Endothelial TLR4 and the microbiome drive cerebral cavernous malformations

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Abstract

Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3–KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

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Figure 1: CCM formation is stimulated by GNB infection and intravenous LPS injection.
Figure 2: CCM lesion formation requires endothelial TLR4/CD14 signalling.
Figure 3: Increased TLR4 or CD14 expression is associated with higher lesion number in familial CCM patients.
Figure 4: CCMs fail to form in most germ-free mice.
Figure 5: CCM susceptibility is associated with increased levels of Gram-negative Bacteroidetes s24-7.
Figure 6: Preventing CCM formation by TLR4 antagonism and microbiome manipulation.

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Acknowledgements

We thank L. Goddard, other laboratory members, and K. Szigety for their comments during this work. We appreciate the guidance of our colleagues: G. Wu, R. Bushman, and Y. Choi. We acknowledge valuable technical assistance with B. fragilis culture from O. Jensen and J. Zhu; 16S sequencing and analysis by D. Kim, L. Mattei, and K. Bittinger from the PennCHOP Microbiome Core; germ-free mouse husbandry from K. Rickershauser and the Penn Gnotobiotic Mouse Facility; KRIT1 Q455X screening and Affymetrix genotyping of human samples from D. Guo and L. Pawlikowska; MRI images from M. Bartlett; patient data analysis from J. Nelson; data sorting from Y. Tang; artwork from L. Guo. We thank A. Ackers and Angioma Alliance for patient enrollment. These studies were supported by National Institute of Health grants R01HL094326 (M.L.K.), P01NS092521 (M.L.K. and I.A.A.), R01NS075168 (K.J.W.), T32HL07439 (A.T.T.), F30NS100252 (A.T.T.), T32DK007780 (J.K.), DFG grant SCHWD-416/5-2 (M.S.), U54NS065705 (H.K., L.M., B.H.), a Penn-CHOP Microbiome Pilot & Feasibility Award Grant (M.L.K.), and Australian NHMRC project grant 161558 (X.Z.).

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Contributions

A.T.T. designed and performed most of the experiments. J.P.C. and X.Z. performed parallel studies in Sydney. J.K. and J.H.-M. performed immunophenotyping experiments. Y.Y. and C.C.H. performed lineage tracing experiments. P.M. and M.C. assisted in numerous experimental studies. J.Y. and L.L. performed histological analysis. R.G., H.A.Z., T.M., R.L., Y.C., N.H., R.S. and I.A.A. performed all microCT lesion imaging and measurements in a blinded manner. C.T. performed bioinformatics analysis on 16S sequencing results. D.K. performed germ-free fostering experiments. U.V. and L.F. provided human eQTL data for TLR4 and CD14. K.J.W., D.Y.L., and M.S. provided critical reagents. B.H., L.M. and H.K. provided analysis of KRIT1 Q455X patients. A.T.T., J.P.C., J.K., C.C.H., C.T., U.V., H.K., and M.L.K. designed experiments and wrote the manuscript.

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Correspondence to Mark L. Kahn.

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Extended data figures and tables

Extended Data Figure 1 CCM formation in resistant Ccm2ECKO animals is stimulated by abscess formation and LPS.

a, Resistance to CCM formation is maintained in a C57BL/6J strain background. Ccm2ECKO (iECre;Ccm2fl/fl) animals were backcrossed seven generations onto a C57BL/6J background and gene deletion was induced at P1 with visual hindbrain assessment at P10. n = 7. Scale bars, 1 mm. b, Retinal CCM formation is stimulated by GNB infection. Retinas of P17 resistant Ccm2ECKO littermates are shown. The sample shown below is from an animal that developed the spontaneous Gram-negative abscess shown in Fig. 1c. Scale bars, 500 μm. c, d, Administration of LPS does not drive CCM formation in Cre-negative neonatal mice. LPS was administered intravenously to Ccm2fl/fl and Ccm2ECKO littermates as shown in Fig. 1g, and hindbrains assessed at P17 visually (c) and histologically (H&E staining; d). n ≥ 3 per group. Scale bars, 1 mm (c) and 100 μm (d). e, LPS induces myosin light chain activation in CCM-deficient brain endothelial cells. Phospho-myosin light chain (pMLC) and PECAM staining of hindbrains from P5 LPS- or vehicle-injected resistant Ccm2ECKO littermates. Dotted lines trace the Purkinje cell layer. n ≥ 4 per group. Scale bars, 50 μm. f, Tlr4 expression does not differ between CCM susceptible and resistant animals. Tlr4 expression was measured using qPCR in cerebellar endothelial cells isolated from the indicated animals at P10. Error bars shown as s.e.m. and significance determined by unpaired, two-tailed Student’s t-test. n.s., P > 0.05. Source data

Extended Data Figure 2 Analysis of immune cells in P6 and P11 Krit1fl/fl and Krit1ECKO brains.

