Milk and Meat Allergens from Bos taurus β-Lactoglobulin, α-Casein, and Bovine Serum Albumin: An In-Vivo Study of the Immune Response in Mice

Abstract

The mechanism of food allergy may vary. This study aimed to compare the effects of milk, yogurt, or beef meat supplementation on humoral and cellular immune responses in a mice model. Mice were divided into four groups: The "Milk group" was sensitized with a β-lactoglobulin (β-lg)/α-casein (α-CN) mixture and supplemented cow milk; the "Yogurt group" was sensitized with β-lg/α-CN and supplemented yogurt; the "Beef group" was immunized with bovine serum albumin (BSA) and supplemented beef meat; and the "PBS group" received PBS in all procedures. ELISA was used to measure humoral response, including: Total IgE, specific IgG, and IgA. Cellular response was determined by phenotyping lymphocyte from lymphoid tissue and measuring the Th1/Th2 cytokine concentration with flow cytometry. The qPCR method was used for quantification of the fecal microbiota. The results obtained revealed a lower IgE level for the Yogurt group than for the Milk one. In the Yogurt group, the contribution of regulatory T cells to MLN and PP was higher compared to the other groups. We confirmed that diet supplementation with yogurt modulates the immune response to the prime allergen, and changes the activity of serum antibodies to milk proteins and BSA. Based on a specific antibodies level, we cannot exclude the possibility of CMA mice reaction against BSA.

Keywords: beef; cow milk; cross-reactivity; immune response; mice allergy model.

Figure 1

Experimental setup.

Figure 2

Total serum IgE levels in mice on days 14, 21, and 30 of the experiment. Mice were divided into four treatment groups (n = 10): Mice in the Milk and Yogurt groups were immunized with α-CN/β-lg and fed milk or yogurt, respectively. Mice in the Beef group were immunized with BSA and fed beef meat. Mice in the PBS group were administered PBS. Data are the mean ± SD. * p < 0.05, *** p < 0.001, one-way ANOVA with Tukey post-hoc tests.

Figure 3

Serum levels of IgGs against α-CN (A), β-lg (B), and BSA (C) on day 30 in mice from the Milk, Yogurt, Beef, and PBS groups. Data are the mean (n = 10) ± SD, *** p < 0.001, one-way ANOVA with Tukey post-hoc tests.

Figure 4

Levels of secretory IgA specific to anti-α-CN (A), anti-β-lg (B), and anti-BSA (C) in fecal samples collected on day 30 of the experiment. Data are the mean (n = 10) ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis test and one-way ANOVA with Tukey post-hoc tests.

Figure 4

Levels of secretory IgA specific to anti-α-CN (A), anti-β-lg (B), and anti-BSA (C) in fecal samples collected on day 30 of the experiment. Data are the mean (n = 10) ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis test and one-way ANOVA with Tukey post-hoc tests.

Figure 5

T cell profiles in the MLN (A,B) and PP (C,D) in mice from the PBS, Milk, Yogurt, and Beef groups. Cells were gated as follows: FSC/SSC lymphocytes were gated. Then, CD3⁺ and CD4⁺ or CD8⁺ cells were gated. From CD4⁺CD25⁺, FoxP3⁺ cells were gated. For each sample, 50,000 events were collected. Data are the mean ± SD. ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey post-hoc tests.

Figure 6

FoxP3⁺ cells among CD4⁺CD25⁺ splenocytes. Cells were stimulated with 100 µg/mL of α-CN, β-lg, or BSA and incubated at 37 °C for 120 h. Data are the mean ± SD. ** p < 0.01, one-way ANOVA with Tukey post-hoc tests.

Figure 7

IL-10 (A), TNF (B), IL-6 (C), IL-4 (D) secretion in cultured splenocytes. Cells were stimulated with 100 µg/mL of α-CN, β-lg, or BSA, or grown in medium, and were incubated at 37 °C for 120 h. Data are the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey post-hoc tests.

Figure 8

Effects of antigen treatments on mouse fecal microbiota profile. qPCR results are presented as log₁₀ of bacterial cells per gram of the wet weight of the fecal content of experimental animals. Data are the mean ± SD. * p < 0.05, *** p < 0.001. TBN = total bacteria; CCOC = Clostridium coccoides; CLEPT = Clostridium leptum; BPP = Bacteroides-Prevotella-Porphyromonas; BIFI = Bifidobacterium; LACT = Lactobacillus.