Raw Cow's Milk Reduces Allergic Symptoms in a Murine Model for Food Allergy-A Potential Role For Epigenetic Modifications

Abstract

Epidemiological studies identified raw cow's milk consumption as an important environmental exposure that prevents allergic diseases. In the present study, we investigated whether raw cow's milk has the capacity to induce tolerance to an unrelated, non-milk, food allergen. Histone acetylation of T cell genes was investigated to assess potential epigenetic regulation. Female C3H/HeOuJ mice were sensitized and challenged to ovalbumin. Prior to sensitization, the mice were treated with raw milk, processed milk, or phosphate-buffered saline for eight days. Allergic symptoms were assessed after challenge and histone modifications in T cell-related genes of splenocyte-derived CD4⁺ T cells and the mesenteric lymph nodes were analyzed after milk exposure and after challenge. Unlike processed milk, raw milk decreased allergic symptoms. After raw milk exposure, histone acetylation of Th1-, Th2-, and regulatory T cell-related genes of splenocyte-derived CD4⁺ T cells was higher than after processed milk exposure. After allergy induction, this general immune stimulation was resolved and histone acetylation of Th2 genes was lower when compared to processed milk. Raw milk reduces allergic symptoms to an unrelated, non-milk, food allergen in a murine model for food allergy. The activation of T cell-related genes could be responsible for the observed tolerance induction, which suggested that epigenetic modifications contribute to the allergy-protective effect of raw milk.

Keywords: epigenetics; farming effect; food allergy; histone acetylation; milk processing; raw milk.

Figure 1

Schematic representation of the experimental timeline. For epigenetic measurements, additional groups of mice were killed after the tolerance induction period (day −1) and after both challenges (day 34; as indicated by †). PBS, phosphate-buffered saline; OVA, ovalbumin; CT, cholera toxin; i.d., intradermal; i.g., intragastric.

Figure 2

Reduced acute allergic symptoms upon ovalbumin (OVA) challenge in mice treated with raw milk. (A) The acute allergic skin response measured as Δ ear swelling 1 h after intradermal (i.d.) challenge. (B) Anaphylactic shock scores and (C) body temperature determined 30 min after i.d. challenge. Data are presented as mean ± standard error of the mean for the acute allergic skin response and body temperature and as individual data points for anaphylactic shock scores, n = 6 in PBS group and n = 8 in all other groups. * p < 0.05, *** p < 0.001, **** p < 0.0001, as analyzed with one-way ANOVA followed by Bonferroni’s multiple comparisons test for pre-selected groups (A,C) or Kruskal–Wallis test for non-parametric data followed by Dunn’s multiple comparisons test for pre-selected groups (B). PBS, phosphate-buffered saline; OVA, ovalbumin; raw, raw cow’s milk; shop, shop milk.

Figure 3

Raw milk treatment did not affect ovalbumin (OVA)-specific IgE levels and mouse mast cell protease-1 (mMCP-1) concentrations. (A) OVA-specific IgE levels and (B) mMCP-1 concentrations measured in serum 16 h after intragastric challenge. Data are expressed as box-and-whisker Tukey plot (in which outliers are shown as separately plotted points) for OVA-specific IgE levels and as mean ± standard error of the mean for mMCP-1 concentrations, n = 6 in PBS group and n = 8 in all other groups. * p < 0.05, ** p < 0.01 as analyzed with Kruskal–Wallis test for non-parametric data followed by Dunn’s multiple comparisons test for pre-selected groups (A) or one-way ANOVA followed by Bonferroni’s multiple comparisons test for pre-selected groups (B). PBS, phosphate-buffered saline; OVA, ovalbumin; AU, arbitrary units; raw, raw cow’s milk; shop, shop milk; mMCP-1; mucosal mast cell protease-1.

Figure 4

Increased histone acetylation of several T cell subset genes directly after raw milk exposure, while only Th2-related gene acetylation was reduced in raw milk-treated mice after both challenges. (A) Histone H4 acetylation at Th2 loci after the tolerance induction period (day −1), (B) after both challenges (day 34) and (C) the raw milk/shop milk ratio. (D) Histone H4 acetylation at Th1/Treg/Th17 loci after the tolerance induction period (day −1), (E) after both challenges (day 34) and (F) the raw milk/shop milk ratio. Histone H4 acetylation status was determined by means of chromatin immunoprecipitation in CD4⁺ T cells derived from splenocytes of raw milk and shop milk-treated mice. Results are expressed as relative enrichment after normalization to ribosomal protein L32 (RPL32) as mean ± standard error of the mean, n = 6/group. * p < 0.05 as analyzed with an unpaired two-tailed Student’s t-test. A Mann–Whitney test was used for T-bet, IFNγ, FoxP3, RORγ (after tolerance), T-bet, IL-17 (after model), and T-bet, IFNγ, RORγ (ratio raw/shop) since data did not obtain normality. Raw, raw cow’s milk; shop, shop milk; AT, after tolerance; AC, after challenge.

Figure 5

Raw milk-induced acetylation pattern of Th2-related genes observed in splenocyte-derived CD4⁺ T cells also visible locally in mesenteric lymph nodes (MLN). (A) Histone H4 acetylation at Th2 loci after the tolerance induction period (day −1), (B) after both challenges (day 34) and (C) the raw milk/shop milk ratio. (D) Histone H4 acetylation at Th1/Treg/Th17 loci after the tolerance induction period (day −1), (E) after both challenges (day 34) and (F) the raw milk/shop milk ratio. Histone H4 acetylation status was determined by means of chromatin immunoprecipitation in MLN of raw milk- and shop milk-treated mice. The results are expressed as relative enrichment after normalization to ribosomal protein L32 (RPL32) as mean ± standard error of the mean, n = 4–6/group. * p < 0.05, ** p < 0.01 as analyzed with an unpaired two-tailed Student’s t-test. A Mann–Whitney test was used for GATA3, IL-10 (after tolerance), IL-10 (after model) and GATA3 (ratio raw/shop) since data did not obtain normality. Raw, raw cow’s milk; shop, shop milk; AT, after tolerance; AC, after challenge; MLN; mesenteric lymph nodes.

Figure 6

Cytokine concentrations produced by ovalbumin (OVA)-stimulated splenocytes corresponded with observed histone acetylation. (A) IL-5, (B) IL-13, (C), IFNγ, (D) IL-10 and (E) IL-17 concentrations measured in supernatant after ex vivo stimulation of splenocytes with OVA for four days (37 °C, 5% CO₂). Data are presented as box-and-whisker Tukey plot (in which outliers are shown as separately plotted points) for IL-5 and IL-13 concentrations and as mean ± standard error of the mean for IFNγ, IL-10 and IL-17 concentrations after subtracting baseline cytokine levels, n = 8/group. * p < 0.05 as analyzed with a Mann-Whitney test (A,B) or an unpaired two-tailed Student’s t-test (C–E). OVA, ovalbumin; raw, raw cow’s milk; shop, shop milk.