lu for providing the BR1 viral plasmid; X. Wang for providing hBMECs; the CIBR genomics centre for scRNA-seq experiments; the NIBS electron microscopy centre for electron microscopy sample preparation; J. D. Buxbaum for CASP4Tg mice; and BioRender.com for generating cartoon illustrations. We also thank the Laboratory Animal Resource Centers of CIBR and NIBS for rapid re-derivation and breeding of the mice and the Vector Core of CIBR for helping with packing AAV viruses. The work was supported by the Basic Science Center Project (82388201) of National Natural Science Foundation of China (NSFC), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB37030202 and XDB29020202), the National Key Research and Development Program of China (2022YFA1304700), and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-084) to F.S.; M.L. is supported by the Beijing Municipal Government, the Research Unit of Medical Neurobiology at Chinese Academy of Medical Sciences (2019RU003), and the STI2030-Major Projects+2021ZD0202803. M.L. and F.S. are also supported by the Tencent New Cornerstone Investigator Program.
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Contributions
C.W., W.J., M.L. and F.S. conceived the study. C.W. and W.J. performed the experiments. R.W. performed the scRNA-seq experiments and bioinformatics analyses, supervised by L.Z. H.Z. and H.H. constructed the Rosa26LSL-mGsdmd-N/+ and the Gsdmdflox/flox mice, respectively. C.W. and X.G. analysed the imaging data with supervision from Q.G. S.Z. and F.Y. provided technical assistance. L.D.J.S. and F.I.S. generated the nanobodies. B.Z. provided the bEC-cre mice. M.T. contributed the BR1 viral plasmid. C.W., W.J., M.L. and F.S. analysed the data and wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.
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F.S. is the scientific founder and chair of the scientific advisory board of Pyrotech Therapeutics. This relationship did not influence this study. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 The Casp11-Gsdmd non-canonical inflammasome and the Lbp-Cd14 LPS transfer/internalization, but not Tlr4-induced cytokines, are required for circulating LPS-caused BBB disruption.
a, b, WT mice were challenged by intraperitoneal injection of LPS (54 mg kg−1) or PBS for 6 h (a, b), or by retro-orbital injection of mouse TNFα (mTNFα, 25 μg) or IL-6 (mIL-6, 25 μg) for 8 h (b). a, Quantification of serum LPS after 1- or 6-h LPS challenge (n = 3 mice per group). b, Quantification of Evans blue leakage in the mouse brains (n = 5 mice per group). c–h, WT, Tlr4−/−, Casp11−/−, Gsdmd−/−, Cd14−/− or Lbp−/− mice were primed with poly(I:C) and challenged with LPS as in Fig. 1d, and BBB disruption was examined. c, g, Sulfo-NHS-biotin assay. c and lower in g, representative high-magnification views of sulfo-NHS-biotin distribution among collagen IV+ blood vessels in mouse cortices. Upper in g, low-magnification views of sulfo-NHS-biotin distribution within brain parenchyma. d, f, TMR-dextran assay. Upper, low-magnification fluorescence images of representative brain slices. Lower, high-magnification views of the cortices (white arrowheads, dextran-labelled parenchymal cells). e, HRP tracer assay. Upper, low-magnification views of brain coronal sections. Lower, high-magnification views of the cortices. h, Evans blue assay (n = 6 mice per group). Data are mean ± s.e.m. (a, b, h); two-way ANOVA with Tukey’s post-hoc test (a, h) and Brown-Forsythe and Welch ANOVA with Games-Howell’s post-hoc test (b) were used (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant). See Supplementary Table 1 for detailed statistics. All data are representative of three independent experiments.
Extended Data Fig. 2 Dose titration, micro-hemorrhage examination, and spatial distribution of LPS disruption of the BBB.
a, Poly(I:C)-primed WT, Casp11−/−, or Gsdmd −/− mice were challenged with indicated increasing doses of LPS, and BBB disruption was assayed by sulfo-NHS-biotin tracer. Upper, representative low-magnification views of sulfo-NHS-biotin in the mouse brains. Lower, representative high-magnification views of sulfo-NHS-biotin among collagen IV+ vessels (magenta) in mouse cortices. b, Poly(I:C)-primed WT mice were treated with 54 mg kg−1 LPS and BBB disruption was marked by sulfo-NHS-biotin. Examination of micro-hemorrhage around the disrupted BBB. Left, sulfo-NHS-biotin tracer distribution. Right, H&E staining of the same brain section. c, Micro-hemorrhage induced by intracranial implantation of GL261 glioma into WT mice. Black arrowheads, micro-hemorrhage. d–f, WT mice were primed with poly(I:C) and challenged with LPS or PBS as in Fig. 1d. 647-dextran was used to report BBB disruption (magenta). Cleared mouse brains were subjected to light-sheet microscopy, and 3D reconstruction was performed. d, Representative images of 647-dextran distribution in the mouse brain. e, f, 647-dextran distribution along the rostral-caudal (e) or the medial-lateral axis (f) (n = 3 mice per group). Gray triangles in e are stitching traces from the light-sheet microscopy procedure. g, h, 10-μm coronal optic slices of 647-dextran-stained brains at different positions along the rostral-caudal axis (1 to 6). The 647-dextran-positive structures in h are brain regions known to contain permeable blood vessels (SFO, subfornical organ; ME, median eminence; CP, choroid plexus). Data (a–c) are representative of three independent experiments.
