Figure 4.

Evaluation of food matrix effects on food-grade ${\text{SiO}}_2\text{-mediated}$ impairment of oral tolerance. (A) The protocol of oral tolerance induction to ovalbumin (OVA) followed by a de novo challenge to OVA in C57BL/6J mice daily treated for 60 d without (water gavage) or with food-grade ${\text{SiO}}_2$ ($fg\text{-}{\text{SiO}}_2$) ($10\mathrm{mg}/\mathrm{kg}$ BW/d) in water suspension [gastric gavage (gav)] or incorporated into food pellets (food). (B) OVA-specific IgG secretion in OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=7$ mice per group). (C) Fecal lipocalin-2 (Lcn2) levels in OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=7$ mice per group). (D–F) Amounts of $\text{IFN-}\mathrm{\gamma }$ (D), IL-10 (E), and $\text{TGF-}\mathrm{\beta }$ (F) secreted by mesenteric lymph node cells from OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=7$ mice per group). (G,H) The average frequency of T cells (${\text{CD}3}^{+}$ ${\text{CD}4}^{+}$) (G) and T cells ${\text{CD}25}^{+}$ ($\text{CD}{3}^{+}$ $\text{CD}{4}^{+}$ ${\text{CD}25}^{+}$) in colon lamina propria of OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=5$ mice per group). Foxp3 transcript expression in colon of OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=5$ mice per group). (J) The average frequency of ${\text{CD}45}^{+}$ ${\text{CD}11\mathrm{b}}^{+}$ ${\text{CD}103}^{+}$ cells (which fuses DC and macrophage subpopulations) in colon lamina propria of OVA-immunized (PBS) or OVA-tolerized mice exposed to a vehicle or $fg\text{-}{\text{SiO}}_2$ through gavage (gav) or food ($n=5$ mice per group). The data are expressed as median with interquartile range and whiskers extending from minimum to maximum ± SEM. *$p<0.05$; **$p<0.01$; ***$p<0.001$ by one-way ANOVA and post hoc Tukey test (B–I) or Kruskal-Wallis test followed by a post hoc Dunn’s test (J). Data behind graphs are reported in Table S4. Note: ANOVA, analysis of variance; BW, body weight; CFA, complete Freund adjuvant; IgG, immunoglobulin G; $\text{IFN-}\mathrm{\gamma }$, interferon gamma; IL-10, interleukin 10; OD, optical density; PBS, phosphate-buffered saline; SEM, standard error of the mean; $\text{TGF-}\mathrm{\beta }$, transforming growth factor beta.