Improvement of Therapeutic Efficacy of Oral Immunotherapy in Combination with Regulatory T Cell-Inducer Kakkonto in a Murine Food Allergy Model

<div><p>Oral immunotherapy (OIT) has been considered a promising approach for food allergies (FAs). However, the current OIT strategy is limited in terms of the long-term efficacy and safety. We have previously demonstrated that kakkonto, a traditional Japanese herbal medicine, suppresses the occurrence of allergic symptoms in a murine model of ovalbumin (OVA)-induced FA, which is attributed to the induction of the Foxp3⁺ CD4⁺ regulatory T cells. In this study, we established an OIT model using the FA mice with already established allergic symptoms and determined whether kakkonto could improve the efficacy of OIT. The OIT method consisted of initially administrating a very small amount of OVA and slowly increasing the amount. Allergic symptoms decreased in the OIT-treated FA mice. OIT significantly downregulated Th2 immune response-related gene expression in the FA mouse colon, and decreased the level of mouse mast cell protease-1, a marker of mast cell degranulation in the FA mouse plasma. Moreover, the concomitant use of kakkonto significantly enhanced the effectiveness of OIT on the allergic symptoms, and the combination therapy further suppressed the Th2 immune responses and the mast cell degranulation. In addition, OIT significantly increased the population of Foxp3⁺ CD4⁺ regulatory T cells in the FA mouse colon, and this population was further increased by OIT in combination with kakkonto. Furthermore, the combined therapy with kakkonto reduced the expression of RA-degrading enzyme CYP26B1 mRNA in the FA mouse colon. These findings indicated that the combination of OIT with kakkonto represents a promising approach for FA treatment.</p></div

Association of retinoic acid with the combined therapy.

<p>(A) Association between our microarray result and the canonical pathway of “retinoid biosynthesis”. Each value is shown as a -log P value, which was calculated using the right-tailed Fisher’s exact test. The threshold line corresponds to the P value of 0.05. (B) CYP26B1, CRABP1 and ALDH1A1 mRNA expression level in the proximal colon. The segment of the proximal colon was obtained 1 h after the non-heated OVA challenge on day 49 (Normal n = 5, FA, OIT and OIT+kakkonto n = 14, *P<0.05, **P<0.01, ***P<0.001). (C) Dose dependency of RA on Foxp3⁺ CD4⁺ Treg differentiation. Naive CD4⁺ T cells from RAG-2^-/- DO11.10 mice were co-cultured with SpDCs with the presence of OVA peptide and RA (100 nM, 10 nM, 3.3 nM, 1 nM, 0.3 nM and vehicle) (n = 3, *P<0.05 vs vehicle).</p

Differential gene expression of Th2 cell markers.

<p>(A) Heat map of the expression of genes associated with the Th2 response (subset mentioned in IPA dataset). (B) The IL-4, IL-5, IL-13, and GATA3 mRNA expression levels in the proximal colon. The segment of the proximal colon was obtained 1 h after the non-heated OVA challenge on day 49 (Normal n = 5, FA, OIT and OIT+kakkonto n = 14, *P<0.05, **P<0.01, ***P<0.001). (C) The OVA-specific IgE and IgG1 levels in the plasma was measured using an ELISA 1 h after the non-heated OVA challenge to assess the allergic symptoms on day 49 (Normal n = 5, FA, OIT and OIT+kakkonto n = 20).</p

Outline of the experimental design.

<p>OVA-sensitized BALB/c mice were challenged 3 times per week by oral administration of non-heated OVA solution. After day 40, mice were treated with kakkonto, OIT, OIT+kakkonto or placebo (MC: 0.5% methylcellulose solution and sterilized water) daily for 8 days. One hour before each oral heated OVA challenge, kakkonto or placebo (MC) was orally administrated. Before and after the OIT, mice received a non-heated OVA challenge to assess allergic symptoms (day 40 and day 49, respectively).</p

Clinical symptoms in mice under different treatments.

<p>OVA-sensitized mice were treated with OIT, OIT and kakkonto combined therapy (OIT+kakkonto), kakkonto alone (kakkonto) or placebo (FA). One hour after the non-heated OVA challenge on day 49, the severity of allergic diarrhea was assessed (kakkonto n = 18, FA n = 46, OIT n = 48, OIT+kakkonto n = 48, **P<0.01, ***P<0.001).</p

Induction of Th2-mediated food allergy.

