Pentraxin 3 Facilitates Shrimp-Allergic Responses in IgE-Activated Mast Cells

Background Since food avoidance is currently the only way to prevent allergic reactions to shrimp, a better understanding of molecular events in the induction and progression of allergy, including food allergy, is needed for developing strategies to inhibit allergic responses. Pentraxin 3 (PTX3) is rapidly produced directly from inflammatory or damaged tissues and is involved in acute immunoinflammatory responses. However, the role of PTX3 in the development of immediate IgE-mediated shrimp allergy remains unknown. Methods Wild-type BALB/c mice were immunized intraperitoneally and were challenged with shrimp extract. Serum IgE and PTX3 levels were analyzed. RBL-2H3 cells were stimulated with either dinitrophenyl (DNP) or serum of shrimp-allergic mice, and markers of degranulation, proinflammatory mediators, and phosphorylation of signal proteins were analyzed. We further examined the effect of PTX3 in shrimp extract-induced allergic responses in vitro and in vivo. Results Mice with shrimp allergy had increased PTX3 levels in the serum and small intestine compared with healthy mice. PTX3 augmented degranulation, the production of proinflammatory mediators, and activation of the Akt and MAPK signaling pathways in mast cells upon DNP stimulation. Furthermore, the expression of transcription factor CCAAT/enhancer-binding protein delta (CEBPD) was elevated in PTX3-mediated mast cell activation. Finally, the PTX3 inhibitor RI37 could attenuate PTX3-induced degranulation, proinflammatory mediator expression, and phosphorylation of the Akt and MAPK signaling. Conclusions The results suggested that PTX3 can facilitate allergic responses. Our data provide new insight to demonstrate that PTX3 is a cause of allergic inflammation and that RI37 can serve as a therapeutic agent in shrimp allergy.


Background
Among all food allergies, allergy to shellfish such as shrimp is particularly common in Asian countries [1,2]. The main symptoms of a shrimp allergy generally develop within minutes to an hour and vary widely from oral symptoms to life-threatening allergic reactions (such as anaphylaxis).
Immunoglobulin E-(IgE-) mediated shrimp allergy is a leading cause of severe allergic reaction. The high-affinity IgE receptor FcεRI is highly expressed in mast cells and is required for transmitting signals that induce degranulation and allergic reactions [8]. The crosslinking of FcεRI with IgE-antigen complexes induces the activation of proteintyrosine kinases (PTKs) and then PTKs phosphorylate substrates that are involved in the activation of several signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt, RAS/extracellular signal-regulated kinase (ERK), p38, and JNK [9]. Following the activation of the above signaling pathways, a degranulation reaction and the production of histamine and inflammatory cytokines, including TNF-α, IL-4, IL-6, IL-13, and cyclooxygenase-2 (COX-2), are observed in mast cells and basophils [10]. Nevertheless, whether PTX3 is involved in FcεRI-mediated signaling cascades and responses in mast cells remains an open question.
Physiologically, PTX3 confers host resistance against pathogens [11]. Accumulated evidence suggests that PTX3 is a potential marker associated with disease severity and mortality in diverse human pathological conditions, such as cardiovascular diseases [12], rheumatoid arthritis [13], chronic kidney disease [14], sepsis [15], and several types of cancers [16][17][18]. In addition, at normal physiological condition, the expression level of CCAAT/enhancer-binding protein delta (CEBPD) is typically low but is rapidly induced by inflammatory factors, such as TNF-α, IL-1β, IL-6, and COX-2 [19]. Recently, our previous studies have demonstrated that PTX3 is a downstream target of CEBPD and contributes to inflammation-related disorders, such as atherosclerosis [20], Alzheimer's disease [21], rheumatoid arthritis [22], invasion and metastasis of cancer, and drugresistant cancers [16]. However, very little is known about whether PTX3 and CEBPD can participate in crosstalk between the innate and adaptive immune systems in the initiation or aggravation of shrimp-allergic immune response.
In this study, we found an increase in PTX3 levels in the intestine of shrimp-allergic mice and IgE-induced allergic inflammation. PTX3 can result in exaggerated degranulation, the production of inflammatory mediators, and activation of the Akt and MAPK signaling cascades in IgE-antigen complex-(IgE/Ag-) treated mast cells. In addition, we showed that, upon PTX3 treatment, CEBPD is activated in mast cells and participates in shrimp allergy. Moreover, a PTX3 inhibitor, the RI37 peptides, was applied to prevent the PTX3-induced hypersensitivity reaction and reduce the degranulation of mast cells in in vitro and in vivo models. Altogether, the results demonstrated the novel role of PTX3 in regulating the IgE-mediated inflammation of DNP-or shrimp-induced allergy in mast cells.
2.2. Mouse Sensitization and Immunization. Three-fourweek-old female BALB/c mice were handled according to the guidelines of our institute (the Guide for Care and Use of Laboratory Animals, National Cheng Kung University). The animal use protocol listed was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). All mice were maintained on a shrimp-free diet and housed in pathogen-free conditions. To induce shrimp hypersensitivity in mice, sensitization was performed by intraperitoneal injection with 10 mg of shrimp extract plus 1 mg of aluminum hydroxide (Al(OH) 3 ) on days 1, 4, 7, 10, 13, 16, 19, and 22, and mice were challenged with 50 mg of shrimp extract on day 30. Mice administered phosphate-buffered saline (PBS) plus adjuvant were included as controls. Blood samples were collected 2 h after the challenge for antibody and PTX3 analysis. CEBPD -/mice (C57BL/6 background) were a kind gift from Sterneck et al. [23]. In CEBPD -/mice, the coding region of CEBPD was replaced by a neomycin resistance gene.

