Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice

Abstract

The proteolytic digest of milk casein, known as casein phosphopeptide (CPP-III), exhibits diverse biological activities, including calcium absorption and antioxidant activities. We hypothesized that the additional phosphorylation of this peptide can enhance its immunomodulatory activity such as suppression of allergy-associated cytokine and antigen-specific immune response. This study was conducted to assess whether oral intake of additionally phosphorylated CPP-III (P-CPP) attenuates ovalbumin (OVA)-induced IgE-mediated allergic reactions because of the additional phosphate groups. Female BALB/c mice were intraperitoneally sensitized with OVA twice at intervals of 14 days and then orally fed native CPP-III (N-CPP), P-CPP, and dephosphorylated CPP-III (D-CPP) for 6 weeks. Next, the mice were orally challenged with 50 mg of OVA. Oral administration of P-CPP suppressed total and specific IgE levels in the serum. Mice fed P-CPP exhibited low levels of OVA-specific IgG1 and increased OVA-specific IgG2a. P-CPP also suppressed IL-4 production, while D-CPP showed similar a level compared to that of the control. Further, P-CPP increased the population of the T follicular helper (Tfh) cell in the spleen. These results suggest that additional phosphorylation of CPP can enhance the attenuation of allergen-specific IgE-modulated allergic reactions in a murine food allergy model.

Keywords: anti-allergic activity; immunomodulation; phosphopeptide; phosphorylation.

Figure 1

Scheme of in vivo experiment and systemic anaphylactic reactions post-oral challenge with ovalbumin (OVA). Female BALB/c mice were divided into five groups (n = 6). Naïve group was not sensitized, not orally challenged, and fed a commercial mice diet; sham-treated group was sensitized with OVA, orally challenged with OVA, not treated, and fed a commercial diet; native CPP-III (N-CPP), phosphorylated CPP-III (P-CPP), and dephosphorylated CPP-III (D-CPP) were sensitized and orally challenged with OVA and then fed a homemade diet containing 0.05% of N-CPP, P-CPP, and D-CPP, respectively. BALB/c mice were sensitized with 50 μg of OVA plus aluminum hydroxide Al(OH)₃ as an adjuvant. Sensitization was repeated 14 days after the initial sensitization. After 6 weeks, oral challenge with 50 mg/mL OVA in PBS was conducted.

Figure 2

Effects of oral feeding with differently phosphorylated CPP-III on body temperature and allergic score following oral challenge with OVA in OVA-sensitized mice. OVA-sensitized mice were fed differently phosphorylated CPP-III for a period of 6 weeks, and the body temperature (A) and allergic score (B) were determined in the oral challenge test. Body temperature changes were recorded at 0, 10, 20, 30, 40, 50, and 60 min, while allergic scores and anaphylactic symptoms were evaluated and scored from 40–90 min after oral challenge. Data represent the mean ± standard error (SE) of individual mice in the group. * p < 0.05.

Figure 3

Effects of oral feeding with differently phosphorylated CPP-III on the immune response of OVA-sensitized mice. OVA-sensitized mice were fed differently phosphorylated CPP-III for a period of 6 weeks, and the serum levels of total IgE (A), OVA-specific IgE (B), total IgA (C), OVA-specific IgA (D), OVA-specific IgG1 (E), and OVA-specific IgG2a (F) were determined by ELISA. Data represent the mean ± SE of individual mice in the group. * p < 0.05; ** p < 0.01.

Figure 4

Effects of oral feeding with differently phosphorylated CPP-III on cytokine production and gene expression of OVA-sensitized mice. OVA-sensitized mice were fed differently phosphorylated CPP-III for a period of 6 weeks, and the collected spleen (SP) cells were incubated with PBS (unstimulated) for normal group and stimulated with 50 μg/mL (final concentration) of OVA for the control, N-CPP, P-CPP, and D-CPP groups. The levels of IFN-γ (A) and IL-4 (B) in the culture supernatant after incubation for 72 h were determined by sandwich ELISA. Relative mRNA expression levels of IL-4 and GATA-3 (C) were determined by qPCR (C). Data represent the mean ± SE of individual mice in the group. * p < 0.05; ** p < 0.01 vs sham group.

Figure 5

Effects of oral feeding with differently phosphorylated CPP-III on Treg cell population. Percentages of Treg cell populations in cultured SP (A) and payer’s patch (PP) (B). Representative FACS staining for SP (C) and from PP (D). Cells harvested from OVA-sensitized mice were determined by flow cytometry without stimulation with OVA. Data represent the mean ± SE of individual mice in the group. * p < 0.05 and dots represent cell proliferation.

Figure 6

Effects of oral feeding with differently phosphorylated CPP-III on T follicular helper (Tfh) cell population. Percentage of Tfh cell populations in cultured SP (A) and PP (B). Representative FACS staining for SP (C) and PP (D). Cells harvested from OVA-sensitized mice were determined by flow cytometry without stimulation with OVA. Data represent the mean ± SE of individual mice in the group. * p < 0.05; ** p < 0.01 and dots represent cell proliferation.