Abstract
Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 μM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
de Almeida SM, Queiroz-Telles F, Teive HA, Ribeiro CE, Werneck LC (2004) Central nervous system paracoccidioidomycosis: clinical features and laboratorial findings. J Inf Secur 48:193–198
Bassetti M, Garnacho-Montero J, Calandra T, Kullberg B, Dimopoulos G, Azoulay E, Chakrabarti A, Kett D, Leon C, Ostrosky-Zeichner L, Sanguinetti M, Timsit JF, Richardson MD, Shorr A, Cornely OA (2017) Intensive care medicine research agenda on invasive fungal infection in critically ill patients. Intensive Care Med 43:1225–1238. https://doi.org/10.1007/s00134-017-4731-2
Bertossi M, Girolamo F, Errede M, Virgintino D, Roncali L (2003) Effects of 6-aminonicotinamide gliotoxin on blood-brain barrier differentiation in the chick embryo cerebellum. Anat Embryol (Berl) 207:209–219. https://doi.org/10.1007/s00429-003-0335-4
Bossou YM, Serssar Y, Allou A, Vitry S, Momas I, Seta N, Menotti J, Achard S (2017) Impact of mycotoxins secreted by Aspergillus molds on the inflammatory response of human corneal epithelial cells. Toxins (Basel) 9(7). https://doi.org/10.3390/toxins9070197
Brennan L, Alves PM, Hewage C, Malthouse JP, McBean GJ (2006) Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes. Neurochem Int 48:739–745. https://doi.org/10.1016/j.neuint.2005.12.004
Camire RB, Beaulac HJ, Willis CL (2015) Transitory loss of glia and the subsequent modulation in inflammatory cytokines/chemokines regulate paracellular claudin-5 expression in endothelial cells. J Neuroimmunol 284:57–66. https://doi.org/10.1016/j.jneuroim.2015.05.008
Efthymiou A, Shaltouki A, Steiner JP, Jha B, Heman-Ackah SM, Swistowski A, Zeng X, Rao MS, Malik N (2014) Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells. J Biomol Scr 19:32–43. https://doi.org/10.1177/1087057113501869
Eichner RD, Al Salami M, Wood PR, Mullbacher A (1986) The effect of gliotoxin upon macrophage function. Int J Immunopharmacol 8:789–797
Fennelly AM, Slenker AK, Murphy LC, Moussouttas M, DeSimone JA (2013) Candida cerebral abscesses: a case report and review of the literature. Med Mycol 51:779–784. https://doi.org/10.3109/13693786.2013.789566
Jong A, Wu CH, Shackleford GM, Kwon-Chung KJ, Chang YC, Chen HM, Ouyang Y, Huang SH (2008) Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells. Cell Microbiol 10:1313–1326. https://doi.org/10.1111/j.1462-5822.2008.01128.x
Jong A, Wu CH, Gonzales-Gomez I, Kwon-Chung KJ, Chang YC, Tseng HK, Cho WL, Huang SH (2012) Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection. J Biol Chem 287:15298–15306. https://doi.org/10.1074/jbc.M112.353375
Kleinschmidt-DeMasters BK (2002) Central nervous system aspergillosis: a 20-year retrospective series. Hum Pathol 33:116–124
Koh W, Mahan RD, Davis GE (2008) Cdc42- and Rac1-mediated endothelial lumen formation requires PAK2, PAK4 and PAR3, and PKC-dependent signaling. J Cell Sci 121:989–1001. https://doi.org/10.1242/jcs.020693
Lewis RE, Wiederhold NP, Chi J, Han XY, Komanduri KV, Kontoyiannis DP, Prince RA (2005a) Detection of gliotoxin in experimental and human aspergillosis. Infect Immun 73:635–637. https://doi.org/10.1128/IAI.73.1.635-637.2005
Lewis RE, Wiederhold NP, Lionakis MS, Prince RA, Kontoyiannis DP (2005b) Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J Clin Microbiol 43:6120–6122. https://doi.org/10.1128/JCM.43.12.6120-6122.2005
Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A, Palecek SP, Shusta EV (2012) Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783–791. https://doi.org/10.1038/nbt.2247
Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV (2014) A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci Rep 4:4160. https://doi.org/10.1038/srep04160
Liu TB, Perlin DS, Xue C (2012) Molecular mechanisms of cryptococcal meningitis. Virulence 3:173–181. https://doi.org/10.4161/viru.18685
Mantle JL, Min L, Lee KH (2016) Minimum transendothelial electrical resistance thresholds for the study of small and large molecule drug transport in a human in vitro blood-brain barrier model. Mol Pharm 13:4191–4198. https://doi.org/10.1021/acs.molpharmaceut.6b00818
Mathisen G, Shelub A, Truong J, Wigen C (2010) Coccidioidal meningitis: clinical presentation and management in the fluconazole era. Medicine (Baltimore) 89:251–284. https://doi.org/10.1097/MD.0b013e3181f378a8
Mazaheri M, Moosavi-Movahedi AA, Saboury AA, Khodagholi F, Shaerzadeh F, Sheibani N (2015) Curcumin protects beta-lactoglobulin fibril formation and fibril-induced neurotoxicity in PC12 cells. PLoS One 10:e0133206. https://doi.org/10.1371/journal.pone.0133206
Mehrzad J, Malvandi AM, Alipour M, Hosseinkhani S (2017) Environmentally relevant level of aflatoxin B1 elicits toxic pro-inflammatory response in murine CNS-derived cells. Toxicol Lett 279:96–106. https://doi.org/10.1016/j.toxlet.2017.07.902
Menard A, Amouri R, Dobransky T, Charriaut-Marlangue C, Pierig R, Cifuentes-Diaz C, Ghandour S, Belliveau J, Gascan H, Hentati F, Lyon-Caen O, Perron H, Rieger F (1998) A gliotoxic factor and multiple sclerosis. J Neurol Sci 154:209–221
Montesano R, Pepper MS, Mohle-Steinlein U, Risau W, Wagner EF, Orci L (1990) Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell 62:435–445
Murayama T, Amitani R, Ikegami Y, Nawada R, Lee WJ, Kuze F (1996) Suppressive effects of Aspergillus fumigatus culture filtrates on human alveolar macrophages and polymorphonuclear leucocytes. Eur Respir J 9:293–300
Patel R, Alahmad AJ (2016) Growth-factor reduced matrigel source influences stem cell derived brain microvascular endothelial cell barrier properties. Fluids Barriers CNS 13. https://doi.org/10.1186/s12987-016-0030-5
Patel R, Page S, Al-Ahmad AJ (2017) Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality. J Neurochem 142:74–88. https://doi.org/10.1111/jnc.14040
Perriere N, Demeuse P, Garcia E, Regina A, Debray M, Andreux JP, Couvreur P, Scherrmann JM, Temsamani J, Couraud PO, Deli MA, Roux F (2005) Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties. J Neurochem 93:279–289. https://doi.org/10.1111/j.1471-4159.2004.03020.x
Quinn M, McMillin M, Galindo C, Frampton G, Pae HY, DeMorrow S (2014) Bile acids permeabilize the blood brain barrier after bile duct ligation in rats via Rac1-dependent mechanisms. Dig Liver Dis 46:527–534. https://doi.org/10.1016/j.dld.2014.01.159
Qureshi H, Hamid SS, Ali SS, Anwar J, Siddiqui AA, Khan NA (2015) Cytotoxic effects of aflatoxin B1 on human brain microvascular endothelial cells of the blood-brain barrier. Med Mycol 53:409–416. https://doi.org/10.1093/mmy/myv010
Sajja RK, Green KN, Cucullo L (2015) Altered Nrf2 signaling mediates hypoglycemia-induced blood-brain barrier endothelial dysfunction in vitro. PLOS ONE 10(3):e0122358. https://doi.org/10.1371/journal.pone.0122358
Schestatsky P, Chedid MF, Amaral OB, Unis G, Oliveira FM, Severo LC (2006) Isolated central nervous system histoplasmosis in immunocompetent hosts: a series of 11 cases. Scand J Infect Dis 38:43–48. https://doi.org/10.1080/00365540500372895
Schlam D, Canton J, Carreno M, Kopinski H, Freeman SA, Grinstein S, Fairn GD (2016) Gliotoxin suppresses macrophage immune function by subverting phosphatidylinositol 3,4,5-trisphosphate homeostasis. MBio 7:e02242. https://doi.org/10.1128/mBio.02242-15
