A free amino acid-based diet partially prevents symptoms of cow's milk allergy in mice after oral sensitization with whey

Abstract

Background: Amino acid-based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known.

Objective: The present study evaluated the preventive effects of an amino acid (AA)-based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism.

Methods: C3H/HeOuJ mice were sensitized with whey protein or with phosphate-buffered saline as sham-sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein-based diet or an AA-based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey-specific immunoglobulin levels, and FoxP3⁺ cell counts in jejunum sections were assessed.

Results: Compared to mice fed with the protein-based diet, AA-fed mice had significantly lower acute allergic skin responses. Moreover, the AA-based diet prevented the whey-induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA-based diet had no effect on the levels of serum IgE and mucosal mast cell protease-1 (mMCP-1), AA-fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA-based diet prevented the whey-induced decrease in FoxP3⁺ cells. In sham-sensitized mice, no differences between the two diets were observed in any of the tested parameters.

Conclusion: This study demonstrates that an AA-based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3⁺ cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti-inflammatory and tolerizing capacities in AA-fed mice. This, combined with the absence of effects in sham-sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research.

Keywords: allergy; free amino acids; infant milk formula; prevention.

Figure 1

Schematic representation of the experimental model and the experimental groups

Figure 2

The acute allergic skin response, body temperature, and anaphylactic shock severity of mice fed with the control or with the amino acid (AA)‐based diet. A, Ear swelling was measured before and 1 hour after i.d. challenge with whey. Body temperature (B) and anaphylactic shock severity (C) was measured before and 15, 30, and 60 minutes after i.d. challenge. Groups are as follows: sham‐sensitized mice fed with the control diet (n = 10); sham‐sensitized mice fed with the AA diet (n = 10); whey‐sensitized mice fed with the control diet (n = 9); whey‐sensitized mice fed with the AA diet (n = 10). Values are expressed as mean ±  standard error of the mean (SEM). Significant differences between whey‐sensitized and sham‐sensitized mice are indicated by *P < .05, **P < .01, ***P < .001, and ****P < .0001. Differences between whey‐sensitized mice fed with the control diet and those fed with the AA diet are indicated by ^$ P < .05, ^$$ P < .01. Differences are analyzed with a one‐way analysis of variance (ANOVA) followed by a Bonferroni post‐hoc test (A and B) or a Kruskal–Wallis test with a post‐hoc Dunn's test (C)

Figure 3

Serum levels of mouse mast cell protease‐1 (mMCP‐1) and whey‐specific Ig of mice fed a control diet or an AA‐based diet. Serum of all mice (n = 9‐10 per group) was harvested 30 minutes after the oral challenge. Serum levels are shown for (A) mMCP‐1 (ng/mL), (B) whey‐specific IgE (arbitrary unit [AU]), (C) whey‐specific IgG1 (AU), and (D) whey‐specific IgG2a (AU). Values are expressed as mean ± SEM. Significant differences between whey‐sensitized and sham‐sensitized mice are indicated by *P < .05, **P < .01, ***P < .001, and ****P < .0001. Differences between whey‐sensitized mice fed with the control diet and those fed with the AA diet are indicated by ^$ P < .05. Differences were analyzed with a one^‐way ANOVA followed by a Bonferroni post‐hoc test for selected groups after log‐transformation of the data. AA, amino acid; ANOVA, analysis of variance; SEM, standard error of the mean

Figure 4

Correlations between Ig serum levels and the acute allergic skin response. Correlations are shown for ear swelling (µm) and serum levels of whey‐specific IgG1 (A), IgG2a (B), and IgE (C) in mice fed the control diet (n = 19). Moreover, correlations are shown for ear swelling (µm) and serum levels of whey‐specific IgG1 (D), IgG2a (E), and IgE (F) in all whey‐sensitized mice (n = 19). Levels of Igs were log‐transformed before the testing of correlations by Pearson correlation. AU, arbitrary unit

Figure 5

Immunohistochemical staining of Forkhead box P3 (FoxP3) in jejunum sections of mice fed with the control diet and mice fed with the AA diet. A, Cell counts of FoxP3⁺ cells in jejunum sections (n = 9‐10 per group) expressed as number of positive cells per villi. Values are expressed as mean ± SEM. Significant differences between whey‐sensitized and sham‐sensitized mice are indicated by *P < .05 and **P < .01. Differences between whey‐sensitized mice fed with the control diet and those fed with the AA diet are indicated by ^$ P < .05. Differences are analyzed by one‐way ANOVA followed by a Bonferroni post‐hoc test. Representative images of the immunohistochemical staining are shown for (B) whey‐sensitized mice fed with the control diet and (C) whey‐sensitized mice fed with the AA diet. Arrows () indicate positive intracellular staining for FoxP3. ANOVA, analysis of variance; SEM, standard error of the mean

Figure 6

The body weight (A) and food intake (B) of the mice throughout the study protocol. Groups are as follows: sham‐sensitized mice fed with the control diet (n = 10); sham‐sensitized mice fed with the AA diet (n = 10); whey‐sensitized mice fed with the control diet (n = 9); whey‐sensitized mice fed with the AA diet (n = 10). Food intake was measured per cage (n = 2 per group). Differences are analyzed with a one‐way ANOVA followed by a Bonferroni post‐hoc test (A) or a Kruskal–Wallis test with a post‐hoc Dunn's test (B). Values are expressed as mean ± SEM. ANOVA, analysis of variance; SEM, standard error of the mean