Differences in Regulatory Mechanisms Induced by β-Lactoglobulin and κ-Casein in Cow's Milk Allergy Mouse Model-In Vivo and Ex Vivo Studies

Abstract

The presence of various proteins, including modified ones, in food which exhibit diverse immunogenic and sensitizing properties increases the difficulty of predicting host immune responses. Still, there is a lack of sufficiently reliable and comparable data and research models describing allergens in dietary matrices. The aim of the study was to estimate the immunomodulatory effects of β-lactoglobulin (β-lg) in comparison to those elicited by κ-casein (κ-CN), in vivo and ex vivo, using naïve splenocytes and a mouse sensitization model. Our results revealed that the humoral and cellular responses triggered by β-lg and κ-CN were of diverse magnitudes and showed different dynamics in the induction of control mechanisms. β-Lg turned out to be more immunogenic and induced a more dominant Th1 response than κ-CN, which triggered a significantly higher IgE response. For both proteins, CD4⁺ lymphocyte profiles correlated with CD4⁺CD25⁺ and CD4⁺CD25⁺Foxp3⁺ T cells induction and interleukin 10 secretion, but β-lg induced more CD4⁺CD25⁺Foxp3^- Tregs. Moreover, ex vivo studies showed the risk of interaction of immune responses to different milk proteins, which may exacerbate allergy, especially the one caused by β-lg. In conclusion, the applied model of in vivo and ex vivo exposure to β-lg and κ-CN showed significant differences in immunoreactivity of the tested proteins (κ-CN demonstrated stronger allergenic potential than β-lg), and may be useful for the estimation of allergenic potential of various food proteins, including those modified in technological processes.

Keywords: T cells; cow’s milk hypersensitivity; mouse model; β-lactoglobulin; κ-casein.

Figure 1

Scheme of experiments design (A) in vivo and (B) ex vivo. Used abbreviations: Ag—antigen; cFA—complete Freund adjuvant; iFA—incomplete Freund adjuvant; CT—cholera toxin.

Figure 2

The effect of antigen dose on proliferation index (PI) of naïve splenocytes stimulated with different doses of β-lg (A) or κ-CN (B) after 120 h culture. Cells were obtained from non-immunized mice. PI was determined with MTT method and calculated as the ratio of mean absorbance of antigen stimulated cells to mean absorbance of unstimulated cells. Concanavalin A (Con-A) was used as a positive control. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by post hoc Tukey test. Different letters present statistical differences between means at p ≤ 0.05.

Figure 3

Specific antibody titers induced in mice after immunization with β-lg (black squares) and κ-CN (white circles): (A) Serum anti-Ag IgG, (B) serum anti-Ag IgA, (C) fecal anti-Ag IgA. Each data point corresponds to the mean of the group ± SD. Statistical analysis was performed by t test. Asterisks indicate differences between the two groups at the same data point at p ≤ 0.05.

Figure 4

Total serum IgE concentration in mice immunized with β-lg (black bar) and κ-CN (white bar). The data are expressed as means ± SD. Statistical analysis was performed with one-way ANOVA follow by Tukey’s post hoc test.

Figure 5

The effect of major milk antigens (dose 200 μg/mL) on splenocytes sensitized in vivo to β-lg (A) or κ-CN (B). Proliferation was assessed via flow cytometry using the CFSE method. The data are expressed as means (n = 5) ± SD. Statistical analysis was performed with one-way ANOVA follow by Tukey’s post-hoc test. Different letters present statistical differences between means at p ≤ 0.05. A rectangle around the bar assigns primary antigen used for cells stimulation.

Figure 6

Distribution of CD8⁺, CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁺Foxp3⁺ T cells in mesenteric lymph nodes (MLNs), Peyer’s Patches (PPs), spleen (SPL), head and neck lymph nodes (HNLNs), and peripheral blood mononuclear cells (PBMCs) of mice immunized with β-lg (black bars) or κ-CN (white bars) and control mice treated with PBS only (gray bars). The data are expressed as the means ± SD. Statistical analysis was performed with one-way ANOVA follow by Tukey’s post-hoc test.

Figure 7

The effect of ex vivo stimulation of splenocytes isolated from mice immunized with β-lg (A) or κ-CN (B) with different milk antigens (at a dose 200 µg/mL) on the induction of CD4⁺CD25⁺Foxp3⁺ cells. Data are expressed as a mean ± SD. Statistical analysis was performed with one-way ANOVA follow by Tukey’s post-hoc test. Different letters present statistical differences between means at p ≤ 0.05. A rectangle around the bar assigns primary antigen used for cells stimulation.

Figure 8

Cytokines secreted during splenocyte culture after ex vivo stimulation with different milk antigens (dose 200 µg/mL). Lymphocytes were isolated from mice immunized with β-lg (A) or κ-CN (B). Data are expressed as mean ± SD. Statistical analysis were performed with one-way ANOVA follow by Tukey’s post-hoc test. Means with different letters differ at p < 0.05.