MiR-126 and miR-126* regulate shear-resistant firm leukocyte adhesion to human brain endothelium

Camilla Cerutti, Laura J Edwards, Helga E de Vries, Basil Sharrack, David K Male, Ignacio A Romero, Camilla Cerutti, Laura J Edwards, Helga E de Vries, Basil Sharrack, David K Male, Ignacio A Romero

Abstract

Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Leukocyte adhesion is mediated mainly by selectins, cell adhesion molecules and chemokines induced by pro-inflammatory cytokines such as TNFα and IFNγ, but the regulation of this process is not fully clear. This study investigated the regulation of firm leukocyte adhesion to human brain endothelium by two different brain endothelial microRNAs (miRs), miR-126 and miR-126*, that are downregulated by TNFα and IFNγ in a human brain endothelial cell line, hCMEC/D3. Using a leukocyte adhesion in vitro assay under shear forces mimicking blood flow, we observed that reduction of endothelial miR-126 and miR-126* enhanced firm monocyte and T cell adhesion to hCMEC/D3 cells, whereas their increased expression partially prevented THP1, Jurkat and primary MS patient-derived PBMC firm adhesion. Furthermore, we observed that miR-126* and miR-126 downregulation increased E-selectin and VCAM1, respectively, while miR-126 overexpression reduced VCAM1 and CCL2 expression by hCMEC/D3 cells, suggesting that these miRs regulate leukocyte adhesion by modulating the expression of adhesion-associated endothelial mRNA targets. Hence, human brain endothelial miR-126 and miR-126* could be used as a therapeutic tool to reduce leukocyte adhesion and thus reduce neuroinflammation.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. TNFα + IFNγ increase E-selectin,…
Figure 1. TNFα + IFNγ increase E-selectin, ICAM1 and VCAM1 expression, enhance firm leukocyte adhesion and downregulate miR-126 and miR-126* expression in hCMEC/D3 cells.
hCMEC/D3 cell monolayers were treated with a combination of cytokines (TNFα + IFNγ) (A) at different concentrations (0, 0.1, 1, 10 ng/ml) or (B–D,F) at 1 ng/ml or left without (w/o) for 24 h. (A) VCAM1, ICAM1 and E-selectin expression levels were quantified by ELISA. (B) VCAM1 expression was quantified by immunofluorescence and expressed as Integrated Density in arbitrary units (A.U.). (C) Numbers of shear-resistant firmly adhered Jurkat and THP1 cells to hCMEC/D3 monolayer per field of view (FOV) (640 × 480 μm). (D) Representative images of shear-resistant firmly adhered fluorescently labelled Jurkat T cells (small white rounded cells) to unstimulated (left) or cytokine treated (right) hCMEC/D3 cells (monolayer of spindle shaped cells in bright field) per FOV (640 × 480 μm). (E) Heatmap representing miR-126 and miR-126* expression in unstimulated hCMEC/D3 cells and after stimulation (10 ng/ml of TNFα + IFNγ) for 24 h (n = 3). Individual repeats are shown in the heatmap. Blue indicates under expression, red overexpression, and intensity of color indicates relative change. Rows were colored using a z-score derived from a gene’s expression across all samples (row z-score). Data from Geo accession GSE21350 (Platform GPL14767) of miR-126 and miR-126* are represented. (F) Relative miR-126 and miR-126* level expression measured by qRT2-PCR from total RNA. The small nuclear RNA U6 was used as internal control. Experiments were carried out three times to four times with (A,B,F) three replicates (C) five FOV. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, *compared to untreated (w/o TNFα + IFNγ).
Figure 2. Human brain endothelial miR-126 downregulation…
Figure 2. Human brain endothelial miR-126 downregulation enhances shear-resistant firmly adhered Jurkat and THP1 cells to hCMEC/D3 cells.
hCMEC/D3 cell were transfected with control Scrambled Anti-miR (grey) or with Anti-miR-126 (black) followed by treatment with cytokines (TNFα + IFNγ, 1 ng/ml) for 24 h or left unstimulated. (A) Relative miR-126 level expression measured by qRT2-PCR from total RNA. The small nuclear RNA U6 was used as internal control. (B,C) Numbers of shear-resistant firmly adhered Jurkat T cells and THP1 monocytes to (B) unstimulated and (C) cytokine-stimulated hCMEC/D3 cells per field of view (FOV) (640 × 480 μm). Experiments were carried out three to five times (A) three replicates (B,C) five FOVs. Data are mean ± SEM. #P < 0.05, **P < 0.01, #compared to unstimulated.
Figure 3. miR-126 upregulation decreases monocytic, T…
Figure 3. miR-126 upregulation decreases monocytic, T cell, PBMC and MS-derived PBMC firm adhesion to hCMEC/D3 cells.
