HDM exposure enhances subsequent responses to OVA-induced food allergy

HDM (House dust mites) are important environmental trigger factors of airway allergic diseases, the allergens of HDM were detected in the human gut mucosa, which induces local inammation and increases intestinal permeability. This study tests a hypothesis that house dust mites contribute to the development of OVA (ovalbumin)-induced food allergy. The serum levels of IgE against HDM in patients with food allergy were detected with UniCAP100 (Pharmacia, Uppsala, Sweden); the HDM-induced/the OVA-induced mouse model of food allergy was developed. Difference between 2 groups was determined by Student t test or ANOVA if more than two groups. Compared to the healthy controls, the patients with food allergy have higher levels of serum IgE against HDM. Compared to food allergy alone groups, the levels of IgE against HDM in food allergy with asthma or allergic rhinitis groups were increased signicantly. In mouse models, we found that HDM/OVA induced allergy-like symptoms, lower body temperature, and lower body weight. The levels of IgE, IgG1, mMCP-1 (mouse mast cell protease-1), IL-4 and IL-5 in the HDM and HDM + CT (cholera toxin) groups were higher than the control groups, and the levels of IgE, IgG1, IL-4 and IL-5 in the HDM, OVA and HDM + OVA groups were higher than the control groups. The pathology of intestinal tract in the HDM and HDM + CT/the HDM, OVA and HDM + OVA groups were more severe and exhibited more eosinophils than the control groups. Moreover, the prior exposure to HDM induced intestinal barrier dysfunction, and facilitated the development of intestinal allergy in mice. Based on above data and previous researches, we put forward that HDM exposure enhances subsequent responses to OVA-induced food allergy.


Background
Food allergy is estimated to affect 5% of adults and 8% of young children ; the prevalence of food allergy has been increasing signi cantly in the recent decades. [1][2] Food allergy have become a worldwide health problem, which burdens patients and families by increasing expenses of healthcare and negatively affects the quality of life.
There are 8 most common food allergens proved by the U.S. Food and Drug Administration (FDA), including peanuts, tree nuts, soy, wheat, sh, shell sh, milk and eggs. 3 These food allergens usually go straightly through the mouth into the host, and then induce intestinal sensitization/allergy.
There is a high coincidence between food allergy and other allergic diseases, such as allergic asthma, allergic rhinitis and atopic dermatitis. 4 Asthmatic patients appear to be at signi cantly increased risk of severe allergic reactions induced by food allergens. 5 The cross-reactivity between aeroallergens and food allergens may induce food allergy in patients with airway allergy. 6 Especially, tropomyosin was involved in cross-reactivity between HDM and shrimp, thus shrimp and house dust mite (HDM) allergies usually occur in the same patient, and consequently, the frequency of HDM sensitization in shrimp allergic people is higher. 7 There are many seasonal/outdoor aeroallergens, such as dust mites, pollen, cockroaches, fungi and animal feathers. 8 Nearly 80% of asthmatic patients are sensitized to HDM, the predominant sources of aeroallergens. 9 In recent research, HDM allergen was detected in the human intestine, which was able to induce local in ammation and increased intestinal permeability. 10 Based on the information above, we hypothesized that HDM may be not only an aeroallergens, but also an important role in food allergy. In this study, the levels of speci c IgE against HDM in patients with food allergy were analyzed, and a HDM/OVA-induced mouse model of food allergy was developed. Moreover, the exposure to HDM increased the intestinal barrier permeability and facilitated the development of intestinal allergy in mice. The data demonstrate that HDM not only is an aeroallergen, which induces airway allergy, but also enhances subsequent responses to OVA-induced food allergy.

Human subjects
In total of 825 food allergic patients' serum were collected at the First A liated Hospital, Guangzhou Medical University (Guangzhou, China) during January 2015 to November 2016. The diagnosis was conducted by the doctors of this hospital. The clinical features of human subjects are presented in Table 1. This study has been approved by the Human Ethic Committee at Shenzhen University and Guangzhou Medical University. Informed consent was obtained from all subjects and if subjects are under 18, from a legal guardian. All experiments were performed in accordance with the relevant guidelines and regulations. were induced in Balb/c mice. Mice were continually exposed to HDM (1 mg, i. g)/PBS for a week.
Combined HDM exposure and food allergy was obtained by applying the two protocols successively (Fig. 4A). Control mice were sensitized with PBS alone and challenged with PBS.
The data were analyzed with the software Flowjo.