a, Gating strategy for B cells, natural killer (NK) cells, γδ T cells, CD4 T cells, CD8 T cells, eosinophils, neutrophils and monocytes/macrophages from cerebrum and cerebellum is shown. Cellular surface markers used were as follows: neutrophils (CD45+, CD11b+, Ly6-G+), eosinophils (CD45+, CD11b+, CD11c, Ly6G, Siglec-F+, SSChi), monocyte/macrophage (CD45+, CD11b+, CD11c, Ly6G, Siglec-F, SSClo), NK cells (CD45+, CD11b, CD19, NK1.1+), B cells (CD45+, CD11b, NK1.1, CD19+), γδ T cell (CD45+, CD11b, NK1.1, CD19, CD3+, TCRγδ+), CD4 T cell (CD45+, CD11b, NK1.1, CD19, CD3+, TCRγδ, CD8, CD4+), CD8 T cell (CD45+, CD11b, NK1.1, CD19, CD3+, TCRγδ, CD4, CD8+). b, The number of B cells, NK cells, γδ T cells, CD4 T cells, CD8 T cells, eosinophils, neutrophils and monocytes/macrophages isolated from P6 cerebrum (top) and cerebellum (bottom) is shown for susceptible Krit1fl/fl and Krit1ECKO (iECre;Krit1fl/fl) littermates. n ≥ 6 per group. No significant differences were detected. c, The number of B cells, NK cells, γδ T cells, CD4 T cells, CD8 T cells, eosinophils, neutrophils and monocytes/macrophages isolated from P11 cerebrum (top) and cerebellum (bottom) is shown for susceptible Krit1fl/fl and Krit1ECKO littermates. n ≥ 6 per group. d, e, Frequency of RORγt+ CD4 T cells isolated from P6 and P11 cerebellum. n ≥ 6 per group. Error bars of all graphs shown as s.e.m. and significance determined by unpaired, two-tailed Student’s t-test. *P < 0.05. Note that there is significant immune cell presence in the cerebellum of susceptible Krit1ECKO animals at P11 but not at P6.

Extended Data Figure 3 Changes in the volume of CCM lesions are not accompanied by changes in total brain volume.

The indicated total brain volumes were measured using microCT imaging. a, b, Brain volumes corresponding to the genetic rescue experiments shown in Fig. 2c–f, respectively. c, Brain volumes corresponding to the C-section/germ-free fostering experiment shown in Fig. 4b, c. d, Brain volumes corresponding to the intergenerational antibiotic experiment shown in Fig. 6f–h, i. n.s., P > 0.05. Source data

Extended Data Figure 4 Lineage tracing of the Cdh5(PAC)-CreERT2 transgene in neonatal mice.

ac, R26-LSL-RFP, R26-CreERT2-R26-LSL-RFP, and Cdh5(PAC)-CreERT2-R26-LSL-RFP neonates were induced with doses of tamoxifen on P1+2 (two total doses) and CD45+RFP+ haematopoietic cell numbers in the spleen and peripheral blood were assessed at P10. n ≥ 5 per group. Error bars shown as s.e.m. and significance determined by one-way ANOVA with Holm–Sidak correction for multiple comparisons. ***P < 0.001; n.s., P > 0.05. Note, the number of labelled haematopoietic cells in Cdh5(PAC)-CreERT2-R26-LSL-RFP animals is indistinguishable from R26-LSL-RFP negative control animals, whereas >90% of CD45+ cells were RFP+ in R26-CreERT2-R26-LSL-RFP positive control animals. d, Anti-RFP and anti-PECAM immunostaining of P10 hindbrains from Krit1fl/fl-R26-LSL-RFP-negative control and Krit1ECKO-R26-LSL-RFP was performed to identify Cre+ descendants at the site of CCM formation. Note that all RFP+ cells in Krit1ECKO-R26-LSL-RFP animals are PECAM+, consistent with endothelial-specific Cre activity. Asterisk indicates CCM lesion. Results are representative of ≥3 per group. Scale bars, 100 μm. Source data

Extended Data Figure 5 The Slco1c1(BAC)-CreERT2 transgene is selectively expressed in brain endothelial cells and confers CCM formation when used to drive deletion of Krit1 in neonatal mice.

a, R26-LSL-RFP, Cdh5(PAC)-CreERT2-R26-LSL-RFP and Slco1c1(BAC)-CreERT2-R26-LSL-RFP neonates were induced with tamoxifen injection on P1+2 (two total doses). Immunostaining for RFP and PECAM was performed at P10 in the indicated tissues. Results are representative of at least three animals per group and three independent experiments. Scale bars, 100 μm. Note the presence of RFP+PECAM+ cells in the brain, small intestine, caecum, colon and liver of Cdh5(PAC)-CreERT2-R26-LSL-RFP animals, but only in the brain of Slco1c1(BAC)-CreERT2-R26-LSL-RFP animals. b, Visual (top) and corresponding microCT (bottom) images of brains from susceptible Slco1c1(BAC)-CreERT2-Krit1fl/+ and Slco1c1(BAC)-CreERT2-Krit1fl/fl P10 animals. Arrow indicates CCM lesions in the cerebrum. Scale bars, 1 mm. c, H&E staining of cerebellum (hindbrain) from the indicated animals (left). H&E staining of cerebrum (forebrain) from the indicated animals (middle). KLF4 and PECAM immunostaining from the indicated animals (right). Scale bars, 50 μm. Asterisks denote CCM lesions. n ≥ 5 per group.