Extended Data Fig. 3 GSDMD is highly expressed in bECs but not pericytes.
a, Immunoblotting of GSDMD expression in tissues derived from different mouse organs. b, c, Immunofluorescence-histochemical staining of GSDMD in cortical vasculatures of WT or Gsdmd−/− mice. The samples were co-stained with an anti-CD31 (b) or anti-CD13 antibody (c). White arrowheads, microglia/macrophages. d, Colocalization analyses of cortical GSDMD with the endothelial marker CD31 (upper) or the pericyte marker CD13 (lower). Left, representative images of GSDMD expression in the CD31+ or CD13+ vasculatures (green). Middle, high-magnification views of the fluorescent signals in the boxed area in the left panel. Graphs on the right show the distribution of anti-GSDMD/anti-CD31 or anti-GSDMD/anti-CD13 fluorescent intensity (grayscale: 0–255) along the white lines marked in the middle images. All data are representative of three independent experiments.
Extended Data Fig. 4 scRNA-seq of mouse brain responses to systemic LPS.
The analyses were performed with brain motor cortices of WT and Gsdmd−/− mice challenged with PBS or LPS (40 mg kg−1). a, b, FACS counting of 20,000 events from a representative single cell dissociation experiment. a, Gating strategy for collecting target cell populations. P1 gate: remove cell debris and enrich the cell population; P2 and P3 gates: reduce doublets. Viable cells (DAPI-low) in the P4 gate were used for library preparation. b, Event numbers and rates for each gate in a. c–f, Quality metrics of the preprocessed dataset. c, Violin plot of the number of genes detected in each sample. d, Sensitivity profile indicated by the cumulative number of genes detected at different cell abundances for each cell type. For a given abundance, cells were randomly selected 50 times from the total population and the averaged cumulative gene number was computed. e, Stacked bar plot showing the cell type composition in each sample. Astro, astrocytes; Endo, endothelial cells; Ex-Neu., excitatory neurons; Inh-Neu., inhibitory neurons; Oligo, oligodendrocytes; Micro, microglia; OPCs, oligodendrocyte precursors; Prolif. OPCs, proliferating OPCs; Peri, pericytes; PVMs, perivascular macrophages; SMCs, smooth muscle cells; VLMCs, vascular leptomeningeal cells. f, UMAP of the preprocessed dataset. Cell types are colored the same as in e. Populations with the cell number <50 (proliferating OPCs, PVMs, VLMCs, Peri, and SMCs) were excluded from subsequent analyses. g, Violin plots showing the expression patterns of canonical marker genes for each cell type. h, UMAP (left) and volcano plots (right) of transcriptional perturbation in relevant cell types from indicated experimental groups (↑, upregulation; ↓, downregulation). Differentially expressed genes were tested by a two-part hurdle model; p-values were adjusted for multiple comparisons by the Bonferroni’s method.
Extended Data Fig. 5 Transcriptional induction of Casp11 in bECs and gene signatures of LPS-challenged mouse brains.
a–c, RNAscope in situ hybridization analyses of Casp11 mRNA in bECs in PBS and LPS-treated mice. a, Representative staining images of Cd31 and Casp11 mRNA in the mouse cortices. DAPI, the nuclei; white arrowheads, Cd31 mRNA+ endothelial nuclei co-labelled with Casp11 mRNA signals. b, Quantification of Casp11 mRNA signals in cortical Cd31 mRNA+ endothelial nuclei. n = 6,222 and 5,214 Cd31 mRNA+ nuclei from 3 PBS- and 3 LPS-treated mice, respectively. c, Percentages of Casp11 mRNA+ nuclei among Cd31 mRNA+ endothelial nuclei (mean ± s.e.m., n = 3 mice per group). Two-sided Wilcoxon rank-sum test (b) and unpaired two-sided Welch’s t test (c) were used (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant). d, Analyses of the scRNA-seq data (Extended Data Fig. 4) to obtain transcriptional signatures in LPS-challenged WT and Gsdmd−/− mouse brains. Left, heatmaps of gene expression profile in selected cell types (other cell types had little responses, thus not included). Right, bar plots of signature genes in each row of the heatmap, corresponding to a GO term (1st row, upregulated in WT but not Gsdmd−/− cells; 2nd row, upregulated in Gsdmd−/− but not WT cells; 3rd row, downregulated in both WT and Gsdmd−/− cells; 4th row, upregulated in both WT and Gsdmd−/− cells). One-sided Fisher’s exact test with the Benjamini-Hochberg multiple comparisons. See Supplementary Table 1 for detailed statistics. Data (a–c) are representative of three independent experiments.
