KOR knockout mice are more susceptible to EAE

We first examined the mRNA levels of the three opioid receptors (DOR, KOR and MOR) and the precursors of endogenous opioids including proenkephalin (Penk) and prodynorphin (Pdyn) in MOG-EAE mice on various days post immunization. In the brain, the expression levels of these genes did not show a clear time-dependent change (Supplementary Fig. 1a). In the spinal cord, however, all the genes were significantly downregulated from day 15, which is typically after the onset of the disease (Supplementary Fig. 1b), similar as previously reported in a Theiler’s murine encephalomyelitis virus model of MS6. In the lymph node, DOR was downregulated from day 9 to 18, while KOR did not show a time-dependent change. Owing to the low expression level, MOR, Penk and Pdyn were undetectable in the lymph node (Supplementary Fig. 1c). The downregulation of the receptors and ligands after MOG 35–55 immunization indicates that the opioid receptors might be involved in EAE pathogenesis.

We then induced EAE in wild-type (WT), opioid receptors knockout and heterozygous mice. Deletion of MOR did not seem to affect the onset of EAE (Fig. 1a). However, the deletion of DOR led to a slightly severer type of EAE (Fig. 1b), whereas ablation of KOR induced a significant increase in the disease score (Fig. 1c). The disease severity in heterozygotes was similar to their WT littermates (Fig. 1b,c). Histological examination of the spinal cords was performed on day 17 post immunization. Leukocytes infiltration and demyelination could be observed in the spinal cord of the WT (KOR+/+)-EAE animals (Fig. 1d–g). KOR−/−-EAE mice exhibited significantly increased leukocytes infiltration and more extensive demyelination (Fig. 1d–g).

Figure 1: Opioid receptors are involved in the pathogenesis of EAE. (a–c) Clinical scores of EAE induced in WT, MOR+/−, MOR−/− (a), DOR+/−, DOR−/− (b), KOR+/−, KOR−/− (c). Data are means±s.e.m. #P<0.05, ###P<0.001 (two-way analysis of variance (ANOVA) test), *P<0.05, **P<0.01 and ***P<0.001 versus WT (Mann–Whitney U-test). (d) Haematoxylin and eosin and (e) Luxol fast blue staining of the paraffin sections of the spinal cords isolated from naive, WT-EAE or KOR−/−-EAE mice on day 17 post immunization. Scale bars, 200 μm. (f,g) Quantification of CNS infiltrates and the amount of demyelination presented in d,e. Three mice from each group were killed, and 15 sections from each mouse were analysed. Data are means±s.e.m. ***P<0.001 versus naive, #P<0.05, ##P<0.01 versus WT-EAE (Student’s t-test). (h) Clinical scores of WT-EAE mice treated with KOR agonist U50488 (0.5, 1.6 or 5 mg kg−1) or vehicle once daily via intraperitoneal injection from day 3 post immunization till the end of the study. ###P<0.001 (two-way ANOVA test), *P<0.05, **P<0.01 versus WT (Mann–Whitney U-test). (i) Clinical scores of WT- or KOR−/−-EAE mice treated with U50488 (1.6 mg kg−1) or vehicle once daily via intraperitoneal from day 3 post immunization. Data are means±s.e.m. ***P<0.001 (two-way ANOVA test). Full size image

Pharmacological activation of KOR alleviates EAE

Since the knockout of KOR led to a severer type of EAE, we wondered whether activation of KOR confers protection against EAE. U50488 and asimadoline, two selective KOR agonists, were tested. U50488 had significant therapeutic effects at all the three dosages tested, with the best effect observed in the 1.6 mg kg−1 group, which showed the lowest peak severity and cumulative clinical score (Fig. 1h). Asimadoline also showed significant therapeutic effect (Supplementary Fig. 2). Histological analysis revealed that U50488 (1.6 mg kg−1) also significantly reduced leukocytes infiltration into the spinal cord and demyelination in EAE (Supplementary Fig. 3). To avoid the possible off-target effect of the agonist, U50488 (1.6 mg kg−1) was used to treat EAE induced in both the WT and KOR−/− mice. Similar to our previous observations, U50488 significantly reduced EAE severity in the WT mice, and KOR knockout significantly increased the disease score. However, the therapeutic effect of U50488 was completely abolished in the KOR−/−-EAE mice (Fig. 1i). These data demonstrate that KOR signalling contributes to EAE resistance.

