In this study, BALB/c mice were used as a model of food allergy in order to analyze the MLN transcriptome profiles to explore the molecular mechanism(s) associated with sensitization to three of the most common food allergens; BLG, OVA and PNA. Analysis of ear swelling, plasma histamine, total IgG1 and IgE concentrations, and antigen-specific IgE level (PCA) indicated that BALB/c mice developed a type I allergic response to these food allergens. These immunological profiles also indicate that the initial exposure doses without any adjuvants were sufficient to induce allergen sensitization in BALB/c mice.
Although the ear swelling response is typically used to assess contact allergy (type IV hypersensitivity) , tape-stripping prior to topical antigenic challenge enabled us to use this endpoint to measure a type I hypersensitivity response. Massive ear swelling was observed as early as 30 min post challenge and peaked 1 h post challenge. Such rapid swelling is characteristic of IgE-mediated mast cell degranulation, a hallmark of a type I allergic response . The ear swelling test also proved to be sensitive and was able to differentiate the potencies of the antigens that used in this study. For example, the ear swelling data demonstrated that PNA was more potent than BLG and OVA for eliciting a type I allergic response. The concentration of PNA solution (0.2%) that used in this study was 10 times less than the concentrations used for BLG and OVA solution (2%), yet the magnitude of ear swelling observed in PNA sensitized and challenged mice was not significantly different to that observed in BLG or OVA sensitized and challenged mice (Additional file 7). These results demonstrate that this method is capable of differentiating the antigen potency and also able to generate reliable quantitative data for assessing the magnitude a type I allergic response.
IgE antibodies are known to play a central role in mediating type I hypersensitivity reactions. In our study, increased production of IgE in BALB/c mice sensitized and challenged with BLG, OVA or PNA illustrates that these mice experienced a IgE mediated type I allergic reaction. In order to confirm that BALB/c mice used in this study had an IgE mediated type I allergic response and anaphylaxis, plasma histamine concentrations were measured. In this study, rapid release of histamine within 20 min of the i.p. challenge confirmed the induction of type I allergic response and subsequent degranulation of mast cells. Traditionally, it was believed that food allergy-related systemic anaphylaxis was only due to the interaction between IgE and FcεRI on the mast cell surface, and this lead to mast cell degranulation and the subsequent release of histamines and other enzymes . Reports, however, suggest that an alternate pathway exists that is dependent on the interaction of IgG1 and FcγRIII . Mouse FcγRIII is a low affinity receptor, present on the surface of the macrophages, neutrophils and natural killer cells that binds to IgG1. Binding of soluble IgG1 with blood-borne antigens must occur before they bind to macrophage FcγRIII. In contrast to the IgE-mediated pathway, this pathway takes relatively longer to induce anaphylaxis, requires high concentrations of IgG1 for elicitation, and involves platelet activating factors (PAF), rather than histamine, as the primary mediator in this type of anaphylactic shock [34, 35]. In our study, the release of histamine within 20 min of i.p. challenge of BALB/c mice strongly suggests that allergic and anaphylactic responses related to BLG, OVA or PNA sensitization and challenge were IgE mediated.
Finally, the PCA data in this study demonstrated that BALB/c mice responded positively and developed BLG-, OVA- or PNA-specific immunoglobulin IgE. This positive PCA response was exclusively due to the binding of antigen-specific IgEs with FcεRIs on mast cell rather than the binding of IgG1 with FcγRIII, since mice were sensitized with antigen specific anti-sera and challenged with the respective antigens and Evans blue dye after 48 h of anti-sera sensitization; only IgE is capable of remain bound to high affinity FcεRI for this period of time .
Analysis of MLN transcriptome profile of BALB/c mice sensitized with BLG, OVA or PNA revealed the differential expression of numerous genes that are associated with activation of the Th2 response. This method of transcriptome profiling appeared to be very sensitive that identified differential expression of hundreds of genes in BALB/c mice within 24 h of the second sensitization with the given food allergens. Comparison of the differentially expressed genes in BLG, OVA and PNA sensitized groups implicates that a common set of genes involved in the sensitization phase of the immune response to these three food allergens in BALB/c mice. A total of 150 overlapping genes being differentially expressed in the same direction among the three food allergen sensitized groups. Among these genes, the expression of Timp1 was up-regulated, whereas the expression of Stard4 and Igj were down-regulated in all three antigen sensitized groups. The involvement of Timp1 with the allergic response is very well documented; being an inhibitor of matrix metalloproteinases, it plays a central role in degradation of extracellular matrix and remodeling of tissue during inflammatory process , and appears to be involved in asthma [37, 38]. Induction of Timp1 has also been implicated in the pathogenesis of other allergic conditions including atopic dermatitis, allergic contact dermatitis and chronic obstructive pulmonary disease (COPD) [36, 39, 40].
