Effect of extrusion processing on immune activation properties of hazelnut protein in a mouse model.

Although food processing can alter food allergenicity, the impact of extrusion processing on in vivo hazelnut allergenicity is unknown. Here, we tested the hypothesis that extrusion processing will alter the immune activation properties of hazelnut protein (HNP) in mice. Soluble extrusion-processed HNP (EHNP) was prepared and evaluated for immune response using an established transdermal sensitization mouse model. Mice were sensitized with identical amounts of EHNP versus raw HNP. After confirming systemic IgE, IgG1 and IgG2a antibody responses, oral hypersensitivity reaction was quantified by hypothermia shock response (HSR). Mechanism was studied by measuring mucosal mast cell (MMC) degranulation. Compared to raw HNP, the EHNP elicited slower but similar IgE antibody (Ab) response, lower IgG1 but higher IgG2a Ab response. The EHNP exhibited significantly lower oral HSR as well as MMC degranulation capacity. These results demonstrate that the extrusion technology can be used to produce soluble HNP with altered immune activation properties.

An Adjuvant-Free Mouse Model of Transdermal Sensitization and Oral Elicitation of Anaphylaxis to Shellfish.

BACKGROUND: Shellfish (SF) allergy is a leading cause of systemic anaphylaxis in humans. An adjuvant-free mouse model to evaluate allergenicity and oral anaphylaxis to SF is currently unavailable. Here, we tested the hypothesis that transdermal exposure (TDE) to SF protein extract (SFPE) not only elicits a systemic allergic immune response but also will clinically sensitize mice for oral anaphylaxis. METHODS: Adult BALB/c female mice (6-8 weeks of age) were exposed to saline or SFPE once a week for 4 weeks using a transdermal sensitization method. Systemic SF-specific IgE, IgG1 and IgG2a and total (t)IgE responses were measured using ELISA. Systemic anaphylaxis upon oral SFPE administration was assessed according to clinical symptoms and the hypothermia shock response (HSR). Using individual mouse data, the correlation between the readouts of allergenicity was determined using Pearson's analysis. Spleen-cell IL-4 and IFN-x03B3; responses were determined using primary cell culture and ELISA. RESULTS: TDE to SFPE resulted in marked systemic specific (s)IgE, tIgE, IgG1 and IgG2a responses. Oral challenge with SFPE in sensitized mice (but not controls) elicited systemic anaphylactic clinical reactions and HSR. A strong correlation was observed between sIgE, tIgE and HSR. Spleen cells isolated from allergic mice (but not controls) exhibited memory IL-4 and IFN-x03B3; cytokine responses. CONCLUSION: We report a novel adjuvant-free mouse model of SF allergy with robust quantifiable and correlated readouts of allergenicity that may be used in basic biomedical, preclinical and applied food/nutrition research on SF allergy.

Cardiac mMCP-4+ mast cell expansion and elevation of IL-6, and CCR1/3 and CXCR2 signaling chemokines in an adjuvant-free mouse model of tree nut allergy.

BACKGROUND: Nut allergy is a growing and potentially fatal public health problem. We have previously reported a novel mouse model of near-fatal hazelnut (HN) allergy that involves transdermal sensitization followed by oral elicitation of allergic reactions. Here we studied the cardiac mast cell and cardiac tissue responses during oral nut induced allergic reaction in this mouse model. METHODS: Groups of mice were sensitized with HN and specific and total IgE were measured by ELISA. Oral allergic reaction was quantified by rectal thermometry and plasma mouse mast cell protease (mMCP)-1 by ELISA. Cardiovascular functions were determined by a non-invasive tail cuff method. Mucosal mast cells (MMC) and intestinal connective tissue MC (CTMC) were studied by immunohistochemistry (IHC) for mMCP-1 and mMCP-4 protein expression respectively. Cardiac MC were studied by toluidine blue (TB) as well as by the above IHC methods. Cytokines and chemokines in the tissues were quantified by a multiplex protein array method. RESULTS: Oral allergen challenge (OAC) of transdermal sensitized mice results in hypothermia, hypotension, tachycardia and rapid elevation of circulating mMCP-1. The IHC analysis of small intestine found significant expansion of mMCP-1+ MMCs and mMCP-4+ CTMCs. The TB analysis of cardiac tissues showed degranulation of majority of cardiac MCs. The IHC analysis of cardiac tissues showed very little mMCP-1 expression, but marked mMCP-4 expression. Furthermore, repeated OAC resulted in significant expansion of mMCP-4+ cardiac MCs in both the pericardium and the myocardium. Protein array analysis revealed significant elevation of cardiac IL-6 and CCR1/3 and CXCR2 signaling chemokines upon oral elicitation compared to sensitization alone. CONCLUSION: These results demonstrate that: (i) besides the intestine, cardiac mast cells and the cardiac tissue respond during oral nut induced allergic reaction; and (ii) repeated oral elicitation of reaction is associated with cardiac mMCP-4+ mast cell expansion and elevation of cardiac IL-6, and CCR1/3 and CXCR2 signaling chemokines.