KOR regulates EAE pathogenesis in CNS

MS is an autoimmune disease initiated by immune cells in the periphery. T cells, B cells and monocytes have all been reported to participate in MS pathogenesis16,17,18. However, U50488 did not alter the percentage of CD4+ T cells, CD8+ T cells, B cells and CD11b+ cells in the spleen, blood and lymph node of EAE mice (Supplementary Fig. 4a). Similarly, KOR knockout did not significantly affect the percentage of these cells except that the CD8+ T cells were slightly reduced (Supplementary Fig. 4b). The percentage of the major pathogenic Th1 and Th17 subgroups of the CD4+ T cells in EAE mice were also not affected by the treatment of U50488 (Fig. 2a and Supplementary Fig. 5a) or KOR knockout (Fig. 2b and Supplementary Fig. 5b). Cytokine secretion by Th1 and Th17 cells (IFN-γ and IL-17A) after MOG 35–55 restimulation was not affected by U50488 or KOR knockout either (Supplementary Fig. 5c,d). In vitro differentiation of Th1, Th17 and Treg cells were also not affected by KOR activation or knockout (Fig. 2c).

Figure 2: KOR in immune cells does not contribute to EAE pathogenesis. (a) Analysis of CD4+ T cell subtypes in the spleen of naive, WT-EAE and WT-EAE mice treated with U50488 (1.6 mg kg−1) on day 12 post immunization. Th1, Th17 and Treg cells were analysed with FACS by intracellular staining of IFN-γ, IL-17A and Foxp3, respectively. (b) Analysis of Th1 and Th17 cells in the spleen of naive, WT-EAE or KOR−/−-EAE mice on day 12 post immunization. (c) In vitro differentiation of Th1, Th17 and Treg cells from WT or KOR−/− CD4+ T cells in the presence of U50488 (10 μM) or not. Data are means±s.e.m. (n=3), *P<0.05, **P<0.01, ***P<0.001 versus naive mice (Student’s t-test). (d) EAE scores of Rag1−/− mice reconstituted with splenocytes (1 × 107) from WT or KOR−/− mice and immunized with MOG 35–55 . (e) EAE scores of lethally irradiated WT mice reconstituted with WT or KOR−/− bone marrow cells (1 × 107) and immunized with MOG 35–55 . (f) EAE scores of lethally irradiated WT or KOR−/− mice reconstituted with WT bone marrow cells and immunized with MOG 35–55 . ###P<0.001 (two-way analysis of variance (ANOVA) test), *P<0.05, KOR−/− versus KOR+/+ recipients (Mann–Whitney U-test). (g) Clinical scores of passive EAE in WT mice induced by transferring MOG-restimulated splenocytes (1 × 107) isolated from WT or KOR−/− EAE mice. (h) Clinical scores of passive EAE in WT or KOR−/− mice induced by transferring of MOG-restimulated splenocytes (1 × 107) isolated from WT EAE mice. ###P<0.001 (two-way ANOVA test), *P<0.05, **P<0.05, KOR−/− versus KOR+/+ recipients (Mann–Whitney U-test). Data are means±s.e.m. Full size image

These observations led us to speculate that KOR might not function in the immune system during EAE pathogenesis. We induced EAE in Rag1−/− mice reconstituted with splenocytes isolated from WT or KOR−/− animals. KOR deficiency in the splenocytes did not affect the EAE score in these Rag1−/− mice (Fig. 2d). Bone marrow chimeras were generated by transferring WT or KOR−/− bone marrow cells to lethally irradiated WT mice, or transferring WT cells to lethally irradiated WT or KOR−/−mice (Fig. 2e,f). EAE were induced after immune system reconstitution. KOR deletion in the bone marrow cells did not affect the EAE score in these chimeras (Fig. 2e). However, a significant increase in the disease score was observed when KOR was knocked out in the recipient mice (Fig. 2f). Passive EAE was also induced by adoptive transfer of splenocytes isolated from MOG 35–55 immunized WT or KOR−/− mice to WT mice, or from MOG 35–55 immunized WT mice to WT or KOR−/− mice (Fig. 2g,h). KOR deletion in the splenocytes did not affect the passive induction of EAE (Fig. 2g), while KOR deletion in the recipient mice significantly enhanced the disease severity (Fig. 2h). These data demonstrate that KOR in the immune system is not involved in EAE pathogenesis, while KOR in the CNS might play a critical role in controlling the disease severity.