Immunohistochemistry (IHC).
Mice were sacrificed by CO 2 asphyxiation prior to IHC examination. The entire small intestine was dissected longitudinally, cut open along its longitudinal axis, and rinsed briefly in PBS. Intestinal tissues were fixed in 10% formalin in PBS and subsequently embedded in paraffin. Then, 5 μm-thick paraffin sections were cut, and the slides were deparaffinized in xylene and rehydrated in graded alcohol dilutions. The sections were treated with Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, and 0.05% Tween 20; pH 9.0) at 120°C for 10 min in a pressure cooker for antigen retrieval. The sections were incubated with a blocking buffer (2% nonfat dry milk and 0.01% Tween 20 in PBS) for 1 h at room temperature. The slides were then stained using a goat anti-PTX3 antibody (Abcam, 1 : 500 dilution) overnight. The slides were then incubated with polymer and DAB solutions from a Novolink Polymer Detection Systems kit (Leica Biosystems) according to the manufacturer's instructions. The sections were then counterstained with hematoxylin and dehydrated. The sections were mounted in mounting medium (Thermo Scientific) and evaluated with an Olympus BX51 microscope.

Degranulation
Assay. For the degranulation assay, RBL-2H3 cells were loaded onto a 24-well plate (2 × 10 5 cells/ well) and incubated with 0.2 μg/ml anti-DNP-IgE antibody Released histamine was evaluated following a previously reported method. First, 100 μl of cell culture supernatant was mixed with 20 μl of 1 M NaOH, and then 25 μl of the reaction solution (1% (w/v) o-phthalaldehyde dissolved in methanol) was immediately added and incubated for 4 min at room temperature. The reaction was terminated by addition of 10 μl of 3 M HCl. The fluorescence intensity was measured at an excitation wavelength 355 nm and emission wavelength 460 nm.
To measure the amount of β-hexosaminidase activity released from the cells, cell culture supernatant (25 μl) was mixed with an equal volume of a 5 mM substrate solution (5 mM p-NAG dissolved in 0.2 M sodium citrate buffer, pH 4.5) at 37°C for 1.5 h. The reaction was terminated by adding 200 μl of stop solution (0.1 M Na 2 CO 3 /NaHCO 3 , pH 10.0). The β-hexosaminidase activity was determined by measuring the difference in absorbance at 405 nm.
2.6. Quantitative PCR (qPCR). Total RNA was extracted using the TRIsure RNA extraction reagent (Invitrogen). cDNA synthesis was performed with an RT reaction using SuperScript III (Invitrogen). qPCR was conducted using KAPA SYBR FAST qPCR Master Mix (Life Technologies Corporation and Kapa Biosystems Inc.). PCR was conducted using a CFX Connect Real-Time PCR System (Bio-Rad) with the following pairs of specific primers: β-Actin Forty cycles were set for quantitative the PCR program, and the relative expression of target genes was calculated following the formula (2 −ΔΔCt ).

