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Therapeutic Effect of Bifidobacterium Administration on Experimental Autoimmune Myasthenia Gravis in Lewis Rats

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Therapeutic Effect of Bifidobacterium Administration on Experimental Autoimmune Myasthenia Gravis in Lewis Rats

Elena Rinaldi et al. Front Immunol.

Abstract

Beneficial effects of probiotics on gut microbiota homeostasis and inflammatory immune responses suggested the investigation of their potential clinical efficacy in experimental models of autoimmune diseases. Indeed, administration of two bifidobacteria and lactobacilli probiotic strains prevented disease manifestations in the Lewis rat model of Myasthenia Gravis (EAMG). Here, we demonstrate the clinical efficacy of therapeutic administration of vital bifidobacteria (i.e., from EAMG onset). The mechanisms involved in immunomodulation were investigated with ex vivo and in vitro experiments. Improvement of EAMG symptoms was associated to decreased anti-rat AChR antibody levels, and differential expression of TGFβ and FoxP3 immunoregulatory transcripts in draining lymph nodes and spleen of treated-EAMG rats. Exposure of rat bone marrow-derived dendritic cells to bifidobacteria or lactobacilli strains upregulated toll-like receptor 2 mRNA expression, a key molecule involved in bacterium recognition via lipotheicoic acid. Live imaging experiments of AChR-specific effector T cells, co-cultured with BMDCs pre-exposed to bifidobacteria, demonstrated increased percentages of motile effector T cells, suggesting a hindered formation of TCR-peptide-MHC complex. Composition of gut microbiota was studied by 16S rRNA gene sequencing, and α and β diversity were determined in probiotic treated EAMG rats, with altered ratios between Tenericutes and Verrucomicrobia (phylum level), and Ruminococcaceae and Lachnospiraceae (family level). Moreover, the relative abundance of Akkermansia genus was found increased compared to healthy and probiotic treated EAMG rats. In conclusion, our findings confirms that the administration of vital bifidobacteria at EAMG onset has beneficial effects on disease progression; this study further supports preclinical research in human MG to evaluate probiotic efficacy as supplementary therapy in MG.

Keywords: EAMG; MG; immunoregulation; probiotics; therapeutic treatment.

Figures

Figure 1
Figure 1
Therapeutic administration of vital bifidobacteria strains ameliorates EAMG course. (A) Clinical EAMG score (mean ± SEM) of EAMG animals treated with vehicle, LBmix or BBmix (n = 6 rats/group). (B) Clinical EAMG score (mean ± SEM) of EAMG animals treated with vehicle, BBmix heat exposed or BBmix vital (n = 11 rats/group). (C,D) Anti-rat AChR Ab serum titer (pmol/ml of rat serum, mean ± SD) of treated-EAMG rats. Two-way ANOVA test with Tukey's post-hoc test for multiple-comparisons was used for clinical score. One-way ANOVA test with Dunnett's multiple comparison test was used for anti-rat AChR. Corrected p-values are reported.
Figure 2
Figure 2
Differential expression of pro-inflammatory and regulatory transcripts in primary and secondary lymphoid organs of EAMG rats. qRT-PCR analysis of IFNγ, IL6, FoxP3, and TGFβ mRNAs (mean ± SD) in drLNs (A) and spleen (B) of EAMG rats treated with vehicle, BBmix vital or BBmix heat exposed. One-way ANOVA test with Dunnett's multiple comparison test was used to assess statistical significance. Corrected p-values are reported.
Figure 3
Figure 3
Probiotics interact with immune cells located in Peyer's Patches and villi. SIM images of WGA-AF555 (red) labeled-BA (A) and LR (B). (C) Hematoxilin and Eosin staining of rat small intestine. (D) Confocal and (E) SIM images of villi stained for cytokeratin (green) and nuclei (blue). WGA-AF555 LR (red) are located inside villi (confocal image scale bar: 50 μm). (F) Confocal and (G) SIM images of PP stained for vimentin (green) and nuclei (blue). WGA-AF555 LR (red) are located inside the PP (confocal image scale bar: 100 μm). SIM images of CD3+ cell (green) (H) and CD11c+ cell (green) (I). WGA-AF555 LR (red) are detected nearby CD3+ and CD11c+ cells. SIM image scale bar: 2 μm.
Figure 4
Figure 4
In vitro interaction between BMDCs and probiotics. (A) Single frame of BMDCs co-cultured with WGA-AF555 labeled-LR (red). Scale bar 20 μm. (B) SIM image of a BMDC physically interacting with a chain of WGA-AF555 labeled-LR (red). (C,D) SIM images of CD11c+BMDC (green) and WGA-AF555-LC and WGA-AF555-BA (red) respectively. Site of contact between BMDCs and bacterial cells are reported in insets (3D volume). SIM image scale bar: 2 μm.
Figure 5
Figure 5
Differential expression of TLRs in BMDCs exposed to probiotics. (A) qRT-PCR analysis of TLR2, TLR1, and TLR6 in BMDCs exposed to single strains (BA, BL, LC, LR) and combinations (BBmix, LBmix) of probiotics for 4 h (mean ± SD). (B) Confocal and (D) SIM images of untreated CD11c+ cells (green) and TLR2 (red). (C) Confocal and (E) SIM images of LR-exposed CD11c+ cells (green) and TLR2 (red). (F) SIM image of LTA (green) and TLR2 (red) of BMDCs exposed to LC (inset: 3D volume). Statistical significance was assessed by one-way ANOVA test with Dunnett's multiple comparison test. Corrected p-values are reported. Confocal image scale bar: 50 μm; SIM image scale bar: 5 μm.
Figure 6
Figure 6
Treatment of BMDCs with probiotics modifies R97-116 T cell motility. (A) Experimental set-up of live imaging analyses of CFSE-labeled R97-116-specific T cells co-cultured with R97-116-loaded BMDCs, and example of motility analysis. (B) Percentage, (D) velocity and (F) meandering index of motile Teff. (C) Percentage, (E) velocity, and (G) meandering index of stationary Teff. Experimental conditions were: control cultures of BMDC, loaded with R97-116 peptide (empty bars), BMDC exposed to BBmix prior to antigen loading (black bars), BMDC exposed to TGFβ prior to antigen loading (striped bars). Statistical significance was assessed by two-way ANOVA test with Dunnett's multiple comparison test. Corrected p-values are reported.
Figure 7
Figure 7
Treatment with Bifidobacteria modifies alpha and beta diversity of EAMG gut microbiota. (A) Observed OTUs, (B) Shannon index, (C) evenness, and (D) unweighted UniFrac distance PCoA of healthy rats (HD rats; red dots), EAMG rats 30 days after the induction of the disease (EAMG onset; blue dots), EAMG rats in the chronic phase (EAMG chronic; green dots), EAMG rats treated with vital bifidobacteria (EAMG BBmix vital; pink dots), and EAMG rats treated with heat exposed bifidobacteria (EAMG BBmix heat exposed; turquoise dots).
Figure 8
Figure 8
Bifidobacteria reduce the dysbiosis induced by the disease. Stacked barplot representing the taxonomic assignments at phylum level (phyla with relative abundance higher than 0.1%). Major phyla, defining on average the 98% of the bacterial community (A), and minor phyla (B). Stacked barplot representing the taxonomic assignments at family level (families with relative abundance higher than 1%). Dominant families, defining on average the 86% of the bacterial community (C), and less abundant families (D).

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