KOR does not block astrocytes and microglia activation

It has been reported that the brain endothelial cells, astrocytes and microglia participate in the leukocyte trans-migration and inflammatory process in the CNS, leading to the onset of EAE19,20,21. So we explored whether KOR signalling affects the activation of these cells. The mouse brain microvascular endothelial bEnd.3 cells could be activated by TNF-α and INF-γ stimulation22,23, and could upregulate the expression of cell adhesion molecules and chemokines, including ICAM1, VCAM1, CCL2, CCL5 and IP-10, and downregulate the tight junction protein Occludin (Supplementary Fig. 6). These changes may facilitate the breakdown of blood–brain barrier and the infiltration of leukocytes. KOR agonist U50488 could not block TNF-α- and INF-γ-induced downregulation of Occludin, it even enhanced cell adhesion molecule and chemokine expression, indicating that the beneficial effect of KOR activation in EAE may not relate to endothelial cell regulation. The primary astrocytes were isolated and activated in vitro by various cytokine combinations including TNF-α/IL17 and TNF-α/INF-γ (ref. 24). U50488 treatment did not seem to affect the expression of inflammatory cytokines and chemokines in activated astrocytes (Supplementary Fig. 7). Primary microglia were also isolated and activated in vitro by LPS25. U50488 even slightly enhanced LPS-induced inflammatory cytokine expression in microglia (Supplementary Fig. 8). Taken together, these data indicate that KOR signalling does not alleviate EAE by blocking the activation of brain vascular endothelial cells, microglia and astrocytes.

KOR activation promotes remyelination in mouse models

MS and EAE are characterized by autoimmune-mediated demyelination and neurodegeneration. In CNS, myelin is formed by OLs. Current research on MS therapy is directed at three major goals: controlling the inflammatory immune response to prevent the development of new demyelinating lesions, protecting the demyelinated neurons from degeneration, and promoting OL differentiation and remyelination26. Remyelination can occur effectively and spontaneously following demyelination with the migration of OPCs to the sites of injury and subsequent differentiation to mature OLs that remyelinate the damaged axons. However, for reasons yet to be fully elucidated, this process is generally incomplete and often fails in MS27. Since KOR did not seem to play a role in the immune system, we wondered whether it could affect the remyelination process. The OLs and OPCs in the EAE lesions in the spinal cord were assessed by immunostaining using antibodies target MBP (OLs) or NG2 (OPCs) (Fig. 3a). MBP staining was substantially reduced in the EAE animals, indicating severe demyelination. The demyelinating areas were filled with NG2+ OPCs, indicating that the recruitment of OPCs to the lesions was normal but differentiation to OLs might be blocked (Fig. 3a–c). In U50488-treated EAE mice, demyelination was significantly mitigated, and NG2+ OPCs were also reduced (Fig. 3a–c). Furthermore, myelin surrounding spinal cord axons in the remission phase of EAE was observed with electron microscopy. Demyelination was very apparent and g-ratio of the myelinated axons was significantly increased in vehicle-treated EAE mice compared with the naive ones, but g-ratio in U50488-treated mice were significantly lower than that in vehicle-treated mice (Fig. 3d,e), indicating a possible better recovery. In contrast, the g-ratio in KOR−/−-EAE mice was significantly higher than WT-EAE animals, indicating less recovery (Fig. 3f,g). These observations suggested that KOR activation might promote the generation of OLs and remyelination in EAE.

Figure 3: Activation of KOR promotes remyelination in EAE mice. (a) Immunofluorescence staining of OLs (MBP) and OPC (NG2) in the spinal cords isolated on day 22 post immunization from WT-EAE mice treated with vehicle or U50488 (1.6 mg kg−1). Scale bars, 100 μm. (b,c) Statistical analysis of the demyelination area and NG2 positive area in white matter. Three animals from each group were killed and 15 sections of the spinal cord of each animal were analysed. Data are means±s.e.m. ***P<0.001 versus naive group, ##P<0.01, ###P<0.001 versus vehicle group (Student’s t-test). (d) Representative electron microscopy images of cross sections of the spinal cords isolated from naive, vehicle or U50488 (1.6 mg kg−1)-treated EAE mice on day 22 post immunization. Scale bar, 0.2 μm. (e) G-ratios of spinal cord axons in d. Data are means±s.e.m. (n=200), ***P<0.001 versus naive, ###P<0.001 versus vehicle treatment (Student’s t-test). (f) Representative electron microscopy images of spinal cords isolated from WT or KOR−/− EAE mice on day 22 post immunization. Scale bar, 0.2 μm. (g) G-ratios of spinal cord axons in f. Data are means±s.e.m. (n=200), ***P<0.001 versus KOR+/+ (Student’s t-test). Full size image

