IL-17 Inhibits Oligodendrocyte Progenitor Cell Proliferation and Differentiation by Increasing K+ Channel Kv1.3

Abstract

Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. IL-17 level is significantly increased in inflammatory conditions of the CNS, including but not limited to post-stroke and multiple sclerosis. IL-17 has been detected direct toxicity on oligodendrocyte (Ol) lineage cells and inhibition on oligodendrocyte progenitor cell (OPC) differentiation, and thus promotes myelin damage. The cellular mechanism of IL-17 in CNS inflammatory diseases remains obscure. Voltage-gated K⁺ (Kv) channel 1.3 is the predominant Kv channel in Ol and potentially involved in Ol function and cell cycle regulation. Kv1.3 of T cells involves in immunomodulation of inflammatory progression, but the role of Ol Kv1.3 in inflammation-related pathogenesis has not been fully investigated. We hypothesized that IL-17 induces myelin injury through Kv1.3 activation. To test the hypothesis, we studied the involvement of OPC/Ol Kv1.3 in IL-17-induced Ol/myelin injury in vitro and in vivo. Kv1.3 currents and channel expression gradually decreased during the OPC development. Application of IL-17 to OPC culture increased Kv1.3 expression, leading to a decrease of AKT activation, inhibition of proliferation and myelin basic protein reduction, which were prevented by a specific Kv1.3 blocker 5-(4-phenoxybutoxy) psoralen. IL-17-caused myelin injury was validated in LPC-induced demyelination mouse model, particularly in corpus callosum, which was also mitigated by aforementioned Kv1.3 antagonist. IL-17 altered Kv1.3 expression and resultant inhibitory effects on OPC proliferation and differentiation may by interrupting AKT phosphorylating activation. Taken together, our results suggested that IL-17 impairs remyelination and promotes myelin damage by Kv1.3-mediated Ol/myelin injury. Thus, blockade of Kv1.3 as a potential therapeutic strategy for inflammatory CNS disease may partially attribute to the direct protection on OPC proliferation and differentiation other than immunomodulation.

Keywords: IL-17; Kv1.3; inflammation; myelin; oligodendrocyte.

FIGURE 1

Expression of Kv1.3 in OPCs/Ols during development. OPCs were cultured in OPCDM to differentiate into mature Ols. OPCs were transferred into OPCDM for 4 days (DF 4d), 6 days (DF 6d), and 8 days (DF 8d). (A) Representative images of Kv1.3 immunofluorescence staining (red) in cultures. Intact cell nuclei were visualized with DAPI (blue). The immunofluorescence density of Kv1.3 was summarized in a bar graph at the right. With the maturity of OPCs, Kv1.3 decreased in a development-dependent manner. (B) Western blot analysis of MBP expression in cells collected from different periods of OPCs/Ols. Band densitometry data are shown in the bar graph (right). Data are normalized to β-actin shown in each gel. In contrast to the Kv1.3 alterations, the expression of MBP increased with differentiation. (C) Representative current traces of outward K⁺ currents recorded during depolarizing and hyperpolarizing pulses are shown in cells of OPCs, DF 4d, DF 6d, and DF 8d. The whole-cell outward K⁺ currents recorded before (Total) and 15 min after superfusion of 10 nM PAP (PAP perfusion) to the bath. The Kv1.3 currents were then isolated by subtraction of outward K⁺ currents recorded in the presence of PAP from the total currents (Kv1.3). The summary bar graph illustrating average Kv1.3 current density measured at +60 mV (pA/pF) obtained from OPCs and Ols (n = 16). With the maturation of Ols, the Kv1.3-conducted potassium currents decreased. All data expressed were obtained from three independent experiments unless otherwise indicated. ^∗∗P < 0.01 vs. control, ^∗∗∗P < 0.001 vs. control.

FIGURE 2

Kv1.3 blockade prevented OPCs from IL-17–induced inhibition of proliferation and differentiation. OPCs were exposed to IL-17 (200 ng/mL) for 48 h with or without preaddition of PAP (10 nM) for 30 min. (A) The dose of IL-17 was tittered by MTT assay performed in OPCs (n = 6). IL-17 significantly reduced cell viability at a concentration of 200 ng/mL and further reduced at the concentration of 400 ng/mL. (B) MTT assay was performed to detect OPC viability (n = 7). PAP pretreatment counteracted the loss of cell viability induced by IL-17. (C) OPCs were treated with IL-17 with or without PAP in the presence of BrdU (10 μM) for 48 h. Representative images of merged BrdU immunofluorescence staining (red) and DAPI (blue) are shown. Scale bar = 20 μm. The average percentage of BrdU⁺ cells from five independent experiments are summarized in (D) There were 10 randomly selected visual fields counted for each group from three independent experimental treatments. IL-17–induced reduction of BrdU⁺ cells was attenuated by PAP. (E) Western blot analysis of Kv1.3 expression in OPCs. Band densitometry data are shown in the bar graph (below) (n = 4). IL-17 treatment for 48 h elevated the Kv1.3 protein expression in OPCs, whereas the PAP attenuated this elevation. For experiments conducted with Ols in (F,G), Ols were exposed to IL-17 (200 ng/mL) with or without prior addition of PAP (10 nM) for 30 min during the differentiation culture in OPCDM for 6 days. (F,G) Representative images and statistical analyses of MBP and Kv1.3 expression in mature Ols of Western blot (n = 3). The band marked by the arrow is a protein band with a size of 18.5 kDa. Pretreatment with PAP for 30 min counteracted the decrease in MBP and increase in Kv1.3 protein expression in Ols induced by IL-17. ^∗P < 0.05 vs. control, ^∗∗P < 0.01 vs. control, ^∗∗∗P < 0.001 vs. control. ^#P < 0.05 vs. IL-17, ^##P < 0.01 vs. IL-17, ^###P < 0.001 vs. IL-17.