Extended Data Fig. 6 LPS triggers GSDMD-dependent pyroptosis in mouse and human bECs.
a–e, Primary bECs were isolated from WT, Tlr4−/−, or Casp11−/− mice. a, A representative fluorescence image of anti-CD31 and Hoechst-stained primary bECs. b, RT-PCR amplification curves of Tlr4 and Gapdh mRNAs from WT or Tlr4−/− primary bECs. The inset table shows the average Ct value. c, Normalized Casp11 mRNA expression (relative to Gapdh mRNA) in WT and Tlr4−/− primary bECs stimulated with LPS or PBS. b, c, Data are mean ± s.e.m. from four replicates. d, e, Indicated primary bECs were primed with poly(I:C) and transfected with LPS or PBS. d, Representative images of SYTOX green uptake by the transfected cells. e, Numbers of SYTOX green+ nuclei (mean ± s.e.m. from 6 fields of view). f, g, Immortalized hBMECs were transfected with LPS or PBS. f, Anti-GSDMD and anti-caspase-4 immunoblots of cell supernatants or lysates. g, LDH release-based cell death data are shown as mean ± s.e.m. from three replicates. h–j, WT or Gsdmd−/− mice were primed with poly(I:C) and challenged with LPS as in Fig. 1d. SYTOX green and TMR-dextran were co-injected into the mice to label pyroptotic cells and disrupted vasculature in the brain, respectively. h, Representative images of SYTOX green+ vascular nuclei associated with the TMR-dextran tracer. High-magnification views of the boxed area are in the left panels. i, Numbers of SYTOX green+ vascular nuclei associated with disrupted vasculature per brain slice from LPS-challenged mice. j, Percentages of BBB disruption-associated SYTOX green+ nucleus among the total SYTOX green+ nuclei in LPS-challenged WT mice. i, j, Data are mean ± s.e.m. from 12 brain slices (3 mice). Two-way ANOVA with Tukey’s post-hoc test (e) and unpaired two-sided t-test with Welch’s correction (i) were performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant). See Supplementary Table 1 for detailed statistics. All data are representative of three independent experiments.
Extended Data Fig. 7 GSDMD pore formation in bECs causes influx of extracellular molecules and efflux of cytosolic contents.
hBMECs harboring a doxycycline (DOX)-inducible GSDMD-N domain were cultured in SYTOX green-containing media (a, c) or pre-loaded with Calcein-AM (b, d). The cells were stimulated with or without DOX. a, b, Time-lapse fluorescence images recording the influx of SYTOX green (a) or the efflux of intracellular Calcein (b). Corresponding differential interference contrast (DIC) images (to observe cell death morphologically) are also shown. White arrowheads mark the cells that started to show SYTOX green entering and staining of the nuclei (a) or the cells that exhibited initial signs of Calcein leakage (b). c, d, Overall (the left, shown as mean ± s.e.m.) and individual (the right) traces of normalized SYTOX green signal (n = 57 cells for the +DOX group and 105 for the –DOX group) or of normalized Calcein signal (n = 86 cells for the +DOX group and 77 for the –DOX group). Data are representative of three independent experiments.
Extended Data Fig. 8 Electron microscopy of LPS-triggered ultrastructural changes in the disrupted BBB.
a–c, WT or Gsdmd−/− mice were primed with poly(I:C) and challenged with LPS as in Fig. 1d (a, b) or treated with PBS (c). 10-kDa biotin-dextran (a, c) or 443-Da sulfo-NHS-biotin (b) was used to locate the disrupted vasculature (see Supplementary Fig. 7 for details). a, b, The left images show the entire structure of representative large brain blood vessels (a) or capillaries (b). On the right are high-magnification views of BBB ultrastructure marked in the left image. Inset #1: pyroptotic endothelial cell. Inset #2: vasculature detachment, enlarged perivascular space and evident parenchymal edema (asterisks). Inset #3–#5: tight junction of pyroptotic endothelia. Arrowheads, tight junction. c, Images show the normal BBB ultrastructure. E, endothelial cell; L, lumen; PE, pyroptotic endothelial cell; PVS, perivascular space; TJ, tight junction. Asterisk, parenchymal edema. For additional EM images, see Fig. 3 and Supplementary Fig. 8.
