Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS
Hongxia Duan1†, Yongting Luo1,7, Liqun Feng2, Irene Gramaglia3, Ying Zhang1, Di Lu1, Qiqun Zeng1, Kelong Fan1, Shu Xing1, Jing Feng1, Dongling Yang1, Zhihai Qin1, Pierre-Olivier Couraud4, Ignacio A. Romero5, Babette Weksler6 and Xiyun Yan1,8
1Key Laboratory of Protein and Peptide Pharmaceuticals, CAS-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China
2Beijing Anzhen Hospital of the Capital University of Medical Sciences, 2 Anzhen Road, Beijing 100029, China
3La Jolla Infectious Disease Institute, 6044 Cornerstone Court, Suite A2, San Diego, CA 92121
4Institut Cochin, Centre National de la Recherche Scientifique UMR 8104, Institut National de la Santé et de la Recherche Médicale (INSERM) U567, Université René Descartes, Paris, France
5Department of Life Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK
6Weill Cornell Medical College, New York, NY 10065 USA
7These authors contributed equally to this work.
8To whom correspondence should be addressed. E-mail:.
Supplementary Materials
Supplementary methods
Antibodies and reagents
The following antibodies were used in this study: anti-CD146 mAb AA981,2, AA98-PE, AA1-APC, AA1-PE, anti-mouse CD31-APC (Tianjin Sungene Biotech Co., Ltd.), anti-GAPDH (Sigma-Aldrich), anti-caveolin-1 (Santa Cruz Biotechnology), anti-human CD31 (Cymbus Biotechnology), anti-ICAM-1-PE, anti-VCAM-1-PE, anti-mouse CD3-PerCP-eFluor 710, anti-CD8-PE, anti-CD117-PE, anti-IL-17A-APC, anti-IFN-PE (eBioscience), and horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (GE Healthcare). Human TNFwas purchased from Peprotech.Phytohemagglutinin-P (PHA-P), brefeldin-A (BFA) and phorbol myristate acetate (PMA)/ionomycin were purchased from Sigma-Aldrich.
Clinical sample collection
MS patients treated at Beijing Anzhen Hospital were selected on a clinical basis. Prior to sample collection, informed consent was obtained, and approval was obtained from the Ethics Committee of Anzhen Hospital. All patients had obvious lesions visible in their cerebral MRI scans and positive oligoclonal bands in their cerebrospinal fluid, and each patient was diagnosed with relapsing–remitting MS based on the clinical course of the disease3. Brain-mounted specimens were obtained from five MS patients following their postmortem examinations, and brain-mounted specimens from five healthy individuals were purchased from Chaoying Biotechnology Co., Ltd (Shanxi, China).
Immunohistochemistry and immunofluorescence
For 3,3’-diaminobenzidine (DAB) staining, paraffin-embedded tissue sections were deparaffinized and stained first with an antibody specific for CD31 or CD146 (AA4), followed bya biotin-conjugated secondary antibody (1:1000), followed by HRP-conjugated streptavidin (Dianova, Rodeo, CA). Finally, the sections were counterstained with hematoxylin.
For immunofluorescence, 5-m-thick sections were deparaffinized and stained with antibodies specific for mouse caveolin-1 and CD146 (AA4), followed by the appropriate fluorescence-labeled secondary antibodies. The nuclei were counterstained with 4',6-diamidino-2-phenylindol (DAPI). The number of infiltrated cells per unit area was measured in at least 10 random spinal cord lesions.
Mouse BBBEC isolation and culture
Mouse brains and spinal cords were collected in sterile phosphate-buffered saline (PBS) and sterilized with three rinses in 75% ethanol. The CNS tissues were then chopped into pieces in DMEM media and incubated with 0.1% collagenase (w/v) at 37C for 1 h. The suspension was then centrifuged and resuspended inDMEM with 25% BSA, then centrifuged at 1,200 rpm for 40 min. The pellet was digested with 0.1% collagenase for an additional 1 h. The mouse BBBECs were pelleted by centrifugation at 1,200 rpm for 10 min, harvested and then grown in EBM-2 media for 1-2 weeks. For FACS analysis, the cultured cells were harvested and stained with AA98-PE and anti-mouse CD31-APC in PBS with 0.1%BSA.
Supplementary Fig. 1
Supplementary Fig. 1. CD146 is over-expressedin the BBB of clinically active MS patients and EAE mice. (a) Immunohistochemical staining of CD146 and CD31 in paraffin-embedded brain sections from healthy donors (controls) and patients with active MS lesions. The mean density of CD146 was analyzed using Image ProPlus software. The summary data represent triplicate experiments performed using five healthy donors and five MS patients with active lesions. Scale bar, 50 m. (b) Immunofluorescence analysis of CD146 (red) and caveolin-1(green) in paraffin-embedded CNS sections from normal (non-EAE-induced; naïve) and EAE mice (n = 5 mice/group). The mean integral optical density (IOD) of CD146 immunofluorescence was analyzed using Image-Pro Plus. The nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (c) FACS analysis of CD146 expression (labeled with the anti-CD146 AA98-PE antibody) on CD31-marked BBBECs isolated from naïve and EAE mice was quantified from overlays using FACSDiva. In this and subsequent figures, the error bars represent SEM. **, p < 0.01, ***, p < 0.001 (unpaired Student’s t-test). The summary data in (c) represent three independent experiments.
Supplementary Fig. 2
Supplementary Fig. 2. CD146 is deleted in endothelial cells but not lymphocytes in CD146EC-KO mice. (a) Immunofluorescence staining of CD146 (green) and caveolin-1 (Cav-1, red) expression in paraffin-embedded CNS sections from CD146EC-KO mice. The nuclei were counterstained with DAPI (blue). The arrows indicate Cav-1+ CNS vessels, and the triangle arrows indicate CD146+ leukocytes identified based on their morphology and histology. Scale bar, 50 µm. (b) CD146EC-KO and WT mice have similar numbers of peripheral leukocytes (n=10 mice/group). (c) CD146EC-KO and WT mice have similar percentages of CD4+, CD8+, Th1 and Th17 cellsin the spleen, lymph nodes (LN) and peripheral blood (n=10 mice/group). (d) Summary of the percentage of CD117+CD146+ cells in the bone marrow of CD146EC-KO and WTmice (n=10 mice/group). (e) CD146EC-KO and WT mice have similar numbers of CD146+ T cells in the spleen, lymph nodes and peripheral blood (n=10 mice/group).
Supplementary Fig. 3
Supplementary Fig. 3. TNF stimulation (50 ng/ml) upregulates the expression of CD146, ICAM-1 and VCAM-1 in human BBBECs. Flow cytometry analysis of CD146, ICAM-1, and VCAM-1 expression in BBBECs treated with or without TNF (50 ng/ml). A non-specific isotype-matched antibody was used as a negative control.
Supplementary Fig. 4
Supplementary Fig. 4. Western blot analysis of CD146 protein. BBBECs weretransfected withsiRNA-CD146 (to knockdown CD146 expression)or a control siRNA against GFP either with the CD146-expressing vector Flag-CD146 to restore the expression of CD146 or with an empty vector as a negative control. GAPDH served as a loading control.
References
1.Zhang, Y., et al. Generation and characterization of a panel of monoclonal antibodies against distinct epitopes of human CD146. Hybridoma (Larchmt)27, 345-352 (2008).
2.Yan, X., et al. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood102, 184-191 (2003).
3.McDonald, W.I., et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol50, 121-127 (2001).
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