ARTICLE Received 14 Apr 2015 | Accepted 15 May 2015 | Published 10 Jul 2015 DOI: 10.1038/ncomms8514 PGD2 deficiency exacerbates food antigen-induced mast cell hyperplasia Tatsuro Nakamura1, Shingo Maeda1, Kazuhide Horiguchi2, Toko Maehara1, Kosuke Aritake3, Byung-il Choi4, Yoichiro Iwakura5, Yoshihiro Urade3 & Takahisa Murata1 Prostaglandin D2 (PGD2) is a major prostanoid secreted mainly by mast cells. Although PGD2 has been identified as a modulator of allergic inflammation, its precise role remains unclear. Here we investigate the role of PGD2 in food allergy. Oral administration of ovalbumin induces allergic responses in sensitized wild-type (WT) mice. Systemic gene deficiency of haematopoietic PGD synthase (H-PGDS À / À ) exacerbates all of the manifestations accompanying severe mast cell hyperplasia in the intestine. Morphological studies show that c-kit/FceRI-positive WT mast cells strongly express H-PGDS. Transplantation of H-PGDS À / À mast cells also aggravates ovalbumin-induced mast cell hyperplasia and allergic symptoms in mast cell null mice. H-PGDS deficiency accelerates the production of SDF-1a and the activity of MMP-9 in the antigen-stimulated intestine. SDF-1a receptor blockade or MMP-9 inhibition relieves the exacerbated mast cell hyperplasia and manifestations observed in H-PGDS À / À . Thus, PGD2 deficiency results in food antigen-induced mast cell hyperplasia. 1 Department of Animal Radiology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. 2 Department of Morphological and Physiological Sciences, University of Fukui, Fukui 910-1193, Japan. 3 International Institute for Integrative Sleep Medicine, University of Tsukuba, Ibaraki 305-8575, Japan. 4 Department of Anatomy, College of Medicine, Korea University, Seoul 136-701, Korea. 5 Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba 278-0022, Japan. Correspondence and requests for materials should be addressed to T.M. (email: [email protected]). NATURE COMMUNICATIONS | 6:7514 | DOI: 10.1038/ncomms8514 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8514 ood allergy is defined as an abnormal immune response to chemoattractant receptor-homologous molecule expressed on food proteins. The number of patients with food allergies Th2 cells (CRTH2). The dehydration product of PGD2, 15-deoxy- Fhas doubled within the last several decades. Currently, delta PGJ2 (15d-PGJ2), also has bioactivity via peroxisome 8 2–10% of the human population suffers from hypersensitivity to proliferator-activated receptors . Although PGD2 has emerged certain food proteins1. Symptoms affecting the skin, lung as a mast cell-derived allergic mediator, there is uncertainty and gastrointestinal (GI) tract include eczema, laboured regarding its contribution to allergic inflammation. Several breathing and diarrhoea. In serious cases, hypersensitivity to studies have reported a pro-inflammatory role associated with food antigens may lead to systemic anaphylaxis reactions, thus PGD2 signalling. Inhalation of PGD2 induced nasal blockage via 9 threatening the life of the patient. Since few effective treatments the DP receptor in atopic rhinitis patients . In addition, PGD2 are available for these reactions, the remaining option for patients enhanced airway inflammation by accumulation of inflammatory is to avoid the causative antigens (that is, specific foods). cells via the DP receptor on epithelial cells10,11. Conversely, other Therefore, the development of new clinical applications for studies have suggested anti-inflammatory roles for PGD2.In food allergies is urgently needed. mouse asthma model, DP agonism reduced eosinophilia and Mast cells are major players in allergic diseases. Following the airway hyperresponsiveness12, whereas CRTH2 agonism onset of inflammation, several chemoattractants are produced restricted the recruitment of eosinophils by limiting the that stimulate the release of mast cell progenitors (MCps) from production of interleukin (IL)-5 (ref. 13). Thus, the role of 2 bone marrow (BM) into the circulation . Circulating MCps PGD2 signalling in allergic response remains poorly understood. migrate and mature peripherally in connective or mucosal tissues. In this study, we investigated the role of PGD2 in food allergy Stem cell factor (SCF) plays a central role in chemoattraction and using genetically modified mice. We found that lack of PGD2 the proliferation and maturation of MCps3. Stromal-derived results in food antigen-induced mast cell hyperplasia. factor (SDF)-1a and tumour necrosis factor (TNF)-1a are also known to induce migration of MCps into peripheral tissues4. In the sensitization phase of allergy, the