Short
Hairpin RNA (shRNA) Assay. Lentiviruses were produced from Phoenix cells that had been cotransfected with various shRNA expression vectors in combination with pMD2.G and psPAX2. After determining the viral infection efficiency, cells were infected for 48 h with shVoid and shCEBPD lentiviruses, each at a multiplicity of infection (MOI) of 10. The shRNA oligo sequences used in the lentiviral expression vectors were as follows: shVoid: 5′-CCGGAGTTCAGTTACGATATCATGTC TCGAGACATTCGCGAGTAACTGAACTTTTTT-3′.
The lentiviral knockdown expression vectors were purchased from the National RNAi Core Facility located at the Genomic Research Center of the Institute of Molecular Biology, Academia Sinica, Taiwan.
2.10. Statistical Analysis. All experiments were repeated at least three times, and the data were analyzed for statistical significance using a two-tailed unpaired t test (Prism 5 software). One-way ANOVA was performed for multiple comparisons between groups. The data were expressed as the means and standard errors of mean (±SEMs). A statistically significant difference was defined at * p < 0:05, * * p < 0:01, and * * * p < 0:001.

Results
3.1. Elevated Levels of PTX3 in Mice Allergic to Shrimp. As mentioned above, the activation of PTX3 has been observed in many inflammation-associated diseases, including asthma [25]. However, the involvement of PTX3 in food allergy remains unknown. To address this issue, a shrimp allergy system was established in BALB/c mice via the intraperitoneal injection of low-dose shrimp extract and adjuvant, Al(OH) 3 , to induce sensitization and then high-dose shrimp extract to induce hypersensitization (Figure 1   Journal of Immunology Research IgE production in the blood. We found that the level of total IgE in the serum was substantially increased in shrimpallergic mice compared with that in control mice (Figure 1(b). Next, the ELISA results showed that PTX3 concentrations were significantly higher in the serum on the 30 th day of the hypersensitization phase in shrimp extractchallenged mice than in control mice (Figure 1(c). Meanwhile, the results of immunohistochemistry staining showed that positive PTX3 immunoreactivity was detected in all cases in the upper region of the small intestinal villi and lumen after challenge (Figure 1(d). These results show that shrimp allergy induces PTX3 levels higher than those in control mice in the serum and intestinal tracts. Thus, our data imply that PTX3 might play an important role in the allergy to shrimp.

PTX3 Augments Degranulation and the Formation of Proinflammatory Mediators in IgE/Ag-Activated RBL-2H3
Cells. Despite the fact that the clinical symptoms of shellfish allergy are mainly within the gastrointestinal tract, detailed immunological studies in terms of shrimp hypersensitivity are limited. Mast cells and basophils have crucial impacts on the initiation and development of food allergic diseases [26]. The secretion of histamine and β-hexosaminidase has been applied as standard biomarkers for determining the degranulation of mast cells. To test whether PTX3 is involved in the enhancement of the degranulation of mast cells, RBL-2H3 cells were first exposed to PTX3 and then stimulated with 2,4-dinitrophenyl (DNP) hapten. Indeed, our results showed that PTX3 could enhance the levels of histamine and β-hexosaminidase in IgE/DNP-stimulated RBL-2H3 cells (Figures 2(a) and 2(b). In addition, the increase in de novo-synthesized proinflammatory mediators has been observed in hypersensitization [27,28]. Therefore, we further examined the effects of PTX3 on the transcriptional activation of proinflammatory cytokines and enzymes associated with the allergic response in RBL-2H3 cells using fluorescent quantitative PCR. The results showed that PTX3 could slightly induce the levels of TNF-α and IL-13 mRNA in RBL-2H3 cells (Figure 2

