Enteric reovirus infection stimulates peanut-specific IgG2a responses in a mouse food allergy model

Abstract

IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.

Figure 1

Induction of reovirus-specific systemic and intestinal humoral immune responses. A. Total reovirus-specific serum IgG antibody titers from treated mice were determined as described in materials and methods. B. Reovirus-specific IgA concentrations in lamina propria fragments were determined as described in materials and methods and are presented as picograms of specific antibody per milligram of total IgA. Results shown are combined from two independent experiments. Error bars indicate standard error among 7-10 mice per group. Data were analyzed for statistical significance by ANOVA followed by Tukey’s post hoc test, (**) p<0.01.

Figure 2

Induction of Th-1 dominant reovirus-specific humoral and cell-mediated immune responses. A. Reovirus-specific serum IgG2a isotype antibody titers from orally treated mice were determined as described in materials and methods. Results shown are combined from two independent experiments. Error bars indicate standard error among 7-10 mice per group. B. Splenic CD8⁺ T-cells were harvested from reovirus infected mice and restimulated in vitro with reovirus. Activation of CD8⁺ effector T-cells was assayed by the production of intracellular IFN-γ when challenged with non-infected (open bars) or reovirus-infected (filled bars) L929 mouse fibroblast cell. Bars represent the percentage of the maximum IFN-γ response of CD8⁺ T-cells following in vitro stimulation. Maximum response of CD8⁺ T-cells was determined through polyclonal activation with anti-CD3ε/CD28 antibodies. Error bars indicate standard deviation of two replicates. Data were analyzed for statistical significance by ANOVA followed by Tukey’s post hoc test, (*) p<0.05, (**) p<0.01.

Figure 3

Effects of reovirus on PE-specific serum antibody production. Mice were orally treated as described in the materials and methods. At the end of the treatment protocol, serum from treated mice was collected and the PE-specific serum IgE, IgG1, and IgG2a antibody titers were analyzed by ELISA. Data were analyzed for statistically significant differences by ANOVA followed by Bonferonni post hoc test (**) p<0.01, (***) p<0.001. Results shown are combined from two independent experiments. Error bars indicate standard error among 7-10 mice per group.

Figure 4

Effects of reovirus on PE-specific IgA production. Lamina propria fragment cultures from treated mice were established in vitro as described in the materials and methods. After 5 days, the culture supernatants were collected and the presence of PE-specific IgA was determined by ELISA. Data were analyzed for statistically significant differences by ANOVA followed by Tukey’s post hoc test. Results shown are combined from two independent experiments. Error bars indicate standard error among 7-10 mice per group. PE-specific IgA concentration is shown as picograms per milligram of total IgA.

Figure 5

Effects of reovirus on IFN-γ, IL-10, IL-4, and IL-5 levels in splenocyte cell culture supernatants. Splenocytes from treated mice were cultured in vitro in the presence (filled bars) or absence (open bars) of 200μg/ml of PE for 96 hours. Following incubation, the cell culture supernatants were harvested and the cytokine levels induced by restimulation were analyzed by ELISA. Cytokine concentration is shown as picograms per milliliter. Data were analyzed for statistically significant differences by ANOVA followed by Bonferonni post hoc test, (*) p<0.05, (**) p<0.01, (***) p<0.001. Results shown are combined from two independent experiments. Error bars indicate standard error among 7-10 mice per group.