uptake of a food Results antigen activates T- or B-lymphocytes to produce immunoglo- H-PGDS deficiency exacerbates food allergy. Naive bulin E (IgE), which binds to mast cells through its high affinity H-PGDS À / À mice with a C57BL/6-background did not receptor FceRI. Upon re-uptake of antigens, IgEs are crosslinked, present any congenital GI tract abnormalities, such as changes which in turn degranulates the mast cells to release a in morphology (Supplementary Fig. 1a,b), epithelial permeability large number of inflammatory mediators, including histamine, (Supplementary Fig. 1c), mRNA expression of inflammatory platelet-activator factor (PAF), cysteinyl leukotrienes (cys-LTs) cytokines (Supplementary Fig. 1d) or tissue serotonin content, 14 and prostaglandin D2 (PGD2). The secreted histamine rapidly which causes oral antigen-induced diarrhoea (Supplementary and strongly increases capillary permeability, whereas PAF Fig. 1e). In addition, both lines of mice showed comparative mainly induces platelet aggregation and degranulation5,6. hypothermia in IgE/mast cell-mediated passive systemic Mast cells strongly express haematopoietic PGD synthase anaphylaxis (Supplementary Fig. 1f). (H-PGDS) and produce a large amount of the arachidonic acid Diarrhoea is a representative symptom in murine models of 15 metabolite PGD2 (ref. 7). PGD2 exerts its biological actions food allergy . We assessed the stool consistency (Fig. 1a) and though two distinct receptors, D prostanoid (DP) and the scored other systemic responses including scratching, immobility a b c ‡ ) H-PGDS –/– –1 1.6 H-PGDS –/– ‡ ‡ 2.5 ‡ WT ‡ 1.6 –/– ‡ ‡ g ml H-PGDS 1.2 2.0 µ ‡ 1.2 ‡ 1.5 0.8 ‡ 0.8 1.0 * Faeces score 0.4 WT 0.4 † Systemic score 0.5 WT 0 0 0 OVA-specific IgE ( 0246810 0246810 Saline ×10 OVA OVA challenge (times) OVA challenge (times) d e f 80 Balb/c H-PGDS –/– ×3 OVA ×5 OVA –/– 3.0 Balb/c H-PGDS ‡ 0.0 60 ‡ 2.0 40 –0.4 1.0 Diarrhoea (%) 20 Balb/c WT Systemic score Balb/c WT –0.8 0 0 WT ‡ –1.2 H-PGDS –/– 0 246810 0246810 Body temperature drop (°C) OVA challenge (times) OVA challenge (times) Figure 1 | H-PGDS deficiency exacerbates OVA-induced allergic reactions. Faeces score (a) and systemic score (b) were monitored in C57BL/6- background mice (n ¼ 8–12). (c) Serum OVA-specific IgE levels in mice following the tenth challenge with either saline or OVA (n ¼ 8–12). The horizontal lines indicate the means. Diarrhoea occurrence (d), systemic score (e) and hypothermia (f) were monitored in Balb/c-background mice (n ¼ 11). Diarrhoea w occurrence is represented as the percentage over the number of OVA challenges. *, , zPo0.05 compared with saline-challenged WT, saline-challenged H-PGDS À / À or OVA-challenged WT, respectively. Data are presented as mean±s.e.m. of 2–3 independent experiments. Mann–Whitney U-test was performed for comparisons in a, b and e, and Tukey’s test for comparison in c and f. 2 NATURE COMMUNICATIONS | 6:7514 | DOI: 10.1038/ncomms8514 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8514 ARTICLE and swelling (bristled fur, oedema around the nose and eyes or regardless of the mouse strain. C57BL/6 mouse lines were utilized laboured breathing; Fig. 1b). In wild-type (WT) mice, both for the subsequent experiments. scores increased after the seventh or eighth oral challenge of 50 mg ovalbumin (OVA), whereas more severe symptoms with earlier onset developed in H-PGDS À / À mice. We next H-PGDS deficiency exacerbates mast cell infiltration. Mast cells investigated whether H-PGDS deficiency affected serum IgE are located at the sites exposed to external environment such as levels. Although OVA challenges significantly increased serum GI tract and skin. We counted the infiltrated mast cells visualized OVA-specific IgE levels in both WT and H-PGDS À / À mice, by chloroacetate esterase (CAE) staining in small and large there was no significant difference between the lines after ten intestine, and also in lung and ear. The number of mast cells in times OVA challenges (Fig. 1c). those tissue was no difference between both lines of mice before Allergic responses are known to vary among mouse strains15. OVA stimulation (Supplementary Fig. 3a). OVA challenge Balb/c WT mice exhibited diarrhoea even in response to 1 mg stimulated mast cell infiltration into the intestinal tract as well as OVA (Supplementary Fig. 2a). At this lower dosage of OVA, in the other sites in WT (Fig. 2a). H-PGDS deficiency was Balb/c H-PGDS À / À showed earlier and more severe observed to accelerate mast cell infiltrations, resulting in mast cell manifestations including diarrhoea (Fig. 1d), systemic reactions hyperplasia in almost all of the