Stard4 is known to be involved in the binding and directional transport of the cholesterol into the liver for the synthesis of bile acid , and its expression is reported to be down-regulated in the presence of cholesterol . It is not clear from available reports whether Stard4 plays any direct role in the allergic response, however, the microarray and real-time RT-PCR data from all antigen sensitized groups in our study demonstrates its involvement in the sensitization of food allergens.
Igj gene products are produced by antibody secreting plasma cells after antigen or cytokine stimulation. They are incorporated into pentameric IgM and dimeric IgA immunoglobulins and play a crucial role in the cellular and mucosal secretion of these molecules [43–45]. Plager et al. reported that the expression of Igj is down-regulated in patients with atopic dermatitis and this may be due to isotype switching of plasma cells from IgM and IgA production to IgG1 and IgE production. Down-regulation of Igj in both microarray and real-time RT-PCR experiments in our study among all antigen sensitized treatments indicates a possible induction of type I allergic reaction, and this was supported by the endpoints that we measured during the challenge phase.
Several uniquely expressed genes were also activated in response to BLG, OVA or PNA sensitization. For example, Itgb1 was up-regulated by BLG sensitization; this plasma membrane receptor is expressed on lymphocytes, monocytes and eosinophils, and may be involved in inducing allergic inflammatory response [46, 47]. Available reports also suggest that Itgb1 may be involved in the airway smooth muscle responsiveness through the association with fibronectin and type I collagen during asthma or allergic inflammation . The observed up-regulation of Itgb1, as indicated by both microarray analysis and real-time RT-PCR, is in agreement with these available reports, and demonstrates its possible role in food allergic process. In contrast, CD79a was down-regulated in BLG sensitized group, and genes Syt4 and Pex13 were up- and down-regulated, respectively in OVA sensitized group. It is not known whether the genes CD79a, Syt4 and Pex13 play any direct role in any allergic conditions. However, microarray analysis and real-time RT-PCR results from this study implicates their involvement in BLG or OVA sensitization.
Gene ontology analysis was also used to reveal the biological roles and molecular functions of the differentially expressed genes that were commonly and uniquely expressed among the allergen sensitized mice. These enriched GO data further validated the involvement of the differentially expressed genes in allergen sensitizations in BALB/c mice. A total of 46 GO biological processes were commonly enriched among all three antigen sensitized groups (p ≤ 0.05) and at least 22 of these processes were related to immune or hypersensitivity related processes (Table 2). The most significant overlapping enriched GO biological process was the response to stimulus [GO:0050896]; this process was enriched with 50 genes from the OVA and PNA sensitized groups, and with 100 genes from BLG sensitized group (Table 2). Within the response to stimulus biological process, was a subcategory biological process referred to as immune response [GO:0006955]. A total of 30 genes from BLG, 12 genes from OVA and 13 genes from PNA sensitized groups enriched this biological process. In addition to the immune response biological process other biological processes that were subcategories within the response to stimulus biological process were enriched with genes from BLG, OVA and PNA sensitized groups. These subcategories included the acute inflammatory response [GO:0002526], humoral immune response [GO:0006959], regulation of immunoglobulin mediated immune response [GO:0002889] and regulation of B cell-mediated immunity [GO:0002712]. Enrichment of these biological processes indicates that the genes differentially expressed in response to BLG, OVA or PNA sensitization are involved in the process of immune regulation or hypersensitivity responses.
