Prevotella histicola, A Human Gut Commensal, Is as Potent as COPAXONE® in an Animal Model of Multiple Sclerosis - PubMed (original) (raw)

Comparative Study

Prevotella histicola, A Human Gut Commensal, Is as Potent as COPAXONE® in an Animal Model of Multiple Sclerosis

Shailesh K Shahi et al. Front Immunol. 2019.

Abstract

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that there is enrichment or depletion of some gut bacteria in MS patients compared to healthy controls (HC), suggesting an important role of the gut bacteria in disease pathogenesis. Thus, specific gut bacteria that are lower in abundance in MS patients could be used as a potential treatment option for this disease. In particular, we and others have shown that MS patients have a lower abundance of Prevotella compared to HC, whereas the abundance of Prevotella is increased in patients that receive disease-modifying therapies such as Copaxone® (Glatiramer acetate-GA). This inverse correlation between the severity of MS disease and the abundance of Prevotella suggests its potential for use as a therapeutic option to treat MS. Notably we have previously identified a specific strain_, Prevotella histicola_ (P. histicola), that suppresses disease in the animal model of MS, experimental autoimmune encephalomyelitis (EAE) compared with sham treatment. In the present study we analyzed whether the disease suppressing effects of P. histicola synergize with those of the disease-modifying drug Copaxone® to more effectively suppress disease compared to either treatment alone. Treatment with P. histicola was as effective in suppressing disease as treatment with Copaxone®, whereas the combination of P. histicola plus Copaxone® was not more effective than either individual treatment. _P. histicola_-treated mice had an increased frequency and number of CD4+FoxP3+ regulatory T cells in periphery as well as gut and a decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4 T cells in the CNS, suggesting P. histicola suppresses disease by boosting anti-inflammatory immune responses and inhibiting pro-inflammatory immune responses. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as efficiently as Copaxone® and may provide an alternative treatment option for MS patients.

Keywords: Copaxone®; HLA transgenic mice; Prevotella histicola; animal model; experimental autoimmune encephalomyelitis (EAE); gut microbiome; immune response; multiple sclerosis.

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Figures

Figure 1

Figure 1

In a prophylactic setting P. histicola suppressed EAE similar to Copaxone® in HLA-DR3.DQ8 transgenic and C57BL/6 mice (A) In a prophylactic setting, HLA-DR3.DQ8 transgenic mice were divided into five groups (P. histicola alone, Copaxone® alone, P. histicola plus Copaxone®, PBS alone, and media alone). Copaxone® alone, P. histicola plus Copaxone® received 2 mg of Copaxone® in 200 μl of IFA 10 days before EAE induction. PBS alone group received 200 μl of a PBS/IFA emulsion 10 days before EAE induction. EAE was induced by immunization with PLP91−110/CFA (pertussis toxin (PTX) was administered on day of EAE induction and 2 days post-EAE induction) and 7 days post EAE induction P. histicola alone and P. histicola plus Copaxone® combination group of mice were orally gavaged with live P. histicola (108 CFUs) every other day for a total of seven doses. Mice in the control group (media only) were orally gavaged with TSB media every other day for a total of seven doses. Clinical scores were assessed daily for the duration of the experiment. (B) Cumulative EAE scores of mice treated as in (A). The data presented represent 1 of 3 experiments performed at different time points (n ≥ 7 mice per group). (C) C57BL/6 mice were treated with Copaxone®, P. histicola, or a combination of both starting day 14 prior to immunization with MOG35−55 CFA/PTX (with treatment administered on alternate days for a total of 14 doses, 7 doses of Copaxone® and 7 doses of P. histicola). Clinical scores were assessed daily for the duration of the experiment. A *p ≤ 0.05, ***p ≤ 0.0005 and ****p ≤ 0.00005 when compared to the PBS medium treated group. 2-way ANOVA Dunnett's multiple comparison test were used to calculate _p_-value among different treatment groups for average daily clinical score (A) and Mann-Whitney unpaired _U_-test were used to calculate _p-_value in cumulative EAE score (B).

Figure 2

Figure 2

In therapeutic setting P. histicola suppresses PLP91−110-Induced EAE in HLA-DR3.DQ8 transgenic mice similar to therapeutic treatment of Copaxone®. (A) Mice were immunized with PLP91−110/CFA plus pertussis toxin on days 0 and 2 of the disease induction and 1 week later mice were treated with Copaxone®, P. histicola or a combination of both (with treatment administered on alternate days for a total of 14 doses, 7 doses of Copaxone® and 7 doses of P. histicola) for 2 weeks. Clinical scores were assessed daily for the duration of the experiment. (B) Cumulative EAE scores of mice treated as in (A). (C) Mice were treated with Copaxone®, P. histicola or a combination of both after 12 days of immunization. Clinical scores were assessed daily for the duration of the experiment. (D) Cumulative EAE scores of mice treated as in (C). The data presented represent 1 of 3 experiments performed at different time points (n ≥8 mice per group). A *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.00005 when compared to the medium treated group. 2way ANOVA Dunnett's multiple comparisons test were used to calculate _p_-value in average clinical EAE score (A,C) and Mann–Whitney unpaired _U_-test were used to calculate _p-_value in cumulative EAE score (B,D).

Figure 3

Figure 3

Treatment with P. histicola alone, Copaxone® alone, or the combination of P. histicola and Copaxone® resulted in decreased inflammation and demyelination in the brain and spinal cord of HLA-DR3.DQ8 transgenic mice induced with EAE. Representative hematoxylin and eosin (H&E)-stained images of the brain or Luxol-fast blue stained images of spinal cords of mice treated with P. histicola alone, Copaxone® alone, P. histicola and Copaxone®, or media. 20X Brain section (inset black boxes in 4X) show regions of inflammation. Similarly spinal cord sections are enlarged at 10X to show regions with inflammation and demyelination. Data are representative of 3 independent experiments (n = 5 mice per group).

Figure 4

Figure 4

Treatment with P. histicola alone or in combination with Copaxone® increases CD4+FoxP3+ regulatory T cells in the spleen and GALT. (A) Mice were immunized with PLP91−110/CFA PTX on days 0 and 2 of the disease induction and 1 week later mice were treated with Copaxone® (7 doses), P. histicola (7 doses), or a combination of both (with treatment administered on alternate days for a total of 14 doses, 7 doses of Copaxone® and 7 doses of P. histicola). Clinical scores were assessed daily for the duration of the experiment. Representative flow cytometric plots to demonstrate CD4+FoxP3+ regulatory T cells in the spleen of mice treated with P. histicola alone, Copaxone® alone, P. histicola and Copaxone®, or media. Cells were previously gated on lymphocytes and singlets. (B) Frequency of CD4+FoxP3+ regulatory T cells from mice treated as in (A). (C) Quantification of the number of CD4+FoxP3+ regulatory T cells in mice treated as in (A). (D) Naïve mice were treated with Copaxone® (7 doses), P. histicola (7 doses), or a combination of both (with treatment administered on alternate days for a total of 14 doses). Gut-associated lymphoid cells were isolated from treated and control group of mice and stained with CD45, CD4 and FoxP3 antibodies. Frequency of CD4+FoxP3+ regulatory T cells from mice treated Copaxone®, P. histicola, P. histicola plus Copaxone®, and media. (E) Quantification of the number of CD4+FoxP3+ regulatory T cells in mice treated as in (D). Error bars are presented as standard error of the mean. The _p_-value determined by Mann–Whitney unpaired _U_-test for comparing each group to media. The data presented represent 1 of 3 experiments performed at different time points (n ≥ 7 mice per group).

