Original article Cryptococcal transmigration across a model brain blood-barrier: evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265 Q3 Q2 Tania C. Sorrell a, *, Pierre Juillard a,b,d , Julianne T. Djordjevic a,b , Keren Francis a,b , Anelia Dietmann c,d , Alban Milonig c,d , Valery Combes a,d , Georges E.R. Grau a,d a Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney and Westmead Millennium Institute for Medical Research, Westmead, New South Wales 2145, Australia b Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Westmead 2145, Australia c Department of Neurology, Innsbruck Medical University, Innsbruck, Austria d Vascular Immunology Unit, Department of Pathology, School of Medical Sciences, University of Sydney, NSW 2006, Australia Received 11 May 2015; accepted 31 August 2015 Available online ▪▪▪ Abstract Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-g stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Cryptococcus; Monocytes; Brain endothelium; Phagocytosis; Transmigration 1. Introduction Despite the known predilection of Cn and Cg for the lung and central nervous system, the emergence of the VGII genotype of Cryptococcus gattii on Vancouver Island several years ago has revealed differences in clinical presentation [1]. In addition, Cn and Cg differ in host preference [2]. For example, Cg VGII most commonly causes lung disease and affects apparently healthy hosts whereas Cn var. grubii (molecular types VNI and VNII predominantly) typically causes meningitis in HIV-infected or otherwise immune- compromised hosts. In rat and mouse models, infection via the respiratory route with the type strain of Cn, H99 and the VGII outbreak strain of Cg (R265), resulted in mortality due * Corresponding author. Marie Bashir Institute for Infectious Diseases & Biosecurity, University of Sydney; Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, 178 Hawkesbury Rd, West- mead NSW 2145, Australia. Tel.: þ61 2 9845 6029. Q1 E-mail address: tania.sorrell@sydney.edu.au (T.C. Sorrell). Please cite this article in press as: Sorrell TC, et al., Cryptococcal transmigration across a model brain blood-barrier: evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265, Microbes and Infection (2015), http://dx.doi.org/ 10.1016/j.micinf.2015.08.017 Microbes and Infection xx (2015) 1e11 www.elsevier.com/locate/micinf + MODEL MICINF4312_proof ■ 16 September 2015 ■ 1/11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 http://dx.doi.org/10.1016/j.micinf.2015.08.017 1286-4579/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.