PTX3 Enhances the FcεRI-Mediated Signaling Pathways in
IgE/Ag-Activated RBL-2H3 Cells. PTX3 contributes to cell proliferation in a cell type-dependent manner [29]. Meanwhile, the proliferation of mast cells has also been suggested to be involved in the enhancement of proallergic activity [30]. Therefore, we were interested in examining the involvement of PTX3 in the proliferation of mast cells. The results showed that PTX3 had no effect on the proliferation of RBL-2H3 cells (Figure 3(a)). Furthermore, the increase in and activation of de novosynthesized FcεRI and FcεRI downstream signaling, respectively, have been suggested to be involved and play a critical role in the IgE-mediated allergic reaction in mast cells [31]. We first tested whether PTX3 increases the level of de novo-synthesized FcεRI mRNA. The results showed that PTX3 had no effect on increasing FcεRI mRNA (Figure 3(b)). We next tested whether PTX3 contributes to the activation of mast cells by enhancing FcεRI downstream signaling, including Akt, ERK, p38, and JNK [10]. The results showed that PTX3 could enhance the activity of Akt, ERK1/2, p38, and JNK1/2 in IgE-stimulated RBL-2H3 cells (Figure 3(c)). These results suggest that PTX3 is able to augment the activation of FcεRI-mediated signaling pathways.  7 Journal of Immunology Research major allergen for shrimp-allergic populations. In addition, pretreatment with tropomyosin peptides that strongly inhibit IgE could attenuate shrimp extract-induced hypersensitization [32]. We further assessed whether the serum of tropomyosin peptide-sensitized mice could attenuate shrimp extractinduced hypersensitization. Following the procedure (Figure 4(a)), we demonstrated a modulation effect of short fragment peptides of tropomyosin (hypoallergens) in the attenuation of shrimp extract-induced hypersensitization (Figure 4(b)). Furthermore, we assessed the levels of PTX3 in the serum of shrimp extract-or tropomyosin peptidesensitized mice. We found that the levels of PTX3 were positively correlated with the levels of IgE in the serum from shrimp extract-(Spearman ' s correlation = 0:8601 and p = 0:0003) or tropomyosin peptide-sensitized (Spearman ' s correlation = 0:7972 and p = 0:0019) mice (Figure 4(c) and Supplementary Figure 1), suggesting that PTX3 may be involved in IgE-mediated allergic reaction. To develop an in vitro model to mimic the immune response of shrimpinduced allergies, the serum from experimental mice was harvested to treat RBL-2H3 cells. Moreover, there were positive correlations among β-hexosaminidase activity, expression of several proinflammatory mediators, including TNF-α, IL-4, and IL-13, and the Akt and MAPK signaling pathways in RBL-2H3 cells challenged with the serum from shrimp extract-or tropomyosin peptide-sensitized mice (Figures 4(d)-4(f)). However, IL-6 and COX-2 expressions in this in vitro system showed no significant change (Figure 4(e)). Taken together, in addition to suggesting that PTX3 could be a diagnostic candidate in shrimp-induced hypersensitization, the results also imply that the inactivation of PTX3 could attenuate shrimpinduced hypersensitization.

