Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jul 23:10:1662.
doi: 10.3389/fimmu.2019.01662. eCollection 2019.

Gut Microbiota Interventions With Clostridium butyricum and Norfloxacin Modulate Immune Response in Experimental Autoimmune Encephalomyelitis Mice

Hao Chen  1 , Xiaomeng Ma  1 , Yingying Liu  1 , Lili Ma  1 , Zhaoyu Chen  1 , Xiuli Lin  1 , Lei Si  1 , Xueying Ma  1 , Xiaohong Chen  1
Affiliations

Gut Microbiota Interventions With Clostridium butyricum and Norfloxacin Modulate Immune Response in Experimental Autoimmune Encephalomyelitis Mice

Hao Chen et al. Front Immunol. .

Abstract

Gut microbiota has been proposed as an important environmental factor which can intervene and modulate central nervous system autoimmunity. Here, we altered the composition of gut flora with Clostridium butyricum and norfloxacin in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We found that appropriate C. butyricum (5.0 × 106 CFU/mL intragastrically daily, staring at weaning period of age) and norfloxacin (5 mg/kg intragastrically daily, 1 week prior to EAE induction) treatment could both ameliorate EAE although there are obvious differences in gut microbiota composition between these two interventions. C. butyricum increased while norfloxacin decreased the abundance and diversity of the gut microbiota in EAE mice, and both of the treatments decreased firmicutes/bacteroidetes ratio. In the genus level, C. butyricum treatment increased the abundance of Prevotella while Akkermansia and Allobaculum increased in norfloxacin treatment. Moreover, both interventions reduced Desulfovibroneceae and Ruminococcus species. Although there was discrepancy in the gut microbiota composition with the two interventions, C. butyricum and norfloxacin treatment both reduced Th17 response and increased Treg response in the gastrointestinal tract and extra-gastrointestinal organ systems in EAE mice. And the reduced activity of p38 mitogen-activated kinase and c-Jun N-terminal kinase signaling in spinal cord could be observed in the two interventions. The results suggested that manipulation of gut microbiota interventions should take factors such as timing, duration, and dosage into consideration. The discrepancy in the gut microbiota composition and the similar protective T cells response of C. butyricum and norfloxacin implies that achieving intestinal microecology balance by promoting and/or inhibiting the gut microbiota contribute to the well-being of immune response in EAE mice.

Keywords: Clostridium butyricum; EAE; Th17/Treg; gut microbiota; multiple sclerosis; norfloxacin.

PubMed Disclaimer

Figures

Figure 1
Figure 1
C.butyricum and norfloxacin treatment both ameliorated clinical severity and neuropathology of EAE mice. (A) Clinical score was assessed daily and shown (n = 8). (B–E) At 21 days post immunization, lumbosacral spinal cords were isolated and performed H&E (n = 6) (D) or Solochrome cyanin impregnation (E) staining for assessment of histopathology. Representative sections (D,E) and statistical analysis (B,C) data are shown. The infiltration of lymphocytes and demyelination are highlighted by arrow. Experiments were repeated three times with similar results. Statistically significant data are indicated by asterisks (**P < 0.01 and ***P < 0.001).
Figure 2
Figure 2
Responses of the diversity, richness, and structure of the gut microbiota to sham, C. butyricum, and norfloxacin treatment in EAE mice. (A) Genus species phylogeny tree of all three groups. (B) Venn diagram of each group and the number of differences between species are shown. (C) Unweighted Unifrac principal coordinates analysis plots 3D of each sample and the distance of each group are shown. Each sample was collected from three mice (n = 3). (D–H) shows the Observed species index, Shannon index, Chao index, Simpson index, and ACE index of each group. (I–J) Relative abundances of the gut microbiota at phylum level and genus level. Statistically significant data are indicated by asterisks (*P < 0.05 and ***P < 0.001). ns, not significant.
Figure 3
Figure 3
Identification of differential microbes in response to C.butyricum and norfloxacin treatment in mice based on the linear discriminant analysis (LDA) and effect size (LEfSe) pipeline. (A) Cladogram using LEfSe method indicated the phylogenetic distribution of gut microbiota associated with mice in three groups. (B) LDA scores showed the significant bacterial differences in three groups.
Figure 4
Figure 4
Gut microbiota intervention diminish Th17 responses in EAE. Lymphocytes from CNS, LN, small intestine, colon, and spleen were isolated 21 days post immunization and used for assessment of different CD4 T cell subsets. (A) Representative staining of different CD4 T cell subsets in CNS and LN, gated on TCRβ+ CD4+. (B) Statistical analysis of the percentages of IL-17 and IFN-γ in (A). Representative staining (D) and statistical analysis (C) of IL-17 and IFN-γ in CD4 T cells isolated from small intestine, colon, and spleen of mice, gated on TCRβ+ CD4+. Statistically significant data are indicated by asterisks (**P < 0.01 and ***P < 0.001). ns, not significant.
Figure 5
Figure 5
Gut microbiota intervention expanded the Treg cells in EAE. (B) Representative staining of different CD4 T cell subsets in inguinal LN, colon and small intestine, gated on TCRβ+ CD4+. Statistical analysis of data in (A). Statistically significant data are indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 6
Figure 6
Gut microbiota intervention suppressed the phosphorylation of p38 MAPK and JNK, but not ERK1/2 in the lumbar spinal cord of EAE mice. The expressions of p38 MAPK, JNK, ERK1/2 and their phosphorylation expression were analyzed by Western blot. Statistically significant data are indicated by asterisks (**P < 0.01 and ****P < 0.0001). ns, not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Koch-Henriksen N, Sorensen PS. The changing demographic pattern of multiple sclerosis epidemiology. Lancet Neurol. (2010) 9:520–32. 10.1016/S1474-4422(10)70064-8 - DOI - PubMed
    1. Chen J, Chia N, Kalari KR, Yao JZ, Novotna M, Paz Soldan MM, et al. . Multiple sclerosis patients have a distinct gut microbiota compared to healthy controls. Sci Rep. (2016) 6:28484. 10.1038/srep28484 - DOI - PMC - PubMed
    1. Jangi S, Gandhi R, Cox LM, Li N, von Glehn F. Alterations of the human gut microbiome in multiple sclerosis. Nat Commun. (2016) 7:12015. 10.1038/ncomms12015 - DOI - PMC - PubMed
    1. Rothhammer V, Quintana FJ. Environmental control of autoimmune inflammation in the central nervous system. Curr Opin Immunol. (2016) 43:46–53. 10.1016/j.coi.2016.09.002 - DOI - PMC - PubMed
    1. Hayashi A, Sato T, Kamada N, Mikami Y, Matsuoka K, Hisamatsu T, et al. . A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice. Cell Host Microbe. (2013) 13:711–22. 10.1016/j.chom.2013.05.013 - DOI - PubMed

Publication types

MeSH terms

Substances