Assessment of the Intestinal permeability in vivo
This measure is based on the intestinal epithelial barrier permeability to 4,000-Da uorescent-dextran (Sigma-Aldrich). 14 6-h-fasted mice were fed with uorescein isothiocyanate (FITC)-dextran at 600 mg/kg body weight (125 mg/ml). After 1 h, the mice were sacri ced. The blood was collected from the tip of the tail vein. The blood was centrifuged t 5000 rpm (

Statistics
Data are presented as mean ± SD. Difference between 2 groups was determined by Student t test or ANOVA if more than two groups. P < 0.05 was set as a signifcant criterion.

Results
Patients with food allergy have higher levels of IgE against HDM in the serum We collected 825 patients at allergy clinic. As shown by Figure 1A, compared to the healthy controls, the patients with food allergy have higher levels of speci c IgE against HDM. Compared to the food allergy alone group (FA), the levels of speci c IgE against HDM in the food allergy with bronchial asthma group (FA+BA) or allergic rhinitis group (FA+AR) were increased signi cantly, while there was no signi cant differance between FA and bronchial asthma alone group (BA) or allergic rhinitis alone group (AR  Figure 1B).
Establishment of a mouse model of food allergy with HDM As illustrated in Figure 2A, mice were sensitized and challenged with HDM following the procedures we previously reported. 15 After sensitization, the mice were challenged intragastrically with HDM. Systemic anaphylactic symptoms were evaluated within 30 to 40 minutes. All the mice in the HDM+CT group and HDM group developed anaphylaxis (median anaphylactic score 3.3 and 2.83 respectively). On the contrary, the control mice showed no anaphylactic reactions ( Figure 2B). There is a decrease in body temperature during systemic anaphylaxis. 16 Twenty-ve minutes after HDM challenge, rectal temperature was measured. As shown in Figure 2C, mice in HDM+CT group and HDM group showed signi cant reductions in rectal temperature than that of control group (P < 0.001). Allergic mice presented a metabolic change that leads to signi cant body weight loss compared with the control group. 17 Consequently, We observed that HDM challenged mice presented body weight loss when compared with control mice ( Figure 2D). What's more, the contour of the jejunum from the control group mice were clear and there was less in ammatory cell in ltration in the submucosa, whereas the in amed jejunum of mice with HDM-treated revealed high levels of in ammatory cells in ltration and sloughing of enterocytes at the tips of the villi ( Figure. 2E-F).
HDM facilitates Th2 immune response Spleen cells labeled with CFSE were cultured in the presence of HDM or saline for 72 h. The result showed that CD4+CSFE-cells in the HDM+CT group and HDM group were more abundant than that in the control group, which indicated that CD4 + T cells markedly proliferated after stimulating with HDM ( Figure 3A). To further investigate whether HDM can enhance Th2 immune response, splenocyte cytokine pro les were analyzed in the present study. The result demonstrated that splenocytes from HDM-treated mice produced signi cantly high levels of Th2 cytokines (IL-4 and IL-5), but the levels of Th1 cytockine (IFN-γ) were not different (P>0.05) (Figure 3 B-D). The speci c immune response to HDM was also measured by testing the Serum speci c immunoglobulin levels. As shown in Figure 3E-F, HDM-speci c IgE and IgG1 were signi cantly increased in the HDM+CT group and HDM group. The mouse mast cell protease-1 (mMCP-1) is a marker of mast cell activation. 18 As shown in Figure 3G, the mMCP-1 concentration in the serum was higher in HDM-sensitized mice than that of the control group (P < 0.001).
Exposure to HDM facilitates development of OVA-induced intestinal allergy.
Prior HDM exposure aggravate allergy-like symptoms, increases permeability of intestinal epithelial barrier, lower body temperature, and lower body weightin OVA-induced intestinal allergy ( Figure 4B-E) . The levels of sIgE, sIgG1, IL-4 and IL-5 in the HDM + OVA groups were higher than the PBS + OVA groups and control groups ( Figure 4F-I). Moreover, the pathology of intestinal tract in HDM +OVA groups were more severe and exhibited more eosinophils than PBS + OVA groups and control groups ( Figure 4J-K).

