Figure 4. Oral Administration of Synthetic miR-30d Ameliorates EAE in a Recipient Gut Microbiome-dependent Manner.

(A-B) synthetic miR-30d or scramble control was orally gavaged to EAE recipients starting at disease onset (day 11, disease score=1) daily at a dose of 250 pmol for 7 consecutive days. (A) Clinical scores of EAE in the recipient mice. Representative data of two independent experiments; H₂O(vehicle) n=8, scrambled miR-30d n=13, miR-30d n=11, Error bars denote mean ± SEM, Friedman test based on scores after the beginning of treatment (12 d.p.i) until the end of experiment followed by Dunn’s multiple comparison test as compared with the H₂O-gavaged group. (B) Quantification of demyelination and axonal loss for individual mice. Data combined from two independent experiments with n=8 mice/group; Error bars denote mean ± SEM, one-way ANOVA Dunnett’s multiple comparisons test. (C-D) Mice were immunized with MOG and orally administered synthetic miR-30d or scramble control daily at a dose of 1000 pmol for 7 consecutive days. Foxp3⁺ T cells in the total CD4⁺ T cell population (C) and in the Vβ11⁺ CD4⁺ T cell population (D) in the spleen were analyzed by FACS. Left panel: Representative FACS plots of Foxp3⁺ CD4⁺ T cells; Right panel: % of CD4⁺ Foxp3⁺ T cells in individual animals (n=4 per group). Error bars denote mean ± SEM; one-way ANOVA Tukey’s multiple comparisons test. (E-G) Effect on EAE of transfer of fecal microbiome from synthetic miR-30d treated mice. Donor mice were immunized with MOG and orally treated with H₂O (vehicle), scrambled miR-30d, or miR-30d for 7 consecutive days. Feces were collected and used to colonize mice that were pre-treated with antibiotics (ABX) for 7 days prior to colonization. Recipient mice were then induced for EAE. (E) Experimental scheme. (F) Clinical scores of EAE in the recipient mice. Combined data of two experiments with H₂O (vehicle) n=19, scramble n=21, miR-30d n=24; Error bars denote mean ± SEM; Friedman test based on scores from the onset of disease (11 d.p.i) until the end of experiment with Dunn’s multiple comparisons as compared with the H₂O-treated donor feces-colonized group. (G) Quantification of demyelination and axonal loss. Values for individual mice are shown, combined from 2 independent experiments with H₂O (vehicle) n=19, scramble n=19, miR-30d n=20; Error bars denote mean ± SEM, one-way ANOVA Dunnett’s multiple comparisons test. (H-I) Antibiotics abrogated therapeutic effect of oral miR-30d on EAE. Synthetic miR-30d, scramble control, or H₂O (vehicle) was orally gavaged to EAE recipients starting at the onset of disease (day 11, disease score =1) at a dose of 250 pmol daily for 7 consecutive days. Mice were simultaneously gavaged with an antibiotics mixture (ABX). (H) Clinical scores of EAE, Combined data from two independent experiments, H₂O (vehicle) n=10, Scramble n=11, miR-30d n=11, Error bars denote mean ± SEM, Friedman test based on scores from the start of the treatment (11 d.p.i) until the end of experiment followed by Dunn’s multiple comparison test as compared with the ABX+H₂O-gavaged group. (I) Quantification of demyelination and axonal loss for individual mice from two independent experiments (n=6 mice/group); Error bars denote mean ± SEM, one-way ANOVA Dunnett’s multiple comparisons test. All panels: n.s.= not significant, ** P<0.01, *** P<0.001, **** P<0.0001.

Related to Figure S2, Figure S3, and Figure S4.