Shi M, Mody CH (2016) Fungal infection in the brain: what we learned from intravital imaging. Front Immunol 7:292. https://doi.org/10.3389/fimmu.2016.00292
Stanzani M, Orciuolo E, Lewis R, Kontoyiannis DP, Martins SL, St John LS, Komanduri KV (2005) Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 105:2258–2265. https://doi.org/10.1182/blood-2004-09-3421
Trebak F, Alaoui A, Alexandre D, El Ouezzani S, Anouar Y, Chartrel N, Magoul R (2015) Impact of aflatoxin B1 on hypothalamic neuropeptides regulating feeding behavior. Neurotoxicology 49:165–173. https://doi.org/10.1016/j.neuro.2015.06.008
Tsunawaki S, Yoshida LS, Nishida S, Kobayashi T, Shimoyama T (2004) Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase. Infect Immun 72:3373–3382. https://doi.org/10.1128/IAI.72.6.3373-3382.2004
Wang ZG, Cheng Y, Yu XC, Ye LB, Xia QH, Johnson NR, Wei X, Chen DQ, Cao G, Fu XB, Li XK, Zhang HY, Xiao J (2016) Bfgf protects against blood-brain barrier damage through junction protein regulation via PI3K-Akt-Rac1 pathway following traumatic brain injury. Mol Neurobiol 53:7298–7311. https://doi.org/10.1007/s12035-015-9583-6
Waring P, Eichner RD, Mullbacher A, Sjaarda A (1988) Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties. J Biol Chem 263:18493–18499
Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1874. https://doi.org/10.1096/fj.04-3458fje
Willis CL, Leach L, Clarke GJ, Nolan CC, Ray DE (2004a) Reversible disruption of tight junction complexes in the rat blood-brain barrier, following transitory focal astrocyte loss. Glia 48:1–13. https://doi.org/10.1002/glia.20049
Willis CL, Nolan CC, Reith SN, Lister T, Prior MJ, Guerin CJ, Mavroudis G, Ray DE (2004b) Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact. Glia 45:325–337. https://doi.org/10.1002/glia.10333
Woollard SM, Li H, Singh S, Yu F, Kanmogne GD (2014) HIV-1 induces cytoskeletal alterations and Rac1 activation during monocyte-blood-brain barrier interactions: modulatory role of CCR5. Retrovirology 11:20. https://doi.org/10.1186/1742-4690-11-20
Wu F, Chen Z, Tang C, Zhang J, Cheng L, Zuo H, Zhang H, Chen D, Xiang L, Xiao J, Li X, Xu X, Wei X (2017) Acid fibroblast growth factor preserves blood-brain barrier integrity by activating the PI3K-Akt-Rac1 pathway and inhibiting RhoA following traumatic brain injury. Am J Transl Res 9:910–925
Yan Y, Shin S, Jha BS, Liu Q, Sheng J, Li F, Zhan M, Davis J, Bharti K, Zeng X, Rao M, Malik N, Vemuri MC (2013) Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells. Stem Cells Transl Med 2:862–870. https://doi.org/10.5966/sctm.2013-0080
Yoshida LS, Abe S, Tsunawaki S (2000) Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils. Biochem Biophys Res Commun 268:716–723. https://doi.org/10.1006/bbrc.2000.2192
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. https://doi.org/10.1126/science.1151526
Acknowledgments
Texas Tech University Health Sciences Center institutional funds to N.G. and A.A.
Funding
This study was supported by Texas Tech University Health Sciences Center institutional funds to N.G. and A.A.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
None.
Electronic supplementary material
ESM 1
(PDF 3030 kb)
Rights and permissions
About this article
Cite this article
Patel, R., Hossain, M.A., German, N. et al. Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro. Mycotoxin Res 34, 257–268 (2018). https://doi.org/10.1007/s12550-018-0320-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12550-018-0320-7