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A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier.

ABSTRACT: The blood-brain barrier (BBB) comprises the foremost protective barrier in the brain and is composed in part of a layer of microvascular endothelial cells that line the capillaries surrounding the brain. Here, we describe a human three-dimensional (3-D) cell-based model of the BBB microvascular endothelium that recapitulates properties of these cells in vivo, including physiologically relevant transcriptional profiles, the capacity to induce potent antimicrobial innate immune signaling, and the ability to resist infection by diverse RNA viruses, including members of the enterovirus (coxsackievirus B, echovirus 11, enterovirus 71, poliovirus) and flavivirus (dengue virus, Zika virus [ZIKV]) families. We show that disruption of apical tight junctions by proinflammatory cytokine tumor necrosis factor alpha (TNF-?) sensitizes 3-D-cultured BBB cells to ZIKV infection and that 3-D derived BBB cells can be used to model the transmigration of ZIKV-infected monocytes across the endothelial barrier to access underlying astrocytes. Taken together, our findings show that human BBB microvascular endothelial cells cultured in 3-D can be used to model the mechanisms by which RNA viruses access the central nervous system (CNS), which could be used for the development and screening of therapeutics to limit this event. IMPORTANCE Neurotropic viral infections are significant sources of global morbidity and mortality. The blood-brain barrier (BBB) is composed in part of a layer of microvascular endothelial cells and functions to restrict viral access to the brain. In vitro models that recapitulate many of the properties of the human BBB endothelium are lacking, particularly with respect to the unique cellular and immunological mechanisms by which these cells restrict viral infections of the brain. Here, we developed a three-dimensional cell culture model that recapitulates many of the morphological and functional properties of the BBB microvasculature and apply this model to the study of RNA virus infections. The model we describe can therefore be used to study a variety of aspects of BBB physiology, including the mechanisms by which viruses might access the CNS, and could be used for the development and screening of antiviral therapeutics to limit this important step in viral pathogenesis.

SUBMITTER: Bramley JC

PROVIDER: S-EPMC5480033 | biostudies-literature | 2017 May-Jun

REPOSITORIES: biostudies-literature

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A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier.

Bramley John C JC Drummond Coyne G CG Lennemann Nicholas J NJ Good Charles A CA Kim Kwang Sik KS Coyne Carolyn B CB

mSphere 20170501 3

The blood-brain barrier (BBB) comprises the foremost protective barrier in the brain and is composed in part of a layer of microvascular endothelial cells that line the capillaries surrounding the brain. Here, we describe a human three-dimensional (3-D) cell-based model of the BBB microvascular endothelium that recapitulates properties of these cells <i>in vivo</i>, including physiologically relevant transcriptional profiles, the capacity to induce potent antimicrobial innate immune signaling, a ...[more]

PMID: 28656176

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Project description:Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening recently entered clinical testing for targeted drug delivery to the brain. Sources of variability exist in the current procedures, motivating the development of real-time monitoring and control techniques to improve treatment safety and efficacy. Here we used three-dimensional (3D) transcranial microbubble imaging to calibrate FUS exposure levels for volumetric BBB opening. Methods: Using a sparse hemispherical transmit/receive ultrasound phased array, pulsed ultrasound was focused transcranially into the thalamus of rabbits during microbubble infusion and multi-channel 3D beamforming was performed online with receiver signals captured at the subharmonic frequency. Pressures were increased pulse-by-pulse until subharmonic activity was detected on acoustic imaging (psub), and tissue volumes surrounding the calibration point were exposed at 50-100%psub via rapid electronic beam steering. Results: Spatially-coherent subharmonic microbubble activity was successfully reconstructed transcranially in vivo during calibration sonications. Multi-point exposures induced volumetric regions of elevated BBB permeability assessed via contrast-enhanced magnetic resonance imaging (MRI). At exposure levels ≥75%psub, MRI and histological examination occasionally revealed tissue damage, whereas sonications at 50%psub were performed safely. Substantial intra-grid variability of FUS-induced bioeffects was observed via MRI, prompting future development of multi-point calibration schemes for improved treatment consistency. Receiver array sparsity and sensor configuration had substantial impacts on subharmonic detection sensitivity, and are factors that should be considered when designing next-generation clinical FUS brain therapy systems. Conclusion: Our findings suggest that 3D subharmonic imaging can be used to calibrate exposure levels for safe FUS-induced volumetric BBB opening, and should be explored further as a method for cavitation-mediated treatment guidance.

Project description:Tembusu Virus (TMUV) is an emerging and re-emerging zoonotic pathogen that adversely affects poultry industry in recent years. TMUV disease is characterized by nonsuppurative encephalitis in ducklings. The duckling infection model was established to study the mechanism of TMUV crossing the blood-brain barrier (BBB) into the central nervous system (CNS). Here, we showed that no obvious clinical symptoms and enhancement of BBB permeability occurred at the early stage of infection (3∼5 dpi). While simultaneously virus particles were observed by transmission electron microscopy in the brain, inducing the accumulation of inflammatory cytokines. Neurological symptoms and disruption of BBB appeared at the intermediate stage of infection (7∼9 dpi). It was confirmed that TMUV could survive and propagate in brain microvascular endothelial cells (BMECs), but did not affect the permeability of BBB in vivo and in vitro at an early date. In conclusion, TMUV enters the CNS then causes encephalitis, and finally destruct the BBB, which may be due to the direct effect of TMUV on BMECs and the subsequent response of "inflammatory storm".IMPORTANCE The TMUV disease has caused huge losses to the poultry industry in Asia, which is potentially harmful to public health. Neurological symptoms and their sequelae are the main characters of this disease. However, the mechanism of how this virus enters the brain and causes encephalitis is unclear. In this study, we confirmed that the virus entered the CNS and then massively destroyed BBB and the BBB damage was closely associated with the subsequent outbreak of inflammation. TMUV may enter the CNS through the transcellular and "Trojan horse" pathways. These findings can fill the knowledge gap in the pathogenesis of TMUV-infected poultry and be benefit for the treatment of TMUV disease. What's more, TMUV is a representative to study the infection of avian flavivirus. Therefore, our studies have significances both for understanding of the full scope of mechanisms of TMUV and other flavivirus infection, and conceivably, for therapeutics.

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A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier.

Publications

A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier.

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