Extended Data Figure 6 CCM formation can be stimulated by IL-1β or poly(I:C) treatment.

a, Schematic of the experimental design in which littermates receive a retro-orbital injection of the indicated cytokine or TLR ligand at P5 and P10 before tissue harvest and analysis at P17. bm, Visual images and volumetric quantification of CCM lesions in the hindbrains of P17 Ccm2ECKO littermates injected with the indicated cytokines, TLR ligands, or vehicle control are shown. Error bars shown as s.e.m. and significance determined by unpaired, two-tailed Student’s t-test. *P < 0.05; n.s., P > 0.05. Scale bars, 1 mm. Source data

Extended Data Figure 7 16S rRNA sequencing results from susceptible and resistant Krit1fl/fl and Ccm2fl/fl dams.

a, Heat map showing relative abundance of bacterial taxa (right) identified in susceptible (blue) and resistant (salmon) Krit1 (ccm1, purple) and Ccm2 (ccm2, green) animals (top). b, Boxplots of bacterial taxa that demonstrated significant differential abundance in susceptible versus resistant animals and the relative abundance of those taxa. c, Boxplot of the Firmicutes (Ruminococcus) taxon that displayed significant differential abundance between Krit1 and Ccm2 genotypes. Note that the relative abundance of Bacteroidetes s24-7 is anywhere from 10-fold to 10,000-fold greater than any other taxon. Significance (P < 0.05) for b and c determined by linear mixed effects modelling with Benjamini–Hochberg correction for multiple comparisons.

Extended Data Figure 8 Blockade of CCM formation by the TLR4 antagonist LPS-RS.

a, Schematic of the experimental design in which Krit1ECKO littermates receive retro-orbital injections of the TLR4 antagonist LPS-RS. b, Visual (left) and microCT (right) images of hindbrains from vehicle or LPS-RS injected animals. c, d, Quantification of CCM lesion and brain volume in Krit1ECKO littermates treated with vehicle or LPS-RS. Error bars shown as s.e.m. and significance determined by unpaired, two-tailed Student’s t-test. **P < 0.01; n.s., indicates P > 0.05. All scale bars, 1 mm. Source data

Extended Data Figure 9 CCM formation is stimulated by spontaneous abscess formation and not blocked by vancomycin.

a, P10 hindbrains from generation 3/post-ABX Krit1ECKO littermates in the longitudinal antibiotic experiment described in Fig. 6e–l. The animal with a large CCM lesion burden on the far right was found to have an abdominal abscess (circle, ‘absc’) and splenomegaly (arrow, lower right). Scale bar, 1 mm. b, Schematic of the experimental design in which cohoused, lesion susceptible Krit1ECKO mating pairs were used to test the acute effect of vancomycin treatment on CCM formation. Offspring were studied after receiving maternal vehicle or vancomycin administered from E14.5 to P11. c, d, Visual images of hindbrains from representative offspring following vehicle or vancomycin antibiotic treatment. Scale bars, 1 mm. e, f, Volumetric quantification of CCM lesions and brain volumes in Krit1ECKO littermates treated with vehicle or vancomycin. g, h, Relative quantification of total neonatal gut bacterial load measured by qPCR of bacterial universal 16S or Firmicutes-specific rRNA gene copies. n ≥ 6 per group. Error bars of all graphs shown as s.e.m. and significance determined by unpaired, two-tailed Student’s t-test. n.s., P > 0.05. ****P < 0.0001. Source data

Extended Data Figure 10 CCM formation is conferred to the offspring of resistant animals by fostering to Swiss Webster mothers.

a, Schematic of the experimental design in which timed matings of resistant Krit1ECKO and resistant Ccm2ECKO mating pairs were used to generate E19.5 offspring delivered by natural birth and raised by the birth mother or C-section/fostered to conventional Swiss Webster foster mothers. b, c, Visual images of hindbrains from P10 resistant Krit1ECKO and Ccm2ECKO offspring following natural delivery and nursing by resistant mothers or after C-section/fostering to Swiss Webster mothers. n ≥ 6 per group.

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Tang, A., Choi, J., Kotzin, J. et al. Endothelial TLR4 and the microbiome drive cerebral cavernous malformations. Nature 545, 305–310 (2017). https://doi.org/10.1038/nature22075

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