Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity

Numbers of Tregs in splenocytes and mesenteric lymph nodes of OVA treated mice.

<p>Typical plots depicting numbers of Tregs in mouse splenocytes (a) and mesenteric lymph node (b) in gated CD3+CD4+CD8– T helper cells after feeding with OVA, h-OVA or PBS, respectively. Numbers in upper quadrants shows proportions (mean ± SD) of either CD25–Foxp3+ or CD25+Foxp3+ Th cells out of all cells. Representative data from one out of three independent experiments. *P≤0.05.</p

Number of Tregs in spleen cell suspensions co-cultured <i>in vitro</i> with OVA digests.

<p>The percentage of Tregs in cell suspension isolated from spleens of non-stimulated (naïve) BALB/c mice cultured <i>in vitro</i> for 48 hours with undigested (0′) and after 20 (20′) and 40 minutes (40′) peptic digest of OVA (white bars), h-OVA (black bars) or b-OVA (grey bars). The data represent the percentage of CD4+Foxp3+ cells out of all live cells as measured by FACS. Representative data from one out of three independent experiments are shown. Data are represented as mean ± SEM.</p

Decreased mast cell protease induction by heated-OVA.

<p>Heated OVA (h-OVA, black bar) induced significantly lower amounts of mast cell protease (MMCP-1), the marker of mast cell activation, compared to mice fed with native OVA (white bar). Data are represented as mean ± SEM (n = 10 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001.</p

Cytokine production after <i>in vitro</i> restimulation with OVA.

<p>The cytokine production from mesenteric lymph nodes (a) and splenocytes (b) of BALB/c mice fed with OVA (white bars) or h-OVA (black bars) and stimulated <i>in vitro</i> with appropriate allergen. Cytokine levels are expressed after subtraction of base line levels of unstimulated lymph node cells or splenocytes. Data shown are mean values ± SEM (n = 4–7 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001, n.d. =  not detectable.</p

RP-HPLC separation profile of native-OVA and heated-OVA peptic digests.

<p>RP-HPLC separation profile monitored at 280 nm corresponds to OVA and OVA heated at 70°C (h)-OVA or boiled at 95°C (b)-OVA undigested (0′) and after 20 (20′) and 40 minutes (40′) of digestion by pepsin. RT – retention time.</p