Role of microRNAs in leukocyte adhesion to human brain microvascular endothelium

Abstract

MicroRNAs (miRs) are small non-coding regulatory RNAs that act through repression of protein translation and/or mRNA degradation at the post-transcriptional level. MiRs are critical players in the pathogenesis of many diseases, including 'neuroinflammatory disorders such as multiple sclerosis (MS), MS is characterized by leukocyte adhesion and infiltration subsequently leading to demyelination of nerve fibres. Leukocyte adhesion on brain endothelial cells (BEC) - the main cellular constituent of the blood-brain barrier (BBB) - is a complex multi-step process where activated BEC overexpress chemokines such as CCL2 and endothelial adhesion molecules (CAM) such as selectins, VCAM1 and ICAM1. Several therapies for MS target the common known mechanisms of leukocyte adhesion. Here, we studied whether specific endothelial miRs act as regulators of leukocyte adhesion to cultured human BEC in vitro, and hence whether they could be a potential therapeutic tool to prevent adhesion to endothelium, First, we characterised leukocyte adhesion using the monocytic (THP1) and T cell (Jurkat) lines under static conditions, interacting with the immortalized hCMEC/ 03 endothelial cell line as an in vitro model of the human BBB. Increased adhesion of both leukocytic cell lines to BEC was observed following treatment with TNFu and IFNy compared to unstimulated cells. Increased expression of both ICAM1 and VCAM1 by hCMEC/D3 cells was also observed following cytokine treatment. Cytokine-induced maximal VCAM1 and ICAM1 expression coincided with the observed maximal leukocyte adhesion to BEC at 24 h, Next, we established a novel flow-based leukocyte adhesion assay coupled with time lapse image acquisition, to mimic more closely the in vivo conditions. We successfully cultured and transfected hCMEC/D3 cells in six-channel chambers, connected to a flow system, to study leukocyte-endothelium interactions and firm adhesion, Second, we performed an initial screening of five cytokine-regulated BEC miRs, Of these five, miR-126 and miR-155 appeared to have the most significant effects on leukocyte adhesion to hCMEC/D3cells, We further investigated the roles of miR-126, miR-126* (the complement of miR-126), and miR-155 in leukocyte adhesion to BEC. MiR-126 and - 126* were down-regulated in cytokine stimulated BEC low levels of miR-126 increased adhesion of both cell lines, while low levels of miR-l26* increased THP-1, but reduced Jurkat adhesion. Elevated miR-l26 and miR-126* levels significantly prevented Jurkat and THP-1 cell adhesion to BEC both in unstimulated and cytokine-treated conditions. Furthermore, elevated miR-126 partially prevented cytokine-induced VCAM1 and CCL2 expression on BEC and an increased level of miR-126* partially prevented cytokine-induced E-selectin expression. In cytokine stimulated-BEC miR-155 was up-regulated, and decreasing the level of miR-155 reduced both T cell and monocyte adhesion to endothelium and VCAMl expression both in basal and in cytokine-stimulated conditions, The opposite effect on leukocyte adhesion was observed when miR-155 expression was increased in unstimulated hCMEC/D3 cells, but not in cytokine-stimulated endothelium. These data suggest that miR-155, miR-126 and miR-126* modulate leukocyte adhesion on human brain microvascular endothelium. To our knowledge, this study is the first to report a role for miR-155 and miR-l26* in the interactions between human brain endothelium and immune cells and the first to confirm the regulation of VCAM1 and CCL2 by miR-126 in brain endothelium.