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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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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.).

Author information

Affiliations

  1. Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA

    • Alan T. Tang
    • , Yiqing Yang
    • , Courtney C. Hong
    • , Mei Chen
    • , Patricia Mericko
    • , Jisheng Yang
    • , Li Li
    •  & Mark L. Kahn
  2. Laboratory of Cardiovascular Signaling, Centenary Institute, Sydney, New South Wales 2050, Australia

    • Jaesung P. Choi
    •  & Xiangjian Zheng
  3. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Jonathan J. Kotzin
    •  & Jorge Henao-Mejia
  4. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Jonathan J. Kotzin
    • , Dmytro Kobuley
    •  & Jorge Henao-Mejia
  5. Neurovascular Surgery Program, Section of Neurosurgery, Department of Surgery, The University of Chicago School of Medicine and Biological Sciences, Chicago, Illinois 60637, USA

    • Nicholas Hobson
    • , Romuald Girard
    • , Hussein A. Zeineddine
    • , Rhonda Lightle
    • , Thomas Moore
    • , Ying Cao
    • , Robert Shenkar
    •  & Issam A. Awad
  6. CHOP Microbiome Center, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA

    • Ceylan Tanes
  7. Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Dmytro Kobuley
  8. Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands

    • Urmo Võsa
    •  & Lude Franke
  9. Division of Cardiovascular Medicine and the Program in Molecular Medicine, University of Utah, Salt Lake City, Utah 84112, USA

    • Kevin J. Whitehead
    •  & Dean Y. Li
  10. Department of Neurology and Pediatrics, University of New Mexico, Albuquerque, New Mexico 87131, USA

    • Blaine Hart
    •  & Leslie Morrison
  11. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, 23562 Lübeck, Germany

    • Markus Schwaninger
  12. Division of Transplant Immunology, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Jorge Henao-Mejia
  13. Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California 94143, USA

    • Helen Kim
  14. Faculty of Medicine, Sydney Medical School, University of Sydney, Sydney, New South Wales 2050, Australia

    • Xiangjian Zheng
  15. Department of Pharmacology, School of Basic Medical Sciences, Tianjian Medical University, Tianjin, China

    • Xiangjian Zheng

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark L. Kahn.

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https://doi.org/10.1038/nature22075

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