CEBPD Is Involved in PTX3-Induced Degranulation and Formation of Proinflammatory Mediators in Mast Cells.
Allergen-reactive T helper 2 (Th2) cells have been demonstrated to play a triggering role in allergic inflammation. Interestingly, our previous research has showed that CEBPD can be activated and function not only in M1-like macrophages [20] but also in M2-like macrophages [33], which mirrors Th1 and Th2 responses, respectively. Additionally, our previous studies have also indicated that PTX3 is a downstream target of CEBPD and that the CEBPD/PTX3 axis is involved in some inflammation-related diseases and even in the tumor progression [16,20,34]. However, whether CEBPD is reciprocally   Journal of Immunology Research regulated by PTX3 and contributes to PTX3-induced shrimp hypersensitization remain unexplored. Here, a shrimp allergy system was tested in Cebpd-deficient (Cebpd -/-) mice via the intraperitoneal injection of shrimp extract to induce sensitization and hypersensitization. ELISA analysis revealed that the loss of Cebpd in mice suppressed shrimp extract-induced total IgE in the serum (Figure 5(a). Surprisingly, the PTX3 concentration was still significantly high in the serum during the hypersensitization phase in Cebpd -/mice ( Figure 5(b)), implying that PTX3 may not be regulated by CEBPD in shrimp allergy. The results prompted us to further examine whether CEBPD is activated by PTX3 and contributes to shrimp allergy. To check the regulation of CEBPD in response to PTX3, IgE/DNP, or allergic serum in mast cells, the expression of CEBPD mRNA and protein were investigated. Upon PTX3 treatment, the CEBPD transcripts and proteins was significantly increased in RBL-2H3 cells (Figures 5(c) and 5(d)). Moreover, RBL-2H3 cells were exposed to PTX3 or not and then stimulated with DNP. The results showed that CEBPD levels were significantly increased in IgE/DNP-stimulated RBL-2H3 cells and were further enhanced under PTX3 treatment ( Figure 5(e)). In addition, CEBPD levels were also increased in allergic serum-activated RBL-2H3 cells ( Figure 5(f)). The PTX3-induced proinflammatory mediators and β-hexosaminidase were attenuated by the knockdown of CEBPD (Figures 5(g) and 5(h)). These results suggest that PTX3-induced degranulation, and the production of proinflammatory mediators is mediated by CEBPD in mast cells.
Following successful demonstration of the suppression effect of RI37 peptides in in vitro system, the effect of RI37 peptides on shrimp extract-induced allergic responses in vivo was examined. The levels of IgE were not significantly different between the negative control and RI37 treatment groups (Figure 6(i), compare group 1 with group 2). Contrary to the elevated levels of IgE in the serum of shrimp-allergic mice, IgE abundance was significantly attenuated in shrimp-allergic mice cotreated with RI37 peptides (Figure 6(i), compare group 3 with group 4). The results suggest that RI37 treatment could be applicable for the amelioration of shrimp allergen-induced inflammation.

Discussion
The prevalence of food allergies is rising, and the strategies for prevention and treatment are not optimal; therefore, the molecular mechanisms behind these allergic reactions require further examination. PTX3 has been suggested to play an 11 Journal of Immunology Research important role in innate immunity and in modulation of the adaptive immune response [35]. Although mast cells are a part of the innate immune system, they can also serve as effectors in the adaptive immune response following IgE/FcεRI interaction. However, the potential involvement and regulation of PTX3 in mast cells remain obscure. Herein, our study is the first evidence to link and evaluate the involvement of PTX3 in shrimp allergy. Moreover, PTX3 has been suggested to be responsive to proinflammatory cytokines, such as TNF-α and IL-1β, in epithelial cells. Our current study further demonstrated that PTX3 contributes to the expression of proinflammatory cytokines in mast cells. This finding raises the speculation that PTX3 can reciprocally regulate proinflammatory cytokines in mast cells.
Previous studies showed that surface FcεRI abundance in mast cells is increased following the increase in serum IgE (e) RBL-2H3 cells were preincubated with RI37 (150 and 300 ng/ml) for 30 min and then stimulated with PTX3 (300 ng/ml) for 30 min. Total lysates were harvested for western blot analysis using antibodies as indicated. The quantitative analysis of phosphorylated proteins is shown in the graph. (f) Serum-sensitized RBL-2H3 cells were preincubated with RI37 (150 and 300 ng/ml) for 30 min and further stimulated with shrimp extract for 15 min. Total lysates were harvested for western blot analysis using antibodies as indicated. The quantitative analysis of phosphorylated proteins is shown in the graph. (g, h) RBL-2H3 cells were preincubated with RI37 (300 ng/ml) for 30 min and further stimulated with PTX3 or allergic serum for 30 min. Total lysates were harvested for western blot analysis using antibodies as indicated. (i) Three-to-fourweek-old female BALB/c mice (n = 6 per group) were intraperitoneally sensitized and challenged with PBS or shrimp extract. Using Al(OH) 3