Extended Data Fig. 9 Brain endothelial Gsdmd determines circulating LPS-caused BBB disruption.
a–e, Mice with indicated genotypes and their control littermates were challenged with PBS or LPS as in Fig. 1d. a, b, BBB disruption was examined by the TMR-dextran (a) or the sulfo-NHS-biotin tracer (b). a, Distribution of TMR-dextran in the mouse brain. Upper, low-magnification views of representative coronal brain sections. Lower, high-magnification views of representative cortex sections. Arrowheads, dextran-labelled parenchymal cells. b, Distribution of sulfo-NHS-biotin in the mouse brain. Upper, representative low-magnification views of brain parenchyma sections. Lower, representative high-magnification views of sulfo-NHS-biotin distribution among anti-collagen IV-stained blood vessels in mouse cortices. c–e, SYTOX green and TMR-dextran were co-injected into the Gsdmdflox/flox or bEC-Cre; Gsdmdflox/flox mice to label pyroptotic cells and disrupted vasculature in the brain, respectively. c, Representative images of SYTOX green+ vascular nuclei associated with the TMR-dextran tracer. d, Numbers of SYTOX green+ vascular nuclei associated with disrupted vasculature per brain slice from LPS-challenged mice. e, Percentages of BBB disruption-associated SYTOX green+ nucleus among the total SYTOX green+ nuclei in the mice. d, e, Data are mean ± s.e.m. from 12 brain slices (3 mice). Unpaired two-sided t-test with Welch’s correction was performed in d (****p < 0.0001; ns, not significant). f–h, AAV-BI30-EGFP or AAV-BI30-Cre viruses were injected into Gsdmdflox/flox mice (f), AAV-BR1-EGFP or AAV-BR1-mGSDMD (WT or D276A) viruses was injected into Gsdmd−/− mice (g), and AAV-BR1-DIO-EGFP or AAV-BRI-DIO-mGSDMD (WT or D276A) viruses were injected into Tie2-Dre; Mfsd2a-CrexER; Gsdmdflox/flox mice (h). All mice were challenged with PBS or LPS as in Fig. 1d. BBB disruption was assayed by the Evens blue tracer. Representative images (left) and quantification (right) of Evans blue leakage in the mouse brains (n = 5 mice per group) are shown. Quantification data are mean ± s.e.m., and two-way ANOVA with Tukey’s post-hoc test (f, g, h) was performed (****p < 0.0001; ns, not significant). See Supplementary Table 1 for detailed statistics. All data are representative of three independent experiments.
Extended Data Fig. 10 GSDMD activation by K. pneumoniae LPS in human cells and inhibition of mGSDMD and BBB disruption by a specific nanobody.
a, LPS from K. pneumoniae was transfected into HeLa cells (WT, CASP4−/− and GSDMD−/−) or hBMECs ( ± zVAD). b, WT, CASP4Tg, or CASP4Tg; Gsdmd−/− mice were challenged with 2 mg kg−1 of LPS as in Fig. 1a. BBB disruption was assayed by the Evens blue tracer. Shown are representative images (upper) and quantification (lower) of Evans blue leakage in the mouse brains (n = 5 mice per group). Data are mean ± s.e.m.; two-way ANOVA with Tukey’s post-hoc test (****p < 0.0001; ns, not significant). See Supplementary Table 1 for detailed statistics. c, Assay of liposome (80% phosphatidylcholine+20% cardiolipin) leakage by interdomain-cleaved mGSDMD-(N + C) in the presence of VHHGSDMD-1 or the control nanobody VHHctrl. Liposome leakage was assessed by measuring the released Tb3+. d, DOX-induced expression of mGSDMD-N domain in HEK 293 T cells harboring VHHGSDMD-1 or VHHctrl. a, d, Upper, LDH release-based cell death data are shown as mean ± s.e.m. from three replicates. Lower, cell lysates were immunoblotted. All data are representative of three independent experiments.
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Uncropped immunoblots for key data presented in the main text and extended data section of the manuscript.
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Supplementary Figs. 1–16.
Supplementary Table 1
This file contains the P values and associated values for all statistic tests performed.
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Wei, C., Jiang, W., Wang, R. et al. Brain endothelial GSDMD activation mediates inflammatory BBB breakdown. Nature (2024). https://doi.org/10.1038/s41586-024-07314-2
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DOI: https://doi.org/10.1038/s41586-024-07314-2
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