<em>Neisseria meningitidis</em> Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

University of Würzburg

University of Alabama

Bacterial meningitis is a serious, life-threatening infection of the central nervous system (CNS). To cause meningitis, bacteria must interact with and penetrate the meningeal blood-cerebrospinal fluid barrier (mB/CSFB), which comprises highly specialized brain endothelial cells. Neisseria meningitidis (meningococcus) is a leading cause of bacterial meningitis, and examination meningococcus' interaction with the BBB is critical for understanding disease progression. To examine specific interactions, in vitro mB/CSFB models have been developed and employed and are of great importance because in vivo models have been difficult to produce considering Neisseria meningitidis is exclusively a human pathogen. Most in vitro blood-brain barrier and mB/CSF models use primary and immortalized brain endothelial cells, and these models have been used to examine bacterial-mB/CSFB interactions by a variety of pathogens. This chapter describes the use of past and current in vitro brain endothelial cells to model Neisseria meningitidis interaction with the mB/CSFB, and inform on the standard operating procedure for their use.

In Vitro Models for Studying the Interaction of Neisseria meningitidis with Human Brain Endothelial Cells.

Meningococcal meningitis is a severe central nervous system infection that occurs when  () penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen,  models are greatly limited and pose a significant challenge.  cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific  interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study  host-pathogen interactions, and provide an overview of host responses to  infection. Using iPSC-BECs, we first confirmed that multiple  strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs.  was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines , , , , and , and the secretion of IFN-γ and RANTES. For the first time, we directly observe that  disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon  challenge. In accordance with tight junction loss, a sharp loss in endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of -responsive host genes. Altogether, this model provides novel insights into  pathogenesis, including an impact of  on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to  traversal of BECs.

Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study  Infection.

The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial - BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the  and  models currently being used to study bacterial brain penetration, and opinion on improved models for the future.

Past and Current Perspectives in Modeling Bacteria and Blood-Brain Barrier Interactions.

Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.

Brandon J. Kim

Institute for Hygiene and Microbiology

University of Würzburg

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Brandon J. Kim

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Hygiene and Microbiology

University of Würzburg

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Institute for Hygiene and Microbiology