Mouse models of food allergy: how well do they simulate the human disorder?

Food allergy is a growing health problem with serious concerns due to high potential for fatality. Rapid advances in the knowledge on causes and mechanisms as well as in developing effective prevention/therapeutic strategies are needed. To meet these goals, mouse models that simulate the human disorder are highly desirable. During the past decade, several mouse models of food allergies have been reported. Here, we briefly reviewed the human disorder and then critically evaluated these models seeking answers to the following important questions: To what extent do they simulate the human disorder? What are the strengths and limitations of these models? What are the challenges facing this scientific area? Our analysis suggest that: (i) the mouse models, with inherent strengths and limitations, are available for many major food allergies; there is scope for additional model development and validation; (ii) models mostly simulate the severe forms of human disorder with similar immune and clinical features; (iii) the approaches used to develop some of the mouse models may be questionable; and (iv) the specific mechanisms of sensitization as wells as oral elicitation of fatal reactions in both humans and mice remains incompletely understood and therefore warrants further research.

Identification of T- and B-Cell Subsets That Expand in the Central and Peripheral Lymphoid Organs during the Establishment of Nut Allergy in an Adjuvant-Free Mouse Model.

Nut allergies are potentially fatal and rarely outgrown for reasons that are not well understood. Phenotype of T- and B-cell subsets that expand during the early stages of nut allergy is largely unknown. Here we studied this problem using a novel mouse model of nut allergy. Mice were rendered hazelnut allergic by a transdermal sensitization/oral elicitation protocol. Using flow cytometry, the T- and B-cell phenotype in the bone marrow (BM), spleen, and the mesenteric lymph node (MLN) of allergic and control mice was analyzed. Nut allergic mice exhibited an expansion of CD4+ CD62L- T cells in BM and spleen; a similar trend was noted in the MLN. There was expansion of CD80+ B cells in BM and spleen and MLN and CD62L- cells in BM and spleen. Interestingly, among CD80+ B cells, significant proportion was CD73- particularly in the MLN. These data demonstrate that during the early establishment of hazelnut allergy there is (i) expansion of CD4+CD62L- T-cell subsets in both the BM and the periphery, (ii) expansion of CD80+ and CD62L- B-cell subsets in BM and the periphery, and (iii) a significant downregulation of CD73 on a subset of B cells in MLN.

Role of myeloid-specific G-protein coupled receptor kinase-2 in sepsis.

Previous studies have implicated a critical role for G-protein coupled receptor kinase-2 (GRK2) in sepsis owing to its ability to regulate inflammatory response and chemotaxis of immune cells. We therefore, hypothesized that deletion of GRK2 in myeloid cells would significantly modulate the pathogenesis of polymicrobial sepsis. To test this hypothesis, we induced cecal ligation and puncture (CLP), in mice with myeloid-specific deletion of GRK2 and the corresponding GRK2 wild type littermates and determined the inflammatory response (IL-6 and IL-10), immune cell infiltration, bacterial load and survival. Six hours after surgery, plasma IL-6 and IL-6:IL-10 ratios were significantly enhanced in the GRK2 knockouts compared to the GRK2 wild type mice. Compared to these effects, IL-6was significantly elevated in the bronchoalveolar lavage but not in the peritoneal fluid of the GRK2 knockout mice. On the other hand, peritoneal IL-10 was significantly elevated in the GRK2 knockout mice compared to the GRK2 wild type. Even though GRK2 knockout mice exhibited an exaggerated cytokine response, there was no difference in immune cell infiltration into the primary site of infection or in bacterial clearance when compared between the GRK2 wild type and GRK2 knockout mice after surgery. Furthermore, in spite of the enhanced pro-inflammatory profile early after surgery, there was only a modest increase in mortality in the GRK2 knockout compared to the GRK2 wild type mice after CLP. Together, our studies demonstrate that myeloid-specific knockout of GRK2 renders the mice more susceptible to an early pro-inflammatory state. However, myeloid-specific GRK2 is not involved in immune cell infiltration to the primary site of infection or in bacterial clearance and does not significantly modulate mortality in the cecal ligation puncture model of polymicrobial sepsis.

Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by beta-arrestins.

Beta-arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that beta-arrestin-1 (beta-arr-1) and -2 knockout (KO) mice are protected from TLR4-mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild-type (WT) and beta-arr-1 and -2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS-induced inflammatory cytokine levels in the plasma were markedly decreased in both beta-arr-1 and -2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11(b+) and CD11(b-) populations) from WT, beta-arr-1, and -2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS-induced inflammatory cytokines were significantly blocked in both splenocyte populations from the beta-arr-2 KO compared to the WT mice. This effect in the beta-arr-1 KO mice, however, was restricted to the CD11(b-) splenocytes. Our studies further indicate that regulation of cytokine production by beta-arrestins is likely independent of MAPK and IkappaBalpha-NFkappaB pathways. Our results, however, suggest that LPS-induced chromatin modification is dependent on beta-arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by beta-arrestins in vivo. Taken together, these results indicate that beta-arr-1 and -2 mediate LPS-induced cytokine secretion in a cell-type specific manner and that both beta-arrestins have overlapping but non-redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice.

Long-term characteristics of hazelnut allergy in an adjuvant-free mouse model.

BACKGROUND: Clinically it is recognized that tree nut allergies such as hazelnut allergy are not usually outgrown. Specific mechanisms underlying the persistence of such food allergies are incompletely understood. Here we studied the natural history and the long-term immune and clinical characteristics of hazelnut allergy in an adjuvant-free mouse model. METHODS: BALB/c mice were sensitized to hazelnut protein using a transdermal sensitization protocol that does not use adjuvant. After establishing sensitization, exposure to hazelnut was withdrawn for 3, 5 or 8 months. The fate of circulating IgE antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE antibody and spleen cell IL-4 responses. Clinical characteristics and hypothermia responses upon oral allergen challenge were studied. RESULTS: Upon allergen withdrawal, circulating hazelnut-specific IgE antibody levels began to drop. Nevertheless, IgE responses once established remained at significantly high levels for up to 8 months (the last time point studied) despite withdrawal of allergen exposure. Memory IgE responses to booster exposures were robust after 3, 5 or 8 months of allergen withdrawal. Furthermore, significant clinical reactivity to oral hazelnut challenge, and hypothermia responses were demonstrable at each of these time points. Long-lasting spleen cell memory IL-4 responses to hazelnut were detectable in these mice explaining the mechanism of sustenance of IgE responses and clinical sensitization. CONCLUSIONS: Hazelnut allergy once established persists for long periods, despite withdrawal of allergen exposure, due to long-lasting, memory IgE and IL-4 responses.

Development of an adjuvant-free cashew nut allergy mouse model.

BACKGROUND: Cashew nut allergy is an emerging food allergy with a high risk of systemic anaphylaxis. Currently, an adjuvant-free animal model to study cashew nut allergy is not available. METHODS: BALB/c mice were exposed to cashew nut protein using a transdermal sensitization protocol that does not use adjuvant. Systemic IgE antibody response, systemic anaphylaxis to oral challenge and allergen-driven, spleen-cell, type-2 cytokine responses were studied. RESULTS: Transdermal exposure to cashew nut resulted in a significant dose-dependent allergic response. Oral challenge of sensitized mice with cashew resulted in severe signs of systemic anaphylaxis and a significant hypothermia. Spleen cell culture with cashew nut protein resulted in allergen-driven IL-4, IL-5 and IL-13 responses only in sensitized but not in saline control mice. CONCLUSIONS: These data demonstrate that (i) transdermal exposure to cashew nut protein elicits a robust IgE response leading to clinical sensitization of mice for systemic anaphylaxis to oral cashew nut challenge; (ii) cashew nut is a potent activator of type-2 cytokines, thus explaining the mechanism of cashew allergy, and (iii) this mouse model may be useful for further basic and preclinical studies on cashew nut allergy.