Fig. 2

Effect of vancomycin on EAE development. A Schematic representation showing the time course for vancomycin/neomycin treatments. B Mean EAE clinical scores overtime. Results are presented as mean ± SEM (n = 10 mice/group). ****p < 0.0001, Freidman test with Dunn correction for multiple comparisons. C Histopathological evaluation of demyelination with Luxol fast blue (LFB), axonal loss with Bielschowsky’s silver (silver) staining, and CD4⁺ cell infiltrates at 15 DPI. Arrows denote demyelination (LFB), axonal loss (silver staining), and CD4⁺ cell infiltrates of representative spinal cord sections from control and vancomycin-treated EAE mice. Scale bars, 500 μm. D Quantification of demyelination, axonal loss, and CD4⁺ cell infiltrates of individual mouse. Representative data of three independent experiments with n = 5 mice per group are shown. Error bars denote mean ± SEM; Mann-Whitney test was performed. *p < 0.05. E Experimental scheme of cohousing experiment. Conventionally raised mice were either untreated or treated with vancomycin daily via oral gavage for 2 weeks. Mice were either housed with the same treatment group (single treatment) or cohoused between treatment groups. Mice were immunized with MOG 1 week post discontinuation of vancomycin for EAE induction. F Mean EAE clinical scores overtime in single-treatment housed untreated mice (control), single-treatment housed vancomycin mice (vancomycin), untreated mice cohoused with vancomycin treated mice (ControlCoho), and vancomycin-treated mice cohoused with untreated mice (VancoCoho). Error bars denote mean ± SEM (n = 11 mice/group); the Friedman test based on scores from 0 DPI until the end of the experiment and Dunn’s multiple-comparison test were performed. *p < 0.05, ***p < 0.001, ****p < 0.0001. G Experimental scheme of fecal transfer experiment in conventionally raised mice. Conventionally raised mice were on drinking water supplemented with ampicillin (1 g/L), neomycin sulfate (1 g/L), metronidazole (1 g/L), and vancomycin (0.5 g/L) for 3 days. Forty-eight hours post antibiotics discontinuation, half of the mice were orally gavage with feces from wild-type B6 mice, and the remaining half was fed feces from vancomycin-treated mice 10 days prior to EAE induction. Mice were immunized with MOG for EAE induction. H Mean EAE clinical scores overtime. Error bars denote mean ± SEM (n = 10 mice/group); the Friedman test based on scores from 0 DPI until the end of the experiment and Dunn’s multiple-comparison test were performed. **p < 0.01 I Experimental scheme of fecal transfer experiment in germ-free mice. Germ-free mice were either fed feces from untreated or vancomycin-treated conventionally raised mice. Four-week postoral gavage, colonized germ-free mice were immunized with MOG for EAE induction. J Mean EAE clinical scores overtime. Error bars denote mean ± SEM (n = 10–12 mice/group); the Friedman test based on scores from 0 DPI until the end of the experiment and Dunn’s multiple-comparison test were performed. *p < 0.05. K Histopathological evaluation of demyelination with Luxol fast blue (LFB) and axonal loss with Bielschowsky’s silver (silver) staining. Arrows denote demyelination (LFB) and axonal loss (silver) staining of representative spinal cord sections. Scale bars, 500 μm. L Quantification of demyelination and axonal loss of individual mouse. Representative data of three independent experiments with n = 5 mice per group are shown. Error bars denote mean ± SEM; Mann-Whitney test was performed. *p < 0.05