African Trypanosome-Induced Blood–Brain Barrier Dysfunction under Shear Stress May Not Require ERK Activation

Abstract

Keywords► African trypanosomes ► blood-brain barrier ► brain microvascular endothelial cells ► trypanosoma brucei rhodesiense ► shear stress ► MAPK AbstractAfrican trypanosomes are tsetse fly transmitted protozoan parasites responsible for human African trypanosomiasis, a disease characterized by a plethora of neurological symptoms and death. How the parasites under microvascular shear stress (SS) flow conditions in the brain cross the blood-brain barrier (BBB) is not known. In vitro studies using static models comprised of human brain microvascular endothelial cells (BMEC) show that BBB activation and crossing by trypanosomes requires the orchestration of parasite cysteine proteases and host calcium-mediated cell signaling. Here, we examine BMEC barrier function and the activation of extracellular signal-regulated kinase (ERK)1/ 2 and ERK5, mitogen-activated protein kinase family regulators of microvascular permeability, under static and laminar SS flow and in the context of trypanosome infection. Confluent human BMEC were cultured in electric cell-substrate impedance sensing (ECIS) and parallel-plate glass slide chambers. The human BMEC were exposed to 2 or 14 dyn/cm 2 SS in the presence or absence of trypanosomes. Real-time changes in transendothelial electrical resistance (TEER) were monitored and phosphorylation of ERK1/2 and ERK5 analyzed by immunoblot assay. After reaching confluence under static conditions human BMEC TEER was found to rapidly increase when exposed to 2 dyn/cm 2 SS, a condition that mimics SS in brain postcapillary venules. Addition of African trypanosomes caused a rapid drop in human BMEC TEER. Increasing SS to 14 dyn/cm 2 , a condition mimicking SS in brain capillaries, led to a transient increase in TEER in both control and infected human BMEC. However, no differences in ERK1/2 and ERK5 activation were found under any condition tested. African trypanosomiasis alters BBB permeability under low shear conditions through an ERK1/2 and ERK5 independent pathway.

New experimental models of the blood-brain barrier for CNS drug discovery

“…Previously, it was shown that intraluminal flow is critical for development and maintenance of BBB phenotype in vivo and in vitro resulting in high barrier tightness [8, 35, 36]. Various in vitro dynamic models (DIV-BBB) were developed to understand the role of laminar or pulsatile shear stress on BBB endothelial function and dysfunction in various pathological conditions including inflammation and stroke [31, 37, 38, 39, 40]. For instance, exposure of immortalized hCMEC/D3 cell line to pulsatile flow strongly potentiates the barrier integrity, with 10-fold increase in TEER values compared to static cultures [41].…”

Biochemical‐ and Biophysical‐Induced Barriergenesis in the Blood–Brain Barrier: A Review of Barriergenic Factors for Use in In Vitro Models

“…[ 272 ] It is thought that these improvements to barrier properties arise from the interplay between the Wnt/B‐catenin signaling cascade with hypoxia‐induced factor 1a (HIF1a) signaling, which Park et al were able to show through upregulation of HIF1a in their hypoxic conditions. [ 274 ] This offers a potential work‐around for the induction of hypoxic conditions, as HIF1a‐mimetic or stabilizing compounds can be used in addition to or instead of the use of a hypoxic chamber.…”

A novel mechanosensitive channel controls osmoregulation, differentiation, and infectivity in Trypanosoma cruzi

“…MSCs in prokaryotes have been postulated to participate in quorum sensing, biofilm formation and regulation of virulence factors [19][20][21][22]. Protozoan pathogens, like trypanosomes and apicomplexans, are subjected to mechanical forces associated with shear stress in the bloodstream [23,24], extravasation [25,26] and invasion of tissues required for establishing persistent infection [27][28][29][30][31]. While it is clear that mechanical cues modulate the parasite life cycle and behavior, the molecular mechanism underlying sensing and triggering of the responses required for survival is unknown.…”

“…MSCs in prokaryotes have been postulated to participate in quorum sensing, biofilm formation, and regulation of virulence factors ( Siryaporn et al, 2014 ; O'Loughlin et al, 2013 ; Thomen et al, 2017 ; Prindle et al, 2015 ). Protozoan pathogens, like trypanosomes and apicomplexans, are subjected to mechanical forces associated with shear stress in the bloodstream ( Harker et al, 2014 ; Sumpio et al, 2015 ), extravasation ( Laperchia et al, 2016 ; Coates et al, 2013 ), and invasion of tissues required for establishing persistent infection ( Barragan et al, 2005 ; Barragan and Sibley, 2002 ; Capewell et al, 2016 ; Trindade et al, 2016 ; Mattos et al, 2019 ). While it is clear that mechanical cues modulate the parasite’s life cycle and behavior, the molecular mechanism underlying sensing and triggering of the responses required for survival is unknown.…”