Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration.

Trafficking of myelin-reactive CD4(+) T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4(+) T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment.

Brain endothelial miR-146a negatively modulates T-cell adhesion through repressing multiple targets to inhibit NF-κB activation.

Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.

MRI of monocyte infiltration in an animal model of neuroinflammation using SPIO-labeled monocytes or free USPIO.

Magnetic resonance imaging (MRI) has been applied to visualize monocyte infiltration with the use of intravenously injected ultrasmall superparamagnetic iron oxide (USPIO). However, USPIO uptake in vivo remains elusive, and the heterogeneous enhancement patterns observed by MRI point to multiple pathophysiological events. This study focused on specific imaging of monocyte infiltration into the brain by transfusion of superparamagnetic iron oxide (SPIO)-labeled monocytes in a rat model of neuroinflammation, experimentally induced photothrombosis (PT). At day 5 after lesion induction, animals were transfused with SPIO-labeled monocytes (5 x 10(6) cells) or free USPIO (17 mg Fe/kg). MRI was performed 24, 72 and, 120 h later. To investigate temporal changes directly after intravenous USPIO administration, MRI was performed repeatedly up to 8 h. Relaxation measurements showed that rat monocytes were efficiently labeled in vitro using SPIO (R2=12+/-0.9 s(-1)). After transfusion of SPIO-labeled monocytes, a significant increase in contrast enhanced area (340%+/-106%) in the PT lesion was observed not before 72 h. Contrast enhancement after USPIO injection increased up to 407%+/-39% at a much earlier point of time (24 h) and diminished thereafter. Repetitive MRI directly after USPIO injection showed significant contrast enhancement in the lesion within 2 h. Our study shows that MRI enables in vivo tracking of SPIO-labeled monocytes longitudinally. Moreover, our data suggest that contrast enhancement after injection of free USPIO does not primarily represent signals from peripherally labeled monocytes that migrated toward the inflammatory lesion. The use of SPIO-labeled monocytes provides a better tool to specifically assess the time window of monocyte infiltration.