Oral pretreatment with β-lactoglobulin derived peptide and CpG co-encapsulated in PLGA nanoparticles prior to sensitizations attenuates cow's milk allergy development in mice

Abstract

Cow's milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected β-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo, 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions.

Keywords: CpG oligodeoxynucleotides; cow’s milk allergy; individual ventilated cages; oral pretreatment; poly (lactic-co-glycolic acid) nanoparticles; toll-like-receptor 9; whey protein; β-lactoglobulin.

Figure 1

Experimental protocol for pre-treatment, sensitizations and challenge in murine model of cow’s milk allergy for the animal study. Three-four-week-old pathogen free female C3H/HeOuJ mice were given 6 daily oral pretreatments starting 2 days after arrival of the mice. Two days after the tolerance induction phase mice received 5-consecutive-weekly sensitizations with 20 mg whey plus 10 µg cholera toxin in 0.5 mL PBS for all groups but sham (only cholera toxin) from day 7 to day 35 to break whey tolerance. Five days after the last whey-sensitization, mice received intradermal challenge with 10 µg whole whey protein in the ears and at t=0 h and t=1 h the acute allergic skin response (ear swelling) was determined. Three hours after the intradermal whey challenge, the mice were orally challenged by means of gavage with 50 mg whey in 0.5 mL PBS. Eighteen hours after the oral whey challenge, mice were anesthetized with isoflurane and euthanized after blood sampling. Subsequently, spleen and mesenteric lymph nodes were collected immediately for further analysis. CT, Cholera toxin; NP, nanoparticles.

Figure 2

Acute allergic skin response and BLG- and whey-specific serum immunoglobulin E. Five days after last sensitization, mice were intradermally challenged in the ear pinnae with 10 µg whey followed by oral challenge. The acute allergic skin response was measured 60 min afterwards in the in vivo study (A). BLG- (B) and whey-specific (C) IgE levels are measured in serum, which were collected 18 h after last oral challenge with whey in mice from all groups of the animal study. Data are presented as box-and-whisker Tukey plots for (A), and mean ± SEM for (B, C) n=9-10 per group except for the sham group, n=4 and whey-tolerant group, n=6. (A–C) are presented with Y axis formatted in two segments to properly show the relevant part for the window of effect (A) or to be able to appreciate the full data set (B, C). (A) is analyzed by one-way ANOVA, followed by Bonferroni’s post hoc test for selected pairs. The outlier (in red) in the BLG-Pep/NP+CpG/NP group is excluded in (A) from statistics; (B, C) are analyzed with the Kruskal-Wallis non-parametric test, followed by Dunn’s post hoc test for selected pairs; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; BLG, β-lactoglobulin; CT, Cholera toxin.

Figure 3

Surface activation markers expression on dendritic cells in mesenteric lymph nodes (MLN). Eighteen hours after oral whey-sensitization, MLN cells were isolated and analyzed using flow cytometry to determine the percentage of CD11c+MHCII+ DC (A) and CD11b+ (D) and CD11b- (G) DC subsets. Surface costimulatory molecules expression of CD80+ (B, E, H) and CD86+ (C, F, I) on CD11c+MHCII+ DC, CD11b+ and CD11b- DC subsets were determined respectively. Data are presented as mean ± SEM for n=9-10 per group except for the sham group, n=4 and whey-tolerant group, n=5. (A) is analyzed with one-way ANOVA for selected pairs after log transformation, followed by Bonferroni’s post hoc test; (B) and (D–I) are analyzed with the Kruskal-Wallis non-parametric test, followed by Dunn’s post hoc test for selected pairs; (C) is analyzed with one-way ANOVA for selected pairs after log transformation, followed by Bonferroni’s post hoc test; *p<0.05, **p<0.01; CT, Cholera toxin. ****p<0.0001.

Figure 4

Expression of PD-L1 on dendritic cells from mesenteric lymph nodes (MLN) and spleen. Eighteen hours after the last oral challenge with whey, cells isolated from MLN and spleen were analyzed by flow cytometry for the frequencies of PD-L1+ on CD11c+MHCII+ DC (A, D), PD-L1+CD11b+ (B, E) and PD-L1+CD11b- (C, F) DC subsets in MLN and spleen respectively. Data are presented as mean ± SEM for n=8-10 per group except for the sham group, n=4 and whey-tolerant group, n=6. (A, B) and (D–F) are analyzed with the Kruskal-Wallis non-parametric test, followed by Dunn’s post hoc test for selected pairs; (C) is analyzed with one-way ANOVA for selected pairs after log transformation, followed by Bonferroni’s post hoc test; *p<0.05, **p<0.01; CT, Cholera toxin. ***p<0.001, ****p<0.0001.

Figure 5

Splenic T-cell subsets and cytokines production of splenocytes upon ex vivo stimulation with whey for 5 days. Eighteen hours after the last oral challenge with whey, splenocytes were isolated and analyzed by flow cytometry for the percentages of T-cell subsets including Treg (A), Th2 (B), Th1 (C), ratio of Treg/Th2 (D), ratio of Treg/Th1 (E) and ratio of Th1/Th2 (F) respectively. Splenocytes were restimulated with whole whey protein for 5 days, Th2- (G), Th1- (H), Th17- (I), and Treg- (J) associated cytokines were measured in supernatants. Ratios of Treg-/Th2- associated (K) and Treg-/Th1-associated (L) cytokines were calculated. (F, I, K, L) are presented with Y axis formatted in two segments to be able to appreciate the full data set. Data are presented as mean ± SEM for n=9-10 per group except for the sham group, n=4 and whey-tolerant group, n=6. (A–C) and (E) are analyzed with one-way ANOVA for selected pairs, followed by Bonferroni’s post hoc test; (D) and (F–K) are analyzed with the Kruskal-Wallis non-parametric test, followed by Dunn’s post hoc test for selected pairs; (L) is analyzed with one-way ANOVA for selected pairs after log transformation, followed by Bonferroni’s post hoc test; *p<0.05, **p<0.01; CT, Cholera toxin.

Figure 6

Graphical Abstract (Created with BioRender.com).