Inhibition of cow's milk allergy development in mice by oral delivery of β-lactoglobulin-derived peptides loaded PLGA nanoparticles is associated with systemic whey-specific immune silencing

Abstract

Background: Two to four percentage of infants are affected by cow's milk allergy (CMA), which persists in 20% of cases. Intervention approaches using early oral exposure to cow's milk protein or hydrolysed cow's milk formula are being studied for CMA prevention. Yet, concerns regarding safety and/or efficacy remain to be tackled in particular for high-risk non-exclusively breastfed infants. Therefore, safe and effective strategies to improve early life oral tolerance induction may be considered.

Objective: We aim to investigate the efficacy of CMA prevention using oral pre-exposure of two selected 18-AA β-lactoglobulin-derived peptides loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in a whey-protein induced CMA murine model.

Methods: The peptides were loaded in PLGA NPs via a double emulsion solvent evaporation technique. In vivo, 3-week-old female C3H/HeOuJ mice received 6 daily gavages with PBS, whey, Peptide-mix, a high- or low-dose Peptide-NPs or empty-NP plus Peptide-mix, prior to 5 weekly oral sensitizations with cholera toxin plus whey or PBS (sham). One week after the last sensitization, the challenge induced acute allergic skin response, anaphylactic shock score, allergen-specific serum immunoglobulins and ex vivo whey-stimulated cytokine release by splenocytes was measured.

Results: Mice pre-treated with high-dose Peptide-NPs but not low-dose or empty-NP plus Peptide-mix, were protected from anaphylaxis and showed a significantly lower acute allergic skin response upon intradermal whey challenge compared to whey-sensitized mice. Compared with the Peptide-mix or empty-NP plus Peptide-mix pre-treatment, the high-dose Peptide-NPs-pre-treatment inhibited ex vivo whey-stimulated pro-inflammatory cytokine TNF-α release by splenocytes.

Conclusion & clinical relevance: Oral pre-exposure of mice to two β-lactoglobulin-derived peptides loaded PLGA NPs induced a dose-related partial prevention of CMA symptoms upon challenge to whole whey protein and silenced whey-specific systemic immune response. These findings encourage further development of the concept of peptide-loaded PLGA NPs for CMA prevention towards clinical application.

FIGURE 1

Amino acids sequence of B variant of β‐lactoglobulin (A). The two selected sequential peptides (Peptide 3 and Peptide 4) used in the study were underlined and italicized. The bold amino acids sequence is the overlapping 12 amino acids. In vitro release of Peptide 3 and Peptide 4 from PLGA NPs in PBS at 37°C was conducted using cumulative method (B). Data are presented as mean ± SEM, n = 3 per formulation

FIGURE 2

Schematic overview of the animal model for CMA prevention and different treatments per group

FIGURE 3

Effect of Peptide‐NPs, Peptide mix, empty‐NP and Peptide mix plus empty‐NP on human moDC activation in vitro. After incubation of human moDC with medium, LPS, Peptide mix, empty‐NP, Peptide mix plus empty‐NP or Peptide‐NPs for 24 h, cell viability (A), % MHC class II expression by human moDC (B) and surface expression of co‐stimulatory molecules in HLA‐DR‐positive moDC (C,D) were evaluated by flow cytometry. IL12p70 (E), IL‐10 (F) and pro‐inflammatory IL‐6 and TNF‐α (G,H) concentrations were measured in the supernatant. Data are presented as mean ± SEM for n = 3 independent experiments per group. Comparison between medium and the other groups were analysed with one‐way ANOVA followed by Bonferroni's post hoc test for selected pairs for (A–D), after square root transformation for (E), (G) and (H) and after log transformation for (F).*p < .05, **p < .01, ***p < .001, ****p < .0001

FIGURE 4

Acute allergic skin response and anaphylaxis score. Five days after last sensitization, mice were i.d. challenged in the ear pinnae with 10 µg whey followed by oral challenge. The acute allergic skin response was measured 60 min afterwards (A), and signs of anaphylaxis were scored after 15 min (B) and 60 min (C). Data are presented as mean ± SEM for n = 9–10 per group except for the non‐sensitized group, n = 3 and whey‐tolerant group, n = 6. (A) is analysed by one‐way ANOVA, followed by Bonferroni's post hoc test for selected pairs; (B) and (C) are analysed with Kruskal–Wallis’ non‐parametric test, followed by Dunn's post hoc test for selected pairs. *p < .05, **p < .01, ***p < .001. CT, Cholera toxin

FIGURE 5

Whey‐ and BLG‐specific serum immunoglobulins levels. Whey‐ (A–C) and BLG‐specific (D,F) IgE, IgG1 and IgG2a levels are measured in sera, which were collected 18 h after last oral challenge with whey in mice from all groups. Data are presented as Tukey box‐ and‐whisker plots for n = 9–10 per group except for the non‐sensitized group, n = 3 and whey‐tolerant group, n = 6. (A–F) are analysed with the Kruskal–Wallis non‐parametric test, followed by Dunn's post hoc test for selected pairs; *p < .05, **p < .01; BLG, β‐lactoglobulin; CT, Cholera toxin

FIGURE 6

Cytokine production after ex vivo re‐stimulation of splenocytes with whole whey protein. Splenocytes were ex vivo re‐stimulated either with whole whey protein for 5 days, Th1‐ (A), Th2‐ (B), regulatory (C) and proinflammatory (D) associated cytokines were measured in supernatants; Data are presented as mean ± SEM for n = 9–10 per group except for the non‐sensitized group, n = 3 and whey‐tolerant group, n = 6. (A)–(D) are analysed with one‐way ANOVA followed by Bonferroni's post hoc test for selected pairs, after log transformation for (A–C) and after square root transformation for (D); *p < .05, **p < .01, ***p < .001; CT, Cholera toxin