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, 3 (7), e479

Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

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Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Dennis J Grab et al. PLoS Negl Trop Dis.

Abstract

Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes.

Methodology/principal findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q) with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified.

Conclusions/significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q)-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. T. b. rhodesiense transmigration across HBMEC silenced for F2RL1 expression by RNAi.
Using laser capture microdissection 15 individual HBMEC expressing GFP in the PAR2-silenced and control siRNA cultures were collected and the RNA extracted. (A) Based on qRT-PCR using pre-designed F2RL1 primers and normalized to ACTB (β-actin transcripts), PAR-2 gene expression was reduced by over 95%. (B) T. b. rhodesiense IL1852 was incubated for 16h in triplicate with HBMEC monolayers silenced for PAR-2 expression (F2RL1 siRNA), with a matched scrambled siRNA control (control siRNA) construct, or with untreated HBMEC (no Rx control) and examined parasite transmigration.
Figure 2
Figure 2. Real-time TEER changes in HBMEC silenced for PAR-2 gene expression.
F2RL1 RNAi transfected HBMEC grown in 8W1E ECIS chambers were incubated overnight with T. b. rhodesiense IL1852. The changes in TEER relative to matched HBMEC control containing scrambled siRNA construct (red line) and HBMEC silenced for F2RL1 (blue line) in the presence of parasites are shown.
Figure 3
Figure 3. PMT from Pasteurella multocida blocks T. b. rhodesiense induced changes in HBMEC TEER.
HBMEC grown in 8W10E+ ECIS chambers were incubated with T. b. rhodesiense IL1852. Shown are the changes in real-time TEER measured by ECIS. A) HBMECs incubated with T. b. rhodesiense IL1852 in the absence (blue line) or continuous presence of PMT (30 ng/ml) (red line). B) HBMECs incubated with T. b. rhodesiense IL1852 was incubated with HBMECs untreated (blue line) or pretreated with PMT (30 ng/ml) (red line). The data are represented as the average change in TEER±EM (n = 3). The changes in TEER are represented as the average change in TEER±SEM (n = 2).
Figure 4
Figure 4. Pathway changes altered by T. b. rhodesiense inhibited for brucipain activity.
Significantly regulated, functional pathway clusters were generated from wild-type (WT Tbr) T. b. rhodesiense or parasites K11777 inhibited for brucipain activity (KT Tbr) gene sets using PAGE gene set analysis. Pathways that were significantly up-regulated (red) or down-regulated (green) are shown. Cluster group numbers are shown on the left, while the grading scale is in the upper right.
Figure 5
Figure 5. Proposed model for African trypanosome-induced BBB dysfunction.
We hypothesize that parasite proteases trigger GPCRs (i.e. PARs?) via Gαq activation, which leads to PLC-mediated Ca2+ release from intracellular stores. The increase in intracellular calcium leads to calmodulin (CaM) activation of myosin light chain kinase (MLCK), ultimately leading to cytoskeletal changes and barrier dysfunction. Ca2+-independent activation of the cytoskeleton mediated by Ras-superfamily GTPases (i.e. RhoA) is also possible via p63RhoGEF. Parasite and/or host-derived proteases may also contribute by degrading or altering adherens junction (AJ) and tight junction (TJ) proteins.

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