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Science 334 (6063): 1727-1731

Copyright © 2011 by the American Association for the Advancement of Science

The Hedgehog Pathway Promotes Blood-Brain Barrier Integrity and CNS Immune Quiescence

Jorge Ivan Alvarez1,*, Aurore Dodelet-Devillers1,*, Hania Kebir1, Igal Ifergan1, Pierre J. Fabre2, Simone Terouz1, Mike Sabbagh1, Karolina Wosik1, Lyne Bourbonnière1, Monique Bernard1, Jack van Horssen3, Helga E. de Vries3, Frédéric Charron2, and Alexandre Prat1,{dagger}

1 Neuroimmunology Unit, Center of Excellence in Neuromics, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Faculty of Medicine, Université de Montréal, Montréal, Quebec, H2L 4M1, Canada.
2 Laboratory of Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal and Department of Medicine, Université de Montréal, Montréal, Quebec, H2W 1R7, Canada.
3 Department of Molecular Cell Biology and Immunology, VU Medical Center, 1007 MB Amsterdam, Netherlands.


Figure 1
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Fig. 1. Shh decreases BBB EC permeability via Smo engagement and induction of Gli-1 and SOX-18. Shown are changes in TEER (A), clearance (B), and permeability to 14C-sucrose (C) and BSA-FITC (D) of human BBB EC monolayers in conditions stimulating or abrogating the Hh pathway. BBB ECs were treated for 24 hours with ACM, hrShh, or ACM plus cyclopamine or purmorphamine, and permeability was recorded over 3 hours (n = 3 to 8 experiments, performed in triplicate). (E) Flow cytometric analysis of Gli-1 expression in human BBB ECs. Percentages and mean fluorescence intensity (MFI) in red refer to values of controls (untreated cells), and in blue refer to values of treated cells. (F) MFI for Gli-1 in BBB ECs shown in (E) (n = 4 experiments). (G) Quantitative real-time fluorescence polymerase chain reaction (QPCR) analysis of Gli-1 (upper panel) and SOX-18 (lower panel) mRNA expression in BBB ECs treated with hrShh (n = 2 experiments in triplicate). (H) Epifluorescence photomicrographs of CNS sections from C57BL/6 mice treated with cyclopamine for 6 hours (upper panels). Dextran-FITC and fibrinogen (arrowheads) accumulate around vessels (asterisks) as compared to vehicle-treated mice (HPβCD, lower panels). Nuclei were stained with 4',6-diamidino-2-phenylindole and are shown in blue. Scale bars, 30 μm. (I) Quantitative analysis of perivascular extravasation of 70-kD dextran-FITC and fibrinogen in cyclopamine- and vehicle-treated animals. For all, mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

 

Figure 2
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Fig. 2. Shh promotes junctional protein expression. (A) QPCR analysis of PTCH1, CLAUDIN5, and OCCLUDIN mRNA extracted from human BBB ECs grown with ACM or hrShh for 24 hours. (B) Western blot (left) and semiquantitative analysis (right) of claudin-5, occludin, JAM-A, claudin-3, and VE-cadherin in human BBB ECs treated with ACM or ACM + cyclopamine for 24 hours. (C) Characterization of CNS vessels in P19 WT and Tie2-Cre; Smoc/c mice. Shown is immunofluorescence for glial fibrillary acidic protein (GFAP) (astrocytes), platelet endothelial cell adhesion molecule–1 (PECAM-1), claudin-5, claudin-3, occludin, ZO1, and p120 (all BBB endothelium markers), fibrinogen, and IgGs (indicators of plasma protein leakage) (left, images) and quantification (right, graphs) (n = 3 to 5 animals). White squares and rectangles indicate areas shown on the right of each panel. Arrowheads on occludin panels indicate vessels. Error bars, mean ± SEM. *P < 0.05; ***P < 0.001. Nuclei were stained with TOPRO-3 and are shown in blue. Scale bars, 30 μm.

 

Figure 3
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Fig. 3. Hh activity promotes BBB immune quiescence. (A) Chemokine secretion (IL-8/CXCL8, left; MCP-1/CCL2, right) by human BBB ECs treated with hrShh, purmorphamine, or both ACM and cyclopamine. (B) ICAM-1 expression by human BBB ECs treated under similar conditions. (C and D) Effect of Hh pathway engagement on human CD4+ T lymphocyte adhesiveness (1 hour) to (C) and migration across (D) human BBB ECs. A similar effect was recorded with the Hh antagonist SANT-1. (E) Adhesion of human TH17, TH1, and TH2 cells polarized in the presence or absence of hrShh to human BBB ECs. (F to I) Effect of pharmacological Hh neutralization with the Smo antagonist GDC-0449 (GDC; 25 mg per kilogram of body weight, injected at days 0, 4, 8, and 12; arrowheads) on the clinical severity of EAE (F), on spinal cord demyelination (arrowheads indicate the border of demyelinating lesions) (G), on the number of leukocytes infiltrating the CNS (H), and on the production of IFN-{gamma} and IL-17 by CNS-infiltrating CD4+ T cells (I), as compared to vehicle (DMSO). (A) to (F) and (H) represent the mean ± SEM (n = 3 to 8 experiments in triplicate). (F) to (I), 25 animals per condition. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 50 μm.

 

Figure 4
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Fig. 4. Hh components are up-regulated under neuroinflammatory conditions in vitro and in situ. (A) Expression of Shh was determined in resting (red histogram) and inflamed (green histogram; TNF + IFN-{gamma} treated for 24 hours) human astrocytes (HFAs) by flow cytometry. Isotype is shown in black. (B) Ptch-1 (left) and Smo (right) expression in human BBB ECs grown with ACM (light gray) or ACM + TNF + IFN-{gamma} (dark gray). (A) and (B), n = 4 experiments). (C) Expression of Shh, Ptch-1, and Smo in CNS sections from a human control (left panels) and MS patients (right panels). Arrowheads indicate astrocyte bodies. Arrows indicate astrocyte endfeet, and asterisks indicate blood vessels. The black dotted line delineates the border between an active MS lesion and the adjacent NAWM. (D) Shh and GFAP (arrowheads) colocalization in NAWM (upper left panels) and active MS lesions (upper right panels). Ptch-1 expression on ECs of NAWM (middle left panel) and active lesions (middle right panel). In active MS lesions, Ptch-1 is also detected in infiltrating cells (arrowheads). The lower panels demonstrate Ptch-1 and Gli-1 expression in ECs of NAWM and active lesions, where arrows indicate ECs and arrowheads indicate infiltrating cells. Nuclei were stained with TOPRO-3 and are shown in blue. Scale bars, 30 μm.

 


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