KOR-promoted remyelination was further evaluated with a non-immune-mediated, cuprizone-induced demyelination model28 (Fig. 4a). Myelin status at the corpus callosum was evaluated with Luxol fast blue staining. All cuprizone-fed groups showed a significant loss of myelin at the corpus callosum region (Fig. 4b). Two weeks after cuprizone withdrawal, both the vehicle and U50488 groups showed limited remyelination (Fig. 4b,c). However, significant spontaneous remyelination could be observed at 3 or 5 weeks after cuprizone withdrawal, and U50488 treatment further enhanced the remyelination process (Fig. 4b,c). We also checked the effect of KOR knockout in the cuprizone model, and the KOR−/− mice showed significantly less remyelination than the WT mice (Fig. 4d–f).

Figure 4: Activation of KOR promotes remyelination in cuprizone-induced demyelination mice model. (a) Demyelination was induced in WT C57BL/6 mice by feeding with a 0.2% cuprizone containing diet for 3 weeks. Following cuprizone withdrawal, the mice were treated with vehicle or U50488 (1.6 mg kg−1) for 2, 3 or 5 weeks (wks). (b) Representative images of the corpus callosum region stained with Luxol fast blue after cuprizone and U50488 treatment. Scale bars, 500 μm. (c) Statistical analysis of the myelinating areas in b. Six animals from each group were killed and six sections of the corpus callosum region of each animal were analysed. ***P<0.001 versus 2 wks treatment group, #P<0.05 versus vehicle group (Student’s t-test). (d) Demyelination was induced in KOR+/+ or KOR−/− C57BL/6 mice by feeding with a 0.2% cuprizone containing diet for 3 wks. Following cuprizone withdrawal, the KOR+/+ or KOR−/− mice were fed with a normal diet for 2 or 3 wks. (e) Representative images of the corpus callosum region from KOR+/+ or KOR−/− mice stained with Luxol fast blue after the cuprizone treatment. Scale bars, 500 μm. (f) Statistical analysis of the myelinating areas in e. Six animals from each group were killed and six sections of the corpus callosum region of each animal were analysed. ***P<0.001 versus KOR+/+ (Student’s t-test). Full size image

KOR promotes OL differentiation and myelination in vitro

To test whether this remyelination promoting effect of KOR is direct or not, and to rule out the possible crosstalk between neurons or other types of the cells in CNS with OPCs or OLs, we performed the in vitro OPC to OL differentiation assay. KOR was indeed expressed by the OPCs (Fig. 5a). And U50488 dose dependently promoted OL differentiation from OPCs, with the best effect appearing at 0.5 and 1 μM (Fig. 5b,c). This KOR agonist-mediated enhancement of OL differentiation was almost completely abolished in the KOR knockout OPCs (Fig. 5d). The downstream pathways linking KOR activation to OL differentiation were further analysed with pathway inhibitors. Blocking Gαi/o pathway with pertussis toxin (PTX), L-type calcium channels with nifedipine, or p38 pathway with SB203580 all significantly reduced U50488-promoted OL differentiation, while other pathway inhibitors showed no significant effect (Fig. 5e). The observation that KOR signalling promotes OPC differentiation towards OL in culture raises the question of whether it also promotes myelin formation. To address this question, we set up an in vitro myelination system29 by co-culturing OPCs isolated from KOR+/+ or KOR−/− mice with dorsal root ganglion (DRG) neurons isolated from KOR+/+ mice. Myelinated axons will be positive for both the OL marker MBP and axon marker NF-200 (neurofilament, 200 kD). Compared with the control, administration of KOR agonist U50488 significantly increased the length of myelinated axons in the co-culture containing the KOR+/+ OPCs (Fig. 5f,g). And the positive effect of U50488 was abolished in the co-culture containing the KOR−/− OPCs (Fig. 5f,h). Taken together, these results suggest that KOR activation enhances OL differentiation and myelination both in vitro and in vivo.