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.2024 Apr;66(2):250-259.
doi: 10.1007/s12016-024-08994-4. Epub 2024 May 22.

Shrimp Extract Exacerbates Allergic Immune Responses in Mice: Implications on Clinical Diagnosis of Shellfish Allergy

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Shrimp Extract Exacerbates Allergic Immune Responses in Mice: Implications on Clinical Diagnosis of Shellfish Allergy

Wai Sze Tong et al. Clin Rev Allergy Immunol.2024 Apr.

Abstract

Tropomyosin has been identified as the major cