Table 4.

Animal studies of commensal therapies: summary of study characteristics and major findings

Study	ROB	Sample	MS Model	Intervention	Timeline	Duration	Major Findings
Montgomery et al.37	Low	M & F C57BL6 & PWD mice 4 wko	2× MOG-induced EAE	T: L. reuteri isolated from PWD cecal contents (100 µL of 109 CFU o.g.) w/ 100 µL cryopreserved C57BL6 cecal microbiota C: 200 µL cryopreserved C57BL6 cecal microbiota	Proph	4 wks (one initial admin)	CD: ↑ CDS & freq. of infiltrating CD4 + T cells in spinal cord; – freq. of infiltrating CD8 + T cells in spinal cord IM: ↑ freq. of GM-CSF- and IFN-γ- producing CD4+ & CD8 + T cells MM: NA MC: NA
Chen et al.65	UN	F C57BL6 mice 3–4 wko n = 8/g	2× MOG-induced EAE	T1: C. butyricum GDBIO1501, 5 × 106 CFU/mL o.g. T2: norfloxacin, 5 mg/kg o.g. C1: PBS w/ EAE C2: normal control	Proph	Daily for 3 wks pre-EAE	CD: ↓ daily CS, lymphocyte infiltration, demyelinating plaques in lumbar spinal cord IM: ↓ TH17 cells in CNS, LN, colon, spleen, & small intestine, IFN-γ-producing CD4 + T cells in spleen; – IL-17A+IFN-γ+ CD4 + T cells; ↑ differentiation of Tregs MM: ↑ # of OTUs, abundance, diversity, and rel. abund. of Prevotella, Bacteriodetes; ↓ rel. abund. of Firmicutes, Desulfovibroneceae, Ruminococcus MC: ↓ phosphorylation of p38 MAPK, ERK1/2, and JNK in lumbar spinal cord
Mangalam et al.56	UN	M & F HLA-DR3.DQ8 transgenic mice 8–12 wko n = 5–11/g	PLP-induced EAE	T1: Proph P. histicola, 108 CFU/mL o.g. T2: Proph P. melaninogenica, 108 CFU/mL o.g. T3: Proph C. sputigena, 108 CFU/mL o.g. All isolated from duodenum of celiac disease patients T4: Proph mouse-specific E. coli, 108 CFU/mL o.g. T5: T1 w/ Abx-depleted flora T6: Thera T1 T7: HK T6 T8: T6 cell-free supernatant T9: T6 107 CFU T10: T6 109 CFU C: medium	Proph & Thera	Every other day for 2 wks (starting 7 d pre-EAE or 7 dpi) Abx depletion for 3 wks AT at 5 dpi	CD: ↓ EAE incidence, CDS, regions of brain & spinal cord inflammation and demyelination, BBB permeability, CNS cellular infiltration (T1); earlier onset (T1); T1 restored gut permeability IM: ↓ IL-23, IL-12, IFN-γ, IL-17, CD4 + T cells, IFN-γ- and IL-17-expressing CD4 + T cells (T1); ↑ IL-10, TGF-β, CD4+ CD25+ Foxp3+ Tregs (T1); ↑ IL-10 (T2) MM: ↑ rel. abund. of Prevotella, Lactobacillus, Sutterella (T1), resembling pre-EAE states MC: Milder EAE (T5); ↑ EAE incidence (T7/T8); ↑ EAE suppression (T6 vs T9/T10); ↓ EAE incidence in AT recipient mice of T1
Shahi et al.57	UN	M & F HLA-DR3.DQ8 double transgenic mice & C57BL6 mice 8–12 wko n ≥ 7/g	PLP-induced EAE (HLA) and MOG-induced EAE (C57BL6)	T1: Proph P. histicola, 108 CFU o.g. in HLA mice T2: Proph Copaxone, 2 mg s.c. in HLA mice T3: T1+ T2 T4: T1 in C57BL6 mice T5: T2 in C57BL6 mice T6: T3 in C57BL6 mice T7: Thera T1 T8: Thera T2 T9: Thera T3 C: PBS or TSB media	Proph & Thera	Every other day for 2 wks (starting 7 d pre-EAE or 7 dpi) T3, T6, T9 = T1 & T2 on alternating days	CD: ↓ average daily scores & CDS (T2,T3,T4-T6,T7-T9); delayed EAE onset (T9); ↓ inflammation, demyelination (T1-T3); effects of T3/T6/T9 not more pronounced IM: ↓ IL-17+ CD4+ & IFN-γ+ CD4 + T cells in brain & spinal cord (T1,T3); ↑ CD4+ Foxp3+ Tregs in splenocytes and GALT (T1,T3); – IL-10-producing CD4 + T cells (T1-T3) MM: ↑ rel. abund. Lactobacillus (T1,C), ↓ (T2,T3) MC: NA
Shahi et al.58	UN	M & F HLA-DR3.DQ8 double transgenic mice 8–12 wko n ≥ 12/g	PLP-induced EAE	T1: Proph P. histicola, 108 CFU o.g T2: Proph IFNβ, 10,000 IU T3: Proph T1+ T2 T4: Thera T1 T5: Thera T2 T6: Thera T3 C: TSB media	Proph & Thera	Every other day for 2 wks pre-EAE (7 doses) or starting 7 dpi (7 doses)	CD: ↓ average daily score & CDS (T4-T6), inflammatory cellular infiltration into brain & spinal cord (T4,T6), spinal cord tissue and meningeal/stratum regions of brain (T4-T6); no additive effects for T6 vs T4/T5 IM: ↓ Iba-1+ microglia & GFAP+ astrocytes in brain & spinal cord white matter, CD4+ IL-17+ & CD4+ IFN-γ + T cells (T1-T3); ↑ CD4+ Foxp3+ Tregs, IL-10-producing CD4 + T cells (T1,T3) MM: NA MC: NA
Takata et al.70	UN	F C57BL6 mice & SJL mice 6 wko n = 17/g	MOG-induced EAE (C57BL6) and PLP-induced EAE (SJL)	T1: HK P. acidilactici R037 in C57BL6 mice, 20 mg/mL o.g. T2: HK P. acidilactici R037 in water in SJL mice, 0.8 mg/mL orally C1: PBS, o.g. C2: PBS in water, orally	Proph & Thera	Daily for 36 d (starting 2 wks pre-EAE)	CD: ↓ CS (T1,T2); ↓ infiltrating MNCs (T1); delayed EAE onset (T2) IM: ↓ IL-17 & IFN-γ in splenocytes & draining LNs; ↑ IL-10 & CD4+ IL-10 + T cells in mesenteric LNs and splenocytes MM: NA MC: NA
Miyauchi et al.38	Low	F GF C57BL6 mice 5–7 wko n = 5–10/g	MOG-induced EAE	T1: OTU0001 (L. reuteri, 100% 16S rRNA match to strains H4 & LMG 18238) T2: OTU0002 (Allobaculum) Both isolated from small intestine of specific PF mice and o.g. at 5–7 wko for stable colonization. T3: T1 + T2 co-colonized T4: T3 with urvA-deficient L. reuteri C: naïve GF	Proph	One admin at 5–7 wko	CD: ↑ CS (T2,T3); – CS (T1); ↓ CS, EAE incidence (T4); ↑ demyelination, spinal cord infiltration, EAE incidence (T3) IM: ↑ IL-17A, TH17 cells in lamina propria of small intestine & splenocytes (T2); ↑ Tregs in small intestine (T2); ↑ TH17 cells (T3); – IL-17A, TH17 cells, Tregs in small intestine (T2/T3); – TH17 cells in small intestine (T1/T4) MM: – rel. abund. in small intestine (T2/T3); – rel. abund. in small intestine (T1/T4) MC: ↑ expression of Saa1, Saa2, Il23a, Il12b, Csf2, Il23r in small intestine (T2); – expression of Saa1, Saa2, Il23a in small intestine (T2/T3)
Ochoa-Reparaz et al.62	UN	F SJL mice 6 wko n = 6–8/g	PLP-induced EAE	T1: 1 wk Abx, recolonize w/ WT B. fragilis NCTC 9343 T2: 1 wk Abx, recolonize w/ PSA-deficient B. fragilis Both at 1010 CFU in 200 µL sterile PBS o.g. C1: 1 wk Abx only C2: sham (no Abx, receive PBS)	Proph	30 d (one initial admin)	CD: ↓ CS, CDS (T1,C1); delayed EAE onset (T1,C1); – EAE incidence IM: ↓ Tbet (C1), IFN-γ (C1), RORγt (T1,C1), IL-17 (T1,C1); ↑ GATA-3 (T1,C1), IL-10 (T1,C1), SMAD-3 (T1), IL-13 (C1), freq. Foxp3+ CD25+ CD4 + T cells in cervical LNs (C1); ↑ IL-17, RORγt, Tbet and ↓ GATA-3, IL-10, IL-13 (T2); ↓ conversion of CD103+ DCs to Foxp3+ Tregs (T2) MM: # of detectable bacteria after Abx restored (T1,T2) (colonization confirmed) MC: Depletion of CD25 + T cells eliminated protective effects (C1,T1); ↓ CS & ↑ IL-10 in AT recipient mice (T1 only)
Liu et al.35	UN	F C57BL6 mice 6–8 wko n = 23–28/g	MOG-induced EAE	T1: Akkermansia muciniphila ATCC BAA-835 T2: E. coli K-12 Strain #7296 C: medium	Thera	Daily for 7 d (starting 11 dpi)	CD: ↓ CS, demyelination, axonal loss (T1) IM: ↑ MOG-specific Foxp3+ Tregs and total Tregs in spleen (T1); ↑ Foxp3+ Tregs from DCs (T1); – direct Foxp3+ Treg induction (T1 vs T2); ↓ IL-6, IL-1b expression in DCs (T1), – TGF-β expression (T1/T2) MM: NA MC: NA

