Fig. 7
Reconstitution of gut microbiota with SCFA-producing bacteria increased humoral responses in an XX-dependent manner. Overview of experimental design (A). Briefly, gut microbiota of FCG mice were depleted using antibiotic oral gavage (3 days). On Day 4, mice were administered one of the following via oral gavage: inulin alone (Group 1); SCFA-producing bacteria alone (Group 2); or Inulin + SCFA-producing bacteria (Group 3). On Day 6, all mice were immunized with 2 × 108 CFU heat-killed Streptococcus pneumoniae. Mice in the inulin alone exposed group received antibiotics by oral gavage every other day through Day 12, while mice receiving SCFA-producing bacteria ± inulin received water. Mice were euthanized on Day 13 and samples collected for immune response evaluations. Fecal pellets were collected on Day 0, Day 4 (prior to gavage treatments), and Day 13. The number of living bacteria per pellet for females (B) and males (C) was assessed by flow cytometry to assess successful gut microbiota depletion and reconstitution. Immune responses were evaluated as numbers of IgM-secreting B cells and analyzed as Inulin alone vs. SCFA-producing bacteria (D) or as Inulin alone vs. Inulin + SCFA-producing bacteria (E). Concentrations of acetate (F), butyrate (G), and propionate (H), three well-studied SCFAs with immunomodulatory function, were assessed by LC–MS/MS (Metabolon) to confirm effective SCFA-producing bacteria recolonization and subsequent SCFA production. The concentrations of additional SCFAs are provided in Additional file 7: Fig. S7. Data point labels in B and C indicate the sex chromosome complement (XX or XY) and the group number as indicated in A. Data are represented as the mean ± SEM with each point representing one mouse. Statistics by three-way ANOVA with Tukey’s multiple comparisons test. Comparisons in graphs are representative of multiple comparisons tests; *p < 0.05. ANOVA main and interactive effects are provided in Additional file 9: Tables S14 and S15