<p>The allergic responses were induced by two instances of systemic OVA priming and repeated oral OVA (50 mg) challenges. <i>A</i>: Systemically primed BALB/c mice (closed circle) and C57BL/6 mice (open circle) and non-systemically primed BALB/c mice (open square) were subjected to food allergy (FA)-inducing oral OVA challenges (<i>n</i> = 5–8 mice per group). <i>B</i>: The level of OVA-specific IgE in the plasma of naïve mice, systemically primed mice, and systemically primed and repeated oral OVA challenged mice is shown. The level of OVA-specific IgE in the plasma was determined using an ELISA kit. Data are expressed as means ±SE. **<i>P</i><0.01 vs. naïve mice. †<i>P</i><0.05 vs. systemically primed mice (<i>n</i> = 5 mice per group).</p

Induction of food allergy in PI3K-deficient mice.

<p>PI3K^−/− mice as a gastrointestinal mast cell-deficient murine model were subjected to FA-inducing oral OVA challenges. <i>A</i>: The occurrence of allergic diarrhea in WT mice (WT FA mice) and PI3K^−/− mice (PI3K^−/− FA mice) after each oral OVA challenge is shown (<i>n</i> = 5 mice per group). <i>B</i>: The proximal colon of FA-induced PI3K^−/− mice after oral OVA challenge was stained with anti- mMCP-1 antibodies. Scale bars represent 200 µm. <i>C</i>: The level of mMCP-1 in the plasma of PI3K^−/− naïve mice and PI3K^−/− FA mice is shown. The level of mMCP-1 in the plasma was determined using an ELISA kit. Data are expressed as means ±SE. **<i>P</i><0.01 vs. WT naïve mice, ††<i>P</i><0.01 vs. WT FA mice (<i>n</i> = 3–5 mice per group). IL-4 and IL-5 cytokine mRNA expression in the spleen (<i>D</i>) and proximal colon (<i>E</i>) from WT naïve mice, WT FA mice, PI3K^−/− naïve mice and PI3K^−/− FA mice were measured by real-time PCR. Relative mRNA levels of cytokines were normalized to GAPDH expression. *<i>P</i><0.05, **<i>P</i><0.01 vs. each naïve mice, ††<i>P</i><0.01 vs. WT FA mice (<i>n</i> = 4–7 mice per group).</p

Effect of mast cell stabilizers on the induction of food allergy in mice.

<p>The occurrence of allergic diarrhea in FA mice (closed circle) and cromolyn-treated mice (open diamond) after each oral OVA challenge is shown (<i>n</i> = 14–15 mice per group). In addition, mice treated with the subcutaneous administration of doxantrazole (open circle, 10 mg/kg) or the oral administration of prednisolone (open square, 10 mg/kg) did not exhibit any sign of allergic responses in the FA model. **<i>P</i><0.01 vs. FA mice (<i>n</i> = 10 mice per each group).</p

Cholinergic control of food allergy in mice.

<p><i>A</i>: The occurrence of allergic diarrhea in FA mice (closed circle), 2-deoxy-d-glucose (2-DG)-treated mice (open circle) and hexamethonium (C6)-treated mice prior to 2-DG administration (open square) after each oral OVA challenge is shown. *<i>P</i><0.05 vs. FA mice. †<i>P</i><0.05 vs. 2-DG mice (<i>n</i> = 11–18 mice per group). <i>B</i>: The occurrence of allergic diarrhea in FA mice (closed circle) and nicotine-treated mice (3.2 mg/kg; open circle, 1.0 mg/kg; open square, and 0.32 mg/kg; open diamond) after each oral OVA challenge is shown. *<i>P</i><0.05, **<i>P</i><0.01 vs. FA mice (<i>n</i> = 10–74 mice per group). <i>C</i>: The occurrence of allergic diarrhea in FA mice (closed circle) and GTS-21-treated mice (open circle) after each oral OVA challenge is shown. *<i>P</i><0.05, **<i>P</i><0.01 vs. FA mice (<i>n</i> = 23–40 mice per group).</p

Mucosal mast cell hyperplasia and degranulation in the colon of food allergy mice.

<p><i>A</i>: The proximal colons of systemically OVA primed mice and oral OVA-challenged mice after each oral OVA challenge were stained with anti-mouse mast cell protease-1 (mMCP-1) antibodies. Scale bar represents 200 µm. <i>B</i>: The concentration of mMCP-1 in the plasma of naïve mice and FA mice is shown. The concentration of mMCP-1 in the plasma was determined using an ELISA kit. Data are expressed as means ±SE. **<i>P</i><0.01 vs. naïve mice (<i>n</i> = 5 mice per group).</p