Two common differentially expressed genes that were involved in the enrichment of several of these immune or hypersensitivity related biological processes were Fcer1γ and C3 (Table 3), whose expression was up-regulated in all of the allergen treated groups of BALB/c mice. Among other genes, Immunoglobulin heavy chain complex (Igh-6), and Signal transducer and activator of transcription 3 (Stat3), and Immunoglobulin joining chain (Igj) also played important role in the enrichment of other immune or hypersensitivity related biological processes (Table 3). The involvement of FcεR1γ, Stat3 and C3 with the allergic responses are very well documented. FcεR1γ for example, is expressed on the surface of mast cell and cross-linking of these IgE bound receptors leads to mast cell degranulation, cytokine production, prostaglandin synthesis, survival and passive systemic anaphylaxis . It has also been reported that elevated level of transcription factor Stat3 is associated with the induction of house dust mite-mediated allergic inflammation and airway hyper-responsiveness in mice . Likewise, Stat3 is required for the active production of IL-21-mediated IgE production by human B cells . Complement C3 plays a central role as a mediator of airway hyper-responsiveness and asthma, and induces the expression of the Th2 phenotype . Anaphylatoxin C3a, an enzymatic derivative of C3 also acts as a chemotactic factor that triggers the release of histamines and cationic proteins from mast cells and eosinophils respectively . C3a also plays a role in smooth muscle contraction that is mediated by leukotrienes, prostanoids, and platelet-activating factor released from mast cells and eosinophils . Other commonly enriched GO biological processes include actin filament-based process [GO:0030029] and muscle development process [GO:0007517]. Enrichment of these two biological processes suggests possible involvement of some of the differentially expressed genes with smooth muscle contraction, bronchoconstriction and vasodilation that are the common phenomena associated with type I allergic responses or anaphylaxis. Some of the differentially expressed genes enriched these processes include Scinderin (Scin), Supervillin (Svil) and Villin 1 (Vil1). Scin is an actin-filament severing and capping protein activated by calcium is known to be differentially expressed in BALB/c mice exposed to OVA, and suggested to be a potential biomarker of asthma, a type I allergy . Similarly, Vil1 is another actin-binding protein that is known for IgE-binding and IgE-cross-reactivity with other plant proteins of the same family . Therefore, differential expression of these genes and enrichment of these GO biological processes illustrate the possible involvement of these genes in food allergic sensitizations.
GO analysis further revealed the molecular functions of some of the differentially expressed genes (Table 4). One significant overlapping enriched GO molecular function was the calcium ion binding [GO: 0005509]. Enrichment of this molecular function is well correlated with other enriched biological processes and emphasized its involvement in the food allergy response. A recent report suggests that the binding of the antigens with the receptors on the T, B or mast cells induces a series of biochemical reactions leading to a transient increase in cytosolic free calcium concentration that leads to other downstream reactions in the process of allergic response . Another report also suggests that antigen-induced FcεRI-mediated cell migration/chemotaxis is dependent on cytosolic free calcium concentration . Therefore, the enrichment of these GO biological processes and molecular functions support the involvement of these differentially expressed genes in the food allergic responses during early sensitization phase. Thus, these gene expression data enabled us to monitor the allergenicity of some of the common food allergens and proved to be sensitive enough to identify the allergic/immune responses within 24 h of the sensitizations in the used animal model.
The experimental mice used in this study were systemically exposed to the food antigens by i.p. injection without any adjuvants, and they responded positively to all of the antigens, as indicated by the increased ear swelling response, elevated serum IgG1, IgE, plasma histamine and a positive PCA test. Oral sensitization was specifically avoided in this study because there is a potential risk of developing oral tolerance when antigens are administered via this route [25, 29]. In order to avoid the development of oral tolerance, adjuvants, such as cholera toxin is being used during oral sensitization processes. However, immune/allergic response developed such a way does not mimic the actual immune/allergic response in humans and this may lead to misinterpretation of the data due to hyperstimulation of the immune system [26, 27]. In our study, we sensitized the BALB/c mice via the i.p. route with only two consecutive injections of allergens on day 0 and 1 without any adjuvants. Use of this route for sensitization not only allowed us to avoid the use of any kind of adjuvants but also allowed us to observe the true effects of the allergens used without having any unwanted external stimulations or influences. Thus, the gene expression data presented here in our study corresponds only to the effects of the food allergens used for sensitizations that may mimic the actual allergic responses in humans. In this study, the PCA data indicated that one mouse in BLG and another mouse in the OVA treated group did not appear to be sensitized. Although they were a different group of animals than those used for the gene expression study, there is the possibility that non-responders may contribute to the variation in gene expression during the sensitization phase.