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Fig. 6 | Acta Neuropathologica Communications

Fig. 6

From: Human olfactory mesenchymal stromal cell transplantation ameliorates experimental autoimmune encephalomyelitis revealing an inhibitory role for IL16 on myelination

Fig. 6

IL-16 expression is upregulated during in vitro demyelination and inhibits OPC differentiation and myelination. a Representative Western blot image of IL-16 expression in CNS control myelinating cultures (Control Dy 0) and 24 h after demyelination with anti-MOG and complement (DeMy 0). After DeMy, cultures were treated for 5 days with hOM-MSC-CM (OM Dy5) or hBM-MSC-CM (BM Dy5) or treated with media alone (Control Dy5). b Western blot quantification revealed the predominant form of IL-16 expressed by the cultures was Pro IL-16, which was significantly upregulated after DeMy. hOM- or hBM-MSC-CM treatment for 5 days post DeMy had no effect on Pro IL-16 expression. The bioactive secreted form of IL-16 (Sec IL-16) was expressed at very low levels in control cultures (Control Dy 0, C Dy 0) but was significantly upregulated after demyelination (DeMy Dy5, D Dy5). hOM-MSC-CM treated cultures (OM Dy5) expressed low levels however, those treated with hBM-MSC-CM (BM Dy5) maintained the higher expression of IL-16 found in DeMy cultures (D Dy5). n = 5, *p < 0.05, **p < 0.01, ANOVA, Tukey’s multiple comparison. c Western blot analysis of Caspase-3 (Cas3) expression in hOM- and hBM-MSC-CM. hOM-MSCs secreted lower amounts compared to hBM-MSCs. Secreted levels were standardised to Ponceau S (PonS) staining of the membrane prior to staining. hOM-MSC-CM, n = 6; hBM-MSC-CM, n = 6 **p < 0.01, Student’s unpaired t test. d IL-16 was expressed in axons and microglia, however astrocytes produced only negligible amounts. e Immunohistochemical images of purified OPCs grown in growth factors (GF) that retain their progenitor state (SATO + GF) or grown in media that allows differentiation (SATO) or after treatment with IL-16 (100 ng/ml in SATO). Proteolipid protein (PLP), a late OPC marker, is shown in green and cell nuclei are stained by DAPI in blue. b Quantification of the percentage of positive OPCs stained for NG2 (early marker), O4 (middle maker) or PLP (late marker). NG2 expression was significantly reduced and PLP was significantly increased in SATO compared to SATO + GF correlating with enhanced OPCs differentiation. IL-16 treatment had no effect on NG2 or O4 expression but caused a significant reduction in the number of mature PLP positive OPCs compared to SATO. There were no significant differences in OPC numbers between treatments (n = 4, all conditions). f Immunohistochemical images of control CNS myelinating cultures or after IL-16 (100 ng/ml) treatment from Day 12. Axons are stained with SMI-31 (shown in red) and myelin stained with PLP (shown in green). Quantification revealed that IL-16 treatment from day 12, significantly reduced the number of myelinated axons compared to control cultures (n = 3, all conditions). g Immunohistochemical images of CD4 expression (shown in green) in mircoglia and OPCs. CD4 was upregulated in microglia after demyelination (Iba-1 shown in red). Purified OPCs (O4 shown in red) expressed CD4 in a peri-nuclear localisation. Scale bars represents 50 μm in all images

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