Figure 5

Figure 5

HLA-DR3.DQ8 transgenic mice induced with EAE and treated with P. histicola alone or in combination with Copaxone® show a decrease in inflammatory cytokine-producing cells in the CNS. (A) Mice were immunized with PLP91−110/CFA plus pertussis toxin on days 0 and 2 of the disease induction and 1 week later mice were treated with Copaxone® (7 doses), P. histicola (7 doses), or a combination of both (with treatment administered on alternate days for a total of 14 doses, 7 doses of Copaxone® and 7 doses of P. histicola). Clinical scores were assessed daily for the duration of the experiment. Flow cytometric plots of IL17+- or IFNγ+-expressing mononuclear cells that were isolated from the brain and spinal cord of mice treated with P. histicola alone, Copaxone® alone, P. histicola and Copaxone®, or media. Cells were isolated and stimulated with antigen (PLP91−110) plus Brefeldin A for 12 h. Plots were previously gated on CD4+ cells. (B–D) Quantification of the frequency of CD4+IL17+T cells (B), CD4+IFNγ+ T cells (C), and CD4+IL17+IFNγ+ T cells (D) from mice treated as in (A). Cells were previously gated on lymphocytes, singlets, and CD4+ cells. The data presented are the average of 2 independent experiments with n = 4 mice per group. The _p_-value determined by Mann–Whitney unpaired _U_-test. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, and “n.s.” indicates not significant when compared to the media-treated group.

Figure 6

Figure 6

P. histicola but not Copaxone® mediates disease suppression through restoration of gut microbiota (A) Fecal samples were collected from pre-immunized HLA-DR3.DQ8 transgenic mice (Naïve/Nor), or mice immunized for EAE (EAE) and treated with Copaxone® (Cop) or P. histicola (PH). Fecal DNA was extracted, 16s rRNA (V3-V5) region was amplified, and sequenced on Illumina MiSeq platform. Two dimension Partial least square- dimension analysis (PLS-DA) scores plot of fecal microbiota from different treatment groups and naïve (Nor) mice with each dot representing a mice. (B) Box plot showing normalized relative abundance of Lactobacillus among different groups. (C) PLS-DA scores plot showing fecal microbiota profile from pre-immunized mice (Nor) or mice immunized for EAE (EAE) or Copaxone® plus P. histicola (Cop_plus_PH). (D) Box plot showing normalized relative abundance of Lactobacillus among different groups. Difference among groups were analyzed using one-way ANOVA (Kruskal–Wallis rank sum test) and FDR-adjusted p < 0.05.

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References

    1. Stinissen P, Raus J, Zhang J. Autoimmune pathogenesis of multiple sclerosis: role of autoreactive T lymphocytes and new immunotherapeutic strategies. Crit Rev Immunol. (1997) 17:33–75. 10.1615/CritRevImmunol.v17.i1.20 - DOI - PubMed
    1. Willer CJ, Dyment DA, Risch NJ, Sadovnick AD, Ebers GC, Canadian Collaborative Study G. Twin concordance and sibling recurrence rates in multiple sclerosis. Proc Natl Acad Sci USA. (2003) 100:12877–82. 10.1073/pnas.1932604100 - DOI - PMC - PubMed
    1. Hollenbach JA, Oksenberg JR. The immunogenetics of multiple sclerosis: a comprehensive review. J Autoimmun. (2015) 64:13–25. 10.1016/j.jaut.2015.06.010 - DOI - PMC - PubMed
    1. Goodin DS. The causal cascade to multiple sclerosis: a model for MS pathogenesis. PLoS ONE. (2009) 4:e4565. 10.1371/journal.pone.0004565 - DOI - PMC - PubMed
    1. Ramagopalan SV, Handel AE, Giovannoni G, Rutherford Siegel S, Ebers GC, Chaplin G. Relationship of UV exposure to prevalence of multiple sclerosis in England. Neurology. (2011) 76:1410–4. 10.1212/WNL.0b013e318216715e - DOI - PMC - PubMed

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