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Journal of Immunology Research concentration and that this effect may enhance the ability of mast cells to sense and bind to allergens during allergic responses [36]. In the present study, our observations showed that the level of FcεRI mRNA is unchanged in RBL-2H3 cells upon PTX3 treatment (Figure 3(b)). In addition, PTX3 has been suggested to bind to FcγRIIa and FcγRIII but not FcεRI [37]. These observations imply that PTX3 may enhance the activity of Akt, ERK1/2, p38, and JNK1/2 through an FcεRI-independent pathway. Moreover, accumulating evidence suggests that osteopontin (OPN) enhances IgE-mediated degranulation of mast cells through binding to CD44 [38] and that CD44 can activate the PI3K/Akt and MAPK/ERK pathways in leukemia cells [39]. Interestingly, CD44 may also be involved in PTX3induced tumorigenesis [16], suggesting that PTX3 could enhance the activity of Akt, ERK1/2, p38, and JNK1/2 through CD44 binding. In addition, the IgE/FcεRI axis triggers production of multiple cytokines, including TNF-α, which could further induce PTX3 production. In this study, our results also showed that, in mast cells, PTX3 can promote the formation of proinflammatory mediators, including IL-4 and IL-13 that have been demonstrated to induce B cell IgE production in a positive-feedback loop [40]. These findings indicate that PTX3 plays a positive role in the IgE-mediated activation of mast cells.
Collectively, in mast cells, upon exposure to allergen, PTX3 may be indirectly activated by TNF-α and enhance allergic responses through CD44 binding in an FcεRI-independent manner. However, this speculation needs further investigation. A recent study reported that CEBPD can induce PTX3 transcription and that the increase in PTX3 further participated in attenuation of the macrophage-mediated phagocytosis of damaged neurons [34]. Moreover, activation of p38 and JNK has been suggested to be involved in the activation of CEBPD transcription [41,42]. Moreover, CEBPD can be regulated or be reciprocally regulated by TNF-α, IL-1β, and IL-6 [43]. The pattern of CEBPD is similar to that of PTX3. Here, we demonstrated that PTX3 treatment can increase TNF-α and IL-6 gene expressions as well as the phosphorylation of MAPK signaling in IgE/Ag-activated RBL-2H3 cells. This finding supports PTX3's role in the enhancement of proinflammatory mediator production and implies that CEBPD could be a PTX3 downstream target in shrimp allergy. Indeed, this study identified CEBPD as a novel PTX3 target. Therefore, several factors, including PTX3 and CEBPD, could be regulated in a positivefeedback loop to enhance allergic inflammation.
It is not currently understood why some individuals develop allergic responses to allergenic foods more easily than healthy individuals, but evidence suggests that the tissue microenvironment is important [44]. It is generally believed that factors from the microenvironment can induce phenotypic changes in mast cells. In this study, we demonstrated that increased shrimp allergy severity is associated with an elevated PTX3 level in the serum. We also showed that PTX3 can augment the allergic responses of IgE/Ag-activated RBL-2H3 cells. These results imply that PTX3 could play a predominant role in the microenvironment for mast cell-dependent shrimp allergy. In addition, recent evidence suggests that prophylactic exposure to hypoallergens may be a promising prevention strategy for food allergy [32,45]. Peptide-based immunotherapy has been proposed as a safe and effective therapeutic strategy. Therefore, a hypoallergen with low/no IgE reactivity is desirable for peptide-based immunotherapy. Tropomyosin is a major allergen in most shrimp species and in other Crustacea species, dust mites, and cockroaches [46]. In this study, we demonstrated that the administration of tropomyosin peptides is able to alleviate the allergic responses to shrimp extract in mice with low PTX3 levels (Figure 4(c)). This finding implies that PTX3 may play a detrimental role under several allergic conditions caused by the panallergen tropomyosin.
Currently, the avoidance of food allergens or treatment of food allergies with antihistamines remains the standard of care. Allergy medications such as antihistamines act by the competitive inhibition of histamine at the H1 receptor on the effector cells [47]. However, antihistamines only control the symptoms of food allergy, and these agents are essentially palliative treatments. Moreover, the common side effects of this type of medication include drowsiness and dizziness through its action on the central nervous system. To address the underlying regulation in allergic immune responses and reduce the side effects of antihistamines, the increased understanding of the biology of mast cells gives rise to PTX3, providing a new target for the treatment of shrimp allergy.

Conclusions
Our data provide the first evidence that PTX3 is activated and contributes to shrimp allergy. The results also provide new insight to suggest that PTX3 participates in the cause of shrimp-allergic inflammation and that the promising application of RI37 peptides as a therapeutic agent.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.