All findings are reported with respect to control group(s) unless otherwise indicated.

Key: ↓ decreased; ↑ increased; – no change or no difference compared to control; NA, not applicable to this study.

Abbreviations: ROB, risk of bias; MS, multiple sclerosis; UN, uncertain; M, male; F, female; wk/wks/wko, week/weeks/weeks old; g, group; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; T, treatment group; C, control; CFU, colony-forming units; o.g., oral gavage; PBS, phosphate-buffered saline; s.c., subcutaneous; Abx, antibiotics; HK, heat-killed; IFNβ, interferon beta; IU, international unit; OTU, operational taxonomic unit; PF, pathogen-free; GF, germ-free; WT, wild-type; PSA, polysaccharide A; Thera, therapeutic; Proph, prophylactic; admin; administration; dpi, days post immunization; CD, clinical disease; IM, immune/metabolic; MM, microbiome/metabolome; MC, mechanistic/correlative; CDS, cumulative disease score; freq., frequency; GM-CSF, granulocyte monocyte colony-stimulating factor; IFN-γ, interferon gamma; CS, clinical score; CNS, central nervous system; LN, lymph node; IL, interleukin; rel. abund., relative abundance; BBB, blood–brain barrier; AT, adoptive transfer; GALT, gut-associated lymphoid tissue; DC, dendritic cell; TGF-β, transforming growth factor beta; MNC, mononuclear cell.