FIGURE 3

Kv1.3 involved in IL-17–induced developmental alterations by diminishing AKT signal but not p38 MAPK. OPCs were treated as described in Figure 2. (A) Representative images and statistical analyses of p-p38 and p-AKT expression in OPCs of Western blot (n = 3). IL-17 suppressed the p-AKT expression, which represented the activation level of AKT pathway in OPCs, but not p-38, whereas PAP relieved this suppression. (B) For experiments conducted with Ols, OPCs were transferred into OPCDM and treated the same as described in Figure 2. Representative images and band densitometry data (right) of p-p38 and p-AKT expression in Ols (n = 4). The attenuation of p-AKT induced by IL-17 was mitigated by PAP, indicating the integral role of Kv1.3 in IL-17–caused decline of AKT activation. ^∗∗P < 0.01 vs. control, ^#P < 0.05 vs. IL-17.

FIGURE 4

Protection of AKT activator on IL-17–induced inhibition of OPC proliferation and differentiation. OPCs were exposed to IL-17 (200 ng/mL) for 48 h with or without AKT activator SC79 (10 μM). (A) MTT assay was performed to detect OPC viability (n = 7). SC79 counteracted the decrease in cell viability induced by IL-17. (B) OPCs were treated as described before in the presence of BrdU (10 μM) for 48 h. Representative images of merged BrdU immunofluorescence staining (red) and DAPI (blue) are shown. Scale bar = 20 μm. The average percentage of BrdU⁺ cells from five independent experiments are summarized in (C) There were 10 randomly selected visual fields counted for each experimental group from three independent treatments. IL-17–induced reduction of BrdU⁺ cell percentage was attenuated by SC79. For experiments conducted with Ols in (D–F), OPCs were exposed to IL-17 (200 ng/mL) with or without SC79 (10 μM) in OPCDM for 6 days. (D–F) Representative images and statistical analyses of MBP (n = 4), p-AKT (n = 4), and Kv1.3 (n = 5) expressions in Ols of Western blot. Band densitometry data are shown in the bar graph (below). SC79 effectively activated AKT signal in our culture system. Similar to the PAP, SC79 counteracted the IL-17–induced decrease in MBP and increase in Kv1.3 expression and mitigated the decrease in p-AKT induced by IL-17. ^∗P < 0.05 vs. control, ^∗∗P < 0.01 vs. control, ^∗∗∗P < 0.001 vs. control. ^##P < 0.01 vs. IL-17, ^###P < 0.001 vs. IL-17.

FIGURE 5

Kv1.3 blockade protected corpus callosum from LPC-induced demyelination in vivo. The frozen sections were obtained from the demyelination model induced by stereotaxic injection of LPC (two points, 1%, 1 μL of each point) in corpus callosum. Mice were treated with or without PAP (6 mg/kg) and accepted BrdU (50 mg/kg) by i.p. injection. (A) Representative images of myelin sheath LFB staining (blue) in coronal sections of animal brain tissues. The low-magnification views (first line) show the complete coronal section, in which the framed parts are enlarged into the high-magnification views showing below. Compared with sham group, the injection site of LPC reduced significantly in overall density of staining for myelin. PAP significantly improved the LPC-induced myelin damage. (B) Western blot analysis of MBP expression in brain tissues around the injected points dissected out from the brain. The band marked by the arrow is a protein band with a size of 18.5 kDa. Blockade of Kv1.3 significantly prevented corpus callosum from LPC-induced MBP reduction (n = 3). (C) Representative images of merged BrdU immunofluorescence staining (red), NG2 (green), and DAPI (blue) in sections. There were 10 randomly selected visual fields counted for each experimental group from three independent treatments. Cell density of BrdU⁺NG2⁺ cells are shown in the bar graph (right). The LPC-induced decrease in BrdU⁺NG2⁺ cells density was mitigated by PAP. ^∗P < 0.05 vs. sham, ^∗∗∗P < 0.001 vs. sham. ^#P < 0.05 vs. LPC, ^###P < 0.001 vs. LPC.