MOG35–55-immunized mice were treated with transduced BMDCs (2 × 106 cells/100 μl) for three time-points on days 3, 5 and 7 post-immunization. Control mice were also treated with PBS or CoLV-DCs. Mice were checked for disease clinical score after cessation of DC treatment. The results showed that BoLV-DC-, p19LV-DC- and CD40LV-DC-treated EAE mice had a significantly milder disease score when compared to PBS-treated (mean clinical scores for whole follow-up were 0·92 ± 0·36, 1·07 ± 0·21 and 1·27 ± 0·11, respectively; P = 0·009, P = 0·018 and P = 0·036, respectively) or CoLV-DC-treated mice (P = 0·012, P = 0·021 and P = 0·037, respectively; Fig. 5). Although the mean clinical score in BoLV-DC-treated mice was almost consistently lower than other treated groups (Fig. 5), that difference was not statistically significant between BoLV-DC-treated EAE mice and p19LV-DC-treated (P = 0·39) or CD40LV-DC-treated mice (P = 0·09).
In order to explore the potential effects of transduced BMDCs on the expression of inflammatory (IFN-γ, IL-17 and GM-CSF) or inhibitory (IL-10) cytokines in EAE mice, mononuclear cells of spleens (Fig. 6a), lymph nodes (Fig. 6b) and central nervous systems (CNS) (Fig. 6c) of treated EAE mice were incubated with MOG35–55 and cytokine levels were determined in their culture supernatants. The IFN-γ levels in the supernatant of MOG35–55-stimulated splenocytes and lymph node cells were significantly lower in CD40LV-DCs-treated mice compared to those of CoLV-DC-treated mice (splenocytes: 502 ± 42 pg/ml and 1072 ± 178 pg/ml, respectively, P < 0·01; lymph nodes: 473 ± 22 pg/ml and 847 ± 116 pg/ml, respectively, P < 0·01). In the same manner, p19LV-DCs-treated mice also showed significantly lower levels of IFN-γ as well as GM-CSF in the culture media of MOG35–55-stimulated splenocytes or lymph node-derived cells compared to those of CoLV-DCs (Fig. 6a,b). In addition, unlike the stimulated splenocytes, the levels of IL-17 were significantly lower in the supernatant of lymph node-derived cells from CD40LV-DC-treated (472 ± 146 pg/ml) or p19LV-DC-treated (387 ± 101 pg/ml) mice compared to PBS-treated (793 ± 110 pg/ml; P = 0·014 and P = 0·002, respectively) or CoLV-DC-treated (734 ± 110 pg/ml; P = 0·031 and P = 0·004, respectively) groups. As shown in Fig. 6, similar to the CD40LV-DC- and p19LV-DC-treated mice, the levels of IFN-γ and GM-CSF in the supernatant of stimulated splenocytes or lymph nodes of BoLV-DC-treated mice were significantly lower than those of CoLV-DC-treated mice (IFN-γ in the splenocyte supernatant: 403 ± 39 pg/ml and 1071 ± 178 pg/ml, respectively, P = 0·004; IFN-γ in lymph node supernatant: 250 ± 23 pg/ml and 847 ± 116 pg/ml, respectively, P = 0·002; GM-CSF in the splenocyte supernatant: 264 ± 64 pg/ml and 405 ± 16 pg/ml, respectively, P = 0·021; GM-CSF in the lymph node supernatant: 232 ± 90 pg/ml and 442 ± 46 pg/ml, respectively, P = 0·011) while IL-17 showed merely a significant decrease in the lymph node-derived cell supernatants of BoLV-DC-treated mice compared to CoLV-DC-treated mice (349 ± 69 pg/ml and 734 ± 110 pg/ml, respectively; P = 0·001). Conversely, mice treated with p19LV-DCs or BoLV-DCs showed a significant increase in IL-10 production by splenocytes compared to those of CoLV-DC-treated mice (for p19LV-DCs: 738 ± 180 pg/ml and 347 ± 81 pg/ml, respectively, P = 0·015; for BoLV-DCs: 901 ± 233 pg/ml and 347 ± 81 pg/ml, respectively, P = 0·012), while those differences did not reach significant levels when the supernatant of lymph node-derived cells were compared in p19LV-DC- or BoLV-DC-treated and CoLV-DC-treated mice (Fig. 6a,b). Regarding CNS, it worth mentioning that on day 35 post-immunization, the mean absolute numbers of mononuclear cells in the CNS of BoLV-DC-, p19LV-DC- and CD40LV-DC-treated mice were significantly lower than in the CoLV-DC-treated group (P = 0·005, P = 0·04 and P = 0·003, respectively). Moreover, comparing the cytokine levels in the supernatant of CNS-derived mononuclear cells from different treatment regimens revealed that the levels of IL-17 were decreased significantly in BoLV-DC-treated mice in contrast to those in CoLV-DC-treated mice (612 ± 47 pg/ml and 809 ± 117 pg/ml, respectively; P = 0·023), while a significant difference in the GM-CSF levels was merely detected when p19LV-DC-treated mice were compared with CoLV-DC-treated mice (407 ± 19 pg/ml and 446 ± 22 pg/ml, respectively; P = 0·038). Although IFN-γ levels were decreased and the levels of IL-10 were increased in all mice treated with CD40LV-DCs, p19LV-DCs or BoLV-DCs compared with CoLV-DC-treated mice, those differences were not significant (Fig. 6c). Overall, our results showed a significant decrease in IL-17 production by mononuclear cells derived from lymph nodes and spinal cords of BoLV-DC-treated EAE mice.
Figure 6. Effects of transduced bone marrow-derived dendritic cells (BMDCs) on interferon (IFN)-γ, interleukin (IL)-17, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL-10 production by splenocytes (a), lymph node cells (b) and cells infiltrating the central nervous system (CNS) (c) in experimental autoimmune encephalomyelitis (EAE)-treated mice. Mononuclear cells isolated from spleens (a), lymph nodes (b) and CNS (c) were stimulated with myelin oligodendrocyte glycoprotein (MOG)35–55 and their supernatants were checked by enzyme-linked immunosorbent assay (ELISA). *P < 0·05; **P < 0·01; ***P < 0·001.
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