Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy
Highlights
► We investigated the impact of endogenous S1P level on food allergy. ► Tight junction integrity and allergen uptake by epithelial cells altered by S1P. ► Sphingosine-kinase deletion impaired food allergy in a mouse model.
Introduction
Food allergies are an increasing and, thus, important health concern in Western societies [1]. Despite substantial research effort in this field, the mechanisms, risk factors and events leading to sensitization towards food compounds are unclear.
Sphingolipids and their metabolites, especially sphingosine-1-phosphate (S1P), were proposed to play a major role in the induction and promotion of allergic diseases [2], [3]. Additionally, single nucleotide polymorphisms of ORMDL3, a gene regulating S1P homeostasis, were recently found to be associated with the occurrence of asthma in childhood [4], [5]. Thus, sphingolipids, which are necessary constituent of membranes and liquid ordered domains and are components of routinely ingested food compounds, gained attention in allergy research [6], [7], [8]. The metabolites of sphingolipid catabolism, ceramide, sphingosine and S1P, have bioactive functions influencing cell motility, calcium homeostasis, cell growth, cell death and differentiation and participate in immune cell activation [6], [9]. S1P is not only an intracellular mediator, but it also functions extracellularly via its five membrane-bound receptors (S1PR1–5) [10]. S1PR1 expression on lymphocytes is required for the egress from thymus and secondary lymphoid organs [11], [12]. While S1PR1 also influences marginal zone B-cell localization, S1PR3 affects B-cell chemotaxis in marginal zones of spleen and DC chemotaxis [13], [14]. These receptors are also found on important effector cells relevant for allergy, eosinophils and mast cells [15]. The chemotaxis and effector responses of mast cells are regulated by S1PR1 and S1PR2, while eosinophils also display S1PR3 on their surface [16].
Production of S1P is regulated by two sphingosine-kinases (SphK1 and SphK2), which phosphorylate sphingosine primarily intracellularly [17]. Upon activation of the cell, S1P can be secreted and act in an autocrine/paracrine manner. Both SphKs have diverse tissue distribution and functions. In mouse tissue SphK activity was found to be high in lung, small intestine and moderate in spleen and stomach. In the small intestine and spleen the contribution of SphK1 to total SphK activity was up to 70%, while SphK2 played only a minor role [18].
Regarding their influence on allergy effector cell functions, mast cells are potent producers of intracellular and secreted S1P [7]. Although both SphK1 and 2 have been proposed to regulate S1P production in mast cells, the circumstances in which one or both may contribute to S1P production in mast cells is not well defined. Our previous work showed that loss of SphK2 in mast cells reduced S1P production and caused a substantial inhibition of FcɛRI mediated degranulation as well as diminished production of IL6, IL13 and TNFα [7]. In contrast, SphK1-deficiency was responsible for lowered circulatory S1P in vivo, which also altered mast cell function [7]. Recently, we found that SphK1 increases the rate of recovery from anaphylactic shock, whereas SphK2 slows the rate of recovery, events associated with the control of blood pressure and subsequent renal clearance of histamine [19]. S1P levels were not only crucial for anaphylactic outcome, but were also found to be increased in bronchial alveolar lavage of asthmatic patients after allergen challenge and were significantly correlated with eosinophil numbers in BAL fluid [2]. Collectively, these findings have led to the conclusion, that circulating S1P and S1P-mediated trafficking may represent a key event in the development of allergic diseases. However, evidence for a role of S1P in food allergy is scarce. A recent study revealed that S1P is important for migration of CD4+ T-cells from the spleen to the intestine, resulting in antigen specific intestinal allergic hypersensitivity in a murine food allergy model [3]. In this study, FTY720, a functional antagonist of S1P receptors, prevented allergic diarrhea and inhibited the infiltration of CD4+T cells as well as mast cells into the large intestine without impairing eosinophil infiltration [3]. Importantly, intestinal epithelial cells express S1PRs [20], which regulate barrier function and, thus, could affect the transport of antigens through the intestinal mucosa.
In the current study we investigated the influence of endogenous S1P production on food allergy induction. First the effects of S1P on epithelial cell integrity and antigen uptake were analyzed in vitro using an established CaCo2 cell model. Based on these findings we studied in vivo the influence of S1P alteration on the development of food allergy in a previously established food allergy model [21] using SphK1 and SphK2 deficient mice. This model is based on recent murine as well as human studies showing that the inhibition of peptic degradation of food allergens by the use of acid-suppressive medication favors the development of IgE mediated food allergy [22], [23], [24]. Feeding OVA as a food model allergen under concomitant acid-suppression was repeatedly shown to be associated with food allergy including elevated allergen specific IgE titers, Th2 cytokines and anaphylactic symptoms after oral allergen provocation [21]. Here, we report that S1P alters tight junction integrity and OVA uptake by epithelial cells in vitro. Furthermore, SphKs are proven as decisive for oral sensitization against food allergens, as SphK1 and SphK2 null mice failed to increase IgE production after oral immunization. While SphK1 seems to be crucial for intestinal mast cell degranulation, SphK2 may be necessary for mast cell accumulation in the gastrointestinal mucosa.
Section snippets
Mice
All experiments were performed in accordance with National Institutes of Health (NIH) guidelines and an animal study proposal A010-04-03 approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases. SphK1 and SphK2 null mice (C57BL/6 × 129 Sv, N5) were generated as described previously [7], [11], [25]. Wild-type mice (WT) were littermates obtained from heterozygous mating pairs. Genotyping was performed as described [7], [25].
Reagents
Transwell filter plates with polycarbonate
S1P affects tight junction integrity and OVA uptake in vitro
To investigate the influence of e.g. autocrine and paracrine S1P secretion, we performed an in vitro transwell study. CaCo2 cells cultured in an inverted orientation (Fig. 1A) for 21 days until the formation of a differentiated intestinal phenotype were stimulated with S1P or medium as control and subsequently evaluated for tight junction integrity and FITC OVA uptake. One-hour stimulation with 0.5 μM S1P significantly decreased the transepithelial resistance (TEER) after 1 and 5 h compared to
Discussion
Food allergy is a disease associated with elevated allergen specific IgE antibody titers, Th2 cytokine preponderance and effector cell migration towards the food allergen after allergen exposure [31]. Here we report that in a food allergy model [21], both SphK1−/− and SphK2−/− mice developed significantly reduced levels of IgE when immunized via the oral route. In contrast, all 3 groups of mice immunized i.p. with OVA showed comparable, high levels of OVA specific IgE and IgG2a, while the IgG1
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the Intramural Research Program of NIAMS, NIH and grants of the Austrian Science fund FWF (P21577 and P21884). Susanne C. Diesner was supported by a short-term fellowship (ASTF 374-2008) of the European Molecular Biology Organization (EMBO). We also acknowledge the support of the Flow Cytometry Section, Laboratory Animal Care and Use Section, and the Light Imaging Section, of the Office of Science and Technology, NIAMS and Dr. Sonja Zehetmayer for statistical advise
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2016, Clinical ImmunologyCitation Excerpt :The advantage of our oral food allergy model in BALB/c mice is the physiological sensitization route applying the allergen intragastrically under acid suppressive medication without further adjuvants [5]. Even though in our animal experiments inbred mice were housed and sensitized under identical conditions, immune responses were not uniform [4–7,9,10,29–32] (and own unpublished data). Thus, in the present study we aimed to phenotype mice being protected from food allergy in comparison to mice being highly sensitized and anaphylactic after immunizations.
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These authors contributed equally.