Involvement of NINJ2 Protein in Inflammation and Blood-Brain Barrier Transmigration of Monocytes in Multiple Sclerosis

Abstract

Multiple sclerosis (MS) is an inflammatory neurodegenerative disorder of the central nervous system (CNS). The migration of immune cells into the CNS is essential for its development, and plasma membrane molecules play an important role in triggering and maintaining the inflammation. We previously identified ninjurin2, a plasma membrane protein encoded by NINJ2 gene, as involved in the occurrence of relapse under Interferon-β treatment in MS patients. The aim of the present study was to investigate the involvement of NINJ2 in inflammatory conditions and in the migration of monocytes through the blood-brain barrier (BBB). We observed that NINJ2 is downregulated in monocytes and in THP-1 cells after stimulation with the pro-inflammatory cytokine LPS, while in hCMEC/D3 cells, which represent a surrogate of the BBB, LPS stimulation increases its expression. We set up a transmigration assay using an hCMEC/D3 transwell-based model, finding a higher transmigration rate of monocytes from MS subjects compared to healthy controls (HCs) in the case of an activated hCMEC/D3 monolayer. Moreover, a positive correlation between NINJ2 expression in monocytes and monocyte migration rate was observed. Overall, our results suggest that ninjurin2 could be involved in the transmigration of immune cells into the CNS in pro-inflammatory conditions. Further experiments are needed to elucidate the exact molecular mechanisms.

Keywords: inflammation; multiple sclerosis; transmigration.

Figure 1

NINJ2 is down-regulated after pro-inflammatory stimulation in monocytes and in THP-1 cells. The relative expression of NINJ2 gene was measured in (A) human monocytes stimulated with LPS (1 μg/mL) for 6 h or 24 h, (B) human monocytes stimulated with IFNγ (100 ng/mL) for 6 h or 24 h, (C) human monocytes stimulated with TNFα (10 ng/mL) for 6 h or 24 h. Data are reported as ratio between stimulated and unstimulated (CTRL) conditions (mean ± SEM), with the number of individuals analyzed for each condition reported. p-values were calculated according to paired t-test starting from the relative expression values for each sample. (D) The relative protein expression of NINJ2 was measured in monocytes collected from 3 HCs and stimulated with LPS (1 μg/mL) for 24 h; data are reported as mean ± SEM from a total of 15 analyzed cells; p values were calculated with unpaired t-test. (E) The relative NINJ2 transcript level was measured in THP-1 cells stimulated with LPS (1 μg/mL) for 24 h; experiments were performed in triplicate and results are reported as mean ± SEM. (F) NINJ2 protein level, expressed as mean fluorescence (arbitrary units, AU), was measured in THP-1 cells stimulated with LPS (1 μg/mL) for 24 h; data are reported as mean ± SEM from a total of 10 analyzed cells for each condition. CTRL: unstimulated condition, LPS: stimulated condition.** p < 0.01, *** p < 0.001.

Figure 2

NINJ2 is up-regulated after LPS stimulation in hCMEC/D3 cells. (A) The relative gene expression of NINJ2 was measured in hCMEC/D3 cells stimulated with LPS (1 μg/mL) for 24 h. (B) The NINJ2 protein level, expressed as mean fluorescence (arbitrary units, AU), was measured in hCMEC/D3 cells stimulated with LPS (1 μg/mL) for 24 h; data are reported as mean ± SEM from a total of 12 analyzed cells for each condition. Data are reported as mean ± SEM. CTRL: unstimulated condition, LPS: stimulated condition. * p < 0.05.

Figure 3

Migration rate of THP-1 and primary monocytes collected from MS and HC subjects. (A) Migrating THP-1 cells (expressed as Relative Fluorescence Units, RFU) through hCMEC/D3 cells’ monolayer were measured without stimulation, pre-stimulating hCMEC/D3 cells with LPS for 24 h or pre-stimulating hCMEC/D3 cells, as well as stimulating THP-1 cells with LPS for 24 h. The experiment was performed in triplicate. Significance comparing the condition in which both cell types were stimulated vs. the condition in which only hCMEC/D3 were stimulated is reported. (B) Migrating cells (expressed as Relative Fluorescence Units) from 7 MS and 7 HC subjects through the hCMEC/D3 cells’ monolayer was measured without stimulation, pre-stimulating hCMEC/D3 cells with LPS for 24 h or pre-stimulating hCMEC/D3 cells and stimulating monocytes with LPS for 24 h. HC and MS subjects showed a different trend (two-way repeated measures ANOVA p = 0.061). For panel (A,B), presence (+) or absence (−) of 24 h LPS stimulation of hCMEC/D3 and/or monocytes is indicated by the +/− table below the graph. (C) The difference in migrating cells between activated endothelium (hCMEC/D3 + LPS) and the unstimulated conditions is plotted for MS and HC subjects; significance comparing the two groups is reported. Data are reported as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 4

Relationship between NINJ2 basal expression and the migration rate. The basal expression of NINJ2 was correlated with the number of monocytes (expressed as Relative Fluorescence Units, RFU) migrating through the surrogate of BBB (hCMEC/D3 cell line) without stimulation (left panel, “unstimulated”), pre-stimulating hCMEC/D3 cells with LPS for 24 h (central panel, “hCMEC/D3 + LPS”) or pre-stimulating hCMEC/D3 cells and stimulating monocytes with LPS for 24 h (right panel, “hCMEC/D3 and monocytes + LPS”). Each dot represents one individual. Regression line is shown (blue line) according to Deming regression model. p values are reported for each panel.

Figure 5

Summary of main findings of the study.