hCMEC/D3 cells were transfected with control Scrambled Pre-miR (grey) or with Pre-miR-126 (black) followed by treatment with cytokines (TNFα + IFNγ, 1 ng/ml) for 24 h or left unstimulated. (A) Relative miR-126 level expression measured by qRT2-PCR from total RNA. The small nuclear RNA U6 was used as internal control. (B,C) Numbers of shear-resistant firmly adhered Jurkat T cells and THP1 monocytes to (B) unstimulated and (C) cytokine-stimulated hCMEC/D3 monolayers per field of view (FOV) (640 × 480 μm). (D) Information of the three PBMC healthy donors and multiple sclerosis (MS) patient PBMC donors; pathological features, relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive (PPMS). (E,F) Numbers of shear-resistant firmly adhered (E) healthy donor PBMC and (F) MS patient derived PBMC to cytokine-stimulated hCMEC/D3 monolayers per field of view (FOV) (640 × 480 μm). (G) Representative images of shear-resistant firmly adhered healthy donor PBMC and MS patient derived PBMC MS to control scrambled Pre-miR or Pre-miR-126 transfected and cytokine-treated hCMEC/D3 cells (monolayer of spindle shaped cells in bright field) per FOV (640 × 480 μm). Experiments were carried out (B,C) three times to five times with five FOVs and (E,F) with ten FOVs. Data are mean ± SEM. *,#P < 0.05, **P < 0.01, ***P < 0.001, #compared to unstimulated.
Figure 4. miR-126* downregulation regulates THP1 monocyte…
Figure 4. miR-126* downregulation regulates THP1 monocyte and Jurkat T cell firm adhesion to hCMEC/D3 cells.
hCMEC/D3 cell were transfected with control Scrambled Anti-miR (grey) or with Anti-miR-126* (black) followed by treatment with cytokines (TNFα + IFNγ, 1 ng/ml) for 24 h or left unstimulated. (A) Relative miR-126* level expression measured by qRT2-PCR from total RNA. The small nuclear RNA U6 was used as internal control. (B,C) Numbers of shear-resistant firmly adhered (B) THP1 monocytes and (C) Jurkat T cells to unstimulated and cytokine-stimulated hCMEC/D3 cells per field of view (FOV) (640 × 480 μm). Experiments were carried out three times with (A) two replicates (B,C) five FOVs. Data are mean ± SEM. #P < 0.05, **P < 0.01, ***,###P < 0.001, #compared to unstimulated.
Figure 5. miR-126* upregulation decreases shear-resistant firmly…
Figure 5. miR-126* upregulation decreases shear-resistant firmly adhered Jurkat and THP1 cells to hCMEC/D3 cells.
hCMEC/D3 cells were transfected with control Scrambled Pre-miR (grey) or with Pre-miR-126 (black) (A) Relative miR-126* level expression measured by qRT2-PCR from total RNA. The small nuclear RNA U6 was used as internal control. (B,C) Numbers of shear-resistant firmly adhered THP1 monocytes and Jurkat T cells to (B) unstimulated and (C) cytokine-stimulated hCMEC/D3 cells per field of view (FOV) (640 × 480 μm). Experiments were carried out three times with five FOVs. Data are mean ± SEM. #P < 0.05, **P < 0.01, ***,###P < 0.001, #compared to unstimulated.
Figure 6. Identification of miR-126 and miR-126*…
Figure 6. Identification of miR-126 and miR-126* adhesion-related putative gene targets in silico.
(A) Stem loop of the primary precursor (Pri-miR) structure of human miR-126 and miR-126* and the mature hsa-miR-126 and has-miR-126* sequences. (B) List of adhesion-associated hsa-miR-126 and hsa-miR-126* predicted targets in silico. (C) Sequence alignment of the predicted duplex formation between mature miRs and their putative gene target mRNA selected for further study in hCMEC/D3 cells. (D) Graphic representation of the experimental design used to study the predicted target at protein level, the same experimental well was used to study membrane associated protein and secreted protein.
Figure 7. Brain endothelial miR-126 modulates VCAM1…
Figure 7. Brain endothelial miR-126 modulates VCAM1 and CCL2 in hCMEC/D3 cells.
(A–D) hCMEC/D3 cells were transfected with control Scrambled Pre-miR or Pre-miR-126 or control Scrambled Anti-miR or Anti-miR-126 (C,D) followed by treatment with cytokines (TNFα + IFNγ, 1 ng/ml) for 24 h or (A,B) left unstimulated. (A,C) Membrane-associated VCAM1 expression was quantified by ELISA and (B,D) secreted CCL2 (MCP1) was measured by capture ELISA. Experiments were carried out (A,C) four or (B,D) three times with three replicates each. Data are mean ± SEM. *P < 0.05, ###P < 0.001, #compared to unstimulated.
Figure 8. Brain endothelial miR-126* modulates E-selectin…
Figure 8. Brain endothelial miR-126* modulates E-selectin in hCMEC/D3 cells.
(A–D) hCMEC/D3 cells were transfected with control Scrambled Pre-miR or Pre-miR-126 or control Scrambled Anti-miR or Anti-miR-126 (C,D) followed by treatment with cytokines (TNFα + IFNγ, 1 ng/ml) for 24 h or (A,B) left unstimulated. (A,C) Membrane-associated E-selectin (CD62E) expression was quantified by ELISA and (B,D) secreted CCL7 (MCP3) was measured by capture ELISA. Experiments were carried out three times with three replicates each. Data are mean ± SEM. *P < 0.05, **P < 0.01, ###P < 0.001, #compared to unstimulated.

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