Butyrate Enhances Desensitization Induced by Oral Immunotherapy in Cow’s Milk Allergic Mice

Figure 2

Enhanced Treg-mediated BMMC suppression after exposure to OIT and butyrate supplementation in vivo. CD4+ cells derived from pooled spleen suspensions were separated based on CD25 positivity. (a) Schematic representation of BMMC-Treg suppression assay. (b) CD4+CD25+ cells were verified for Foxp3 expression with flow cytometry and showed ±70% positivity in all groups. (c) CD4+CD25− cells and (d) CD4+CD25+ cells after MACS separation. Subsequently, CD4+CD25+ cells were cocultured with BMMC sensitized with anti-DNP-IgE. (e) Reduced release of β-hex upon BMMC activation with 25 ng/ml DNP-HSA was observed in the presence of Tregs derived from OIT + butyrate mice compared to Tregs derived from OIT and sensitized control mice. Additional 24 h incubation of BMMC in fresh culture medium indicated (f) reduced production of IL-13 after coculture with OIT + butyrate-Tregs compared to OIT-Tregs and (g) no differences in IL-6 release by activated BMMC. Data are represented as mean ± SD in (e–g) duplicate measurements. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test to compare preselected combinations. , . FSC-A: forward scatter-area; OIT: oral immunotherapy; FOS: fructo-oligosaccharides; but: butyrate; Treg: regulatory T cell; MC: mast cell; BMMC: bone marrow-derived mast cell; β-hex: β-hexosaminidase; DNP-HSA: dinitrophenol hapten conjugated to human serum albumin.

(a)

(b)

(c)

(d)

(e)

(f)

(g)