Discussion
A large amount of studies have been conducted to highlight the critical role of HDM allergen exposure particularly in respiratory allergic diseases. In contrast, HDM were known as aeroallergen and little attention was attracted in the study of food allergy. Tulic et al reported recently that HDM allergen was detected in the human gastrointestinal tract, and the intestinal barrier function was affected directly by the cysteine protease activity of HDM allergen without prior sensitisation. 10 Nevertheless, the contribution of HDM in the pathogenesis of food allergy remains unknown. We carried out this study to elucidate that HDM is a crucial environmental trigger factor for developing food allergy.
Speci c IgE antibodies play an important role in mediating type 1 allergic reaction in human. 19 Speci c IgE that have already bound to the surface of mast cells or basophils can be bound by food allergens to cause the secretion of the allergy-related meditors such as histamines. Subsequently, with the second exposure to the speci c allergens, symptoms were usually triggered soon. In this study, we have observed that among 825 food-allergic patients, 65.9% exhibited IgE reactivity to HDM. Compared to FA, the levels of IgE against HDM in FA + BA or FA + RA were increased signi cantly, while there was no signi cant differance between FA and BA or RA. These data indicate that HDM may be an important contributor to food allergy.
A HDM-induced mouse model of food allergy was developed successfully in the present study, and hypersensitivity was evaluated by using some well established parameters, including allergy-like symptoms scores, decreased body temperatures, heavy in ltration of in ammatory cells in the jejunal mucosa, 20-21 increased serum mMCP-1 levels 22 and decreased body weights, 23 which demonstrate HDM induce an obvious intestinal allergy in mouse model.
Cytokines, secreted by T helper type 2 (Th2) cells, such as IL-4 and IL-5, are the major pathological feature of allergic disease, including food allergy. 23 Especially, IL-4 promotes the production of allergen-speci c IgE and activates mast cells to mediate type 1 in ammation in food allergy. 24 Meanwhile, IL-5 plays an important role in the proliferation, recruitment and activation of eosinophils, and then promotes the development of type 1 in ammation. 25 In this study, we found that mice exposed to HDM showed signi cantly higher levels of Th2 cytokine IL-4 and IL-5, but not T helper type 1 (Th1) cytokine IFN-γ, indicating that HDM facilitates Type 2 in ammation in mice.
CT, secreted by Vibrio cholera, which is a potent mucosal adjuvant for stimulating allergen-speci c immune response, 26 is also considered as a potent Th2 adjuvant because it stimulates production of Th2 cytokines and promotes speci c IgE and IgG1 production. 27 In the present study, we fed the mice with HDM (1 mg/mouse) in the presence of CT as an adjuvant. The group 1 allergen of Dermatophagoides pteronyssinus (Der p1) has been proven as the major allergen, the proteolytic activity of Der p1 resulted in a signi cant reduction in IL-12 production in dendritic cells(DCs), and that DCs induced naive T cells induce the TIM4 gene transcription and expression in DCs, 30 indicating that Der p1 can conduce DCs to produce more TIM4 and induce Th2 polarization subsequently. Tulic et al showed that Der p1 was present in the human intestine, the proteolytic activity of Der p1 resulted in disrupted of TJ proteins, reduced integrity of the mucus barrier, as well as increased permeability of epithelial cells . 10 Therefore, there is a hypothesis that some HDM proteins like Der p1 can act as mucosal adjuvant, which facilitate Th2 polarization, contribute to intestinal barrier dysfunction, and increase the allergen transportation across the intestinal epithelial barrier. In this study, mice were sensitized and challenged without any adjuvant, and the result show that HDM alone induce an intestinal allergy in mice obviously. To further test our hypothesis, an OVA food allergy followed by HDM exposure were induced in mice. Consequently, the result show that the prior exposure to HDM is able to induce intestinal barrier dysfunction, and facilitate the development of intestinal allergy in mice.

Conclusion
In conclusion, based on above data and previous researches, we put forward that HDM contribute to the development of food allergy.    Each group consists of six mice, with *p < 0.05, **p < 0.01, ***p < 0.001, (ANOVA) compared to the control group. &p <0.05, &&p <0.01, &&&p <0.01 (t test) was compared to the PBS + OVA group.