Figure 1.
Oral administration of IRT5 probiotics suppresses EAMG progression.
Oral administration of IRT5 probiotics or PBS as a control was initiated two weeks before immunization of TAChR and continued till the end of experiment (6 weeks after induction of EAMG) (A). Effect of IRT5 probiotics or PBS treatment on EAMG progression was analyzed by monitoring clinical score every other day (B). Mean clinical score was evaluated based on the standard clinical score criteria. The points and bars represent means and standard deviations, respectively. Data are representative of three independent experiments. *p<0.05. Complement deposition (C) and weight change (Table 1) of each treatment group were assessed. To assess the presence of AChR and complement at the NMJ in each treatment group, 10–15 muscle sections were analyzed (AChR: red fluorescence, complement C5b-9: green fluorescence). Result shown is one representative section. Normal non-immunized rats were employed as healthy control (positively stained for AChR but negative for C5b-9).
Table 1.
Effect of IRT5 probiotics treatment on EAMG.
Figure 2.
IRT5 probiotics treatment suppresses AChR-reactive lymphocyte proliferation.
(A) Mixed lymphocytes and (B) CD4+ T cells of draining lymph nodes from each treatment group were cultured for 3 days either alone or presence of TAChR. Proliferation of mixed lymphocytes and CD4+ T cells were determined by assessing [3H]-thymidine incorporation during the last 18 hr of 72 hr culture period to evaluate proliferated status of cells. Results are expressed as CPM. *p<0.05, **p<0.01.
Figure 3.
Treatment with IRT5 probiotics down-regulates expression of pro-inflammatory cytokines in draining LN.
(A) Mixed lymphocytes and (B) CD4+ T cells isolated from the draining lymph nodes (dLN) of each treatment group were cultured for 40 hr in the presence of TAChR, and then total mRNA was isolated. The expression level of cytokines of control PBS group was set at 100% and the relative value of the IRT5 probiotics treated group was shown. (C) IFN-γ, TNF-α and IL-17A producing mixed lymphocytes and CD4+ T cells were analyzed by flow cytometry. (D) Protein levels of TNF-α and IL-17A from mixed lymphocytes were measured by ELISA. Data are representative of three independent experiments. *p<0.05, **p<0.01.
Figure 4.
Treatment with IRT5 probiotics down-regulates AChR specific IgG isotypes.
Sera were collected from EAMG rats treated with either IRT5 probiotics or control PBS for 6 weeks after immunization. Each AChR specific IgG isotype in the PBS-treated group was assigned a value of 1 and the ODs of IRT5 probiotics-treated group were calculated accordingly. Anti-AChR isotypes were determined by ELISA as described in the Material and Method. Data are representative of three independent experiments.*p<0.05, **p<0.01.
Figure 5.
IRT5 probiotics administration induces regulatory DCs.
(A) MLN DCs isolated from IRT5 probiotics or PBS treated groups were stimulated with PMA/Ionomycin and then the expression levels of marker molecules for rDC were measured by RT-PCR. (B) CFSE-labeled CD4+ T cells isolated from EAMG rats were stimulated by DCs isolated from each treatment group in the presence of TAChR and then proliferation (B) and production of cytokine levels (C) by CD4+ T cells were analyzed by flow cytometry and RT-PCR respectively. Proliferation of CD4+ T cells was monitored for 7 days co-culture periods. Cytokine expression in CD4+ T cells of PBS control mice was set at 100%. (D) CD4+ T cells isolated from EAMG rats were stimulated with MLN DCs from each treatment group in the presence of TAChR for 7 days. After co-culture, alteration of CD4+Foxp3+ population was measured by flow cytometry. The data are representative of three independent experiments. *p<0.05.
Figure 6.
IRT5 probiotics administration induces regulatory DCs in the peripheral sites.
(A) dLN DCs and (B) splenic DCs isolated from IRT5 probiotics or PBS treated group were stimulated with TAChR and then the expression levels of marker molecules for rDC or IL-10 protein expression (C) were measured by RT-PCR and ELISA, respectively. (D) Lymphocytes isolated from IRT5 probiotics or PBS treated groups were stimulated with TAChR and the relative expression levels of Foxp3 was measured by RT-PCR. The data are representative of three independent experiments. *p<0.05, **p<0.01.
Table 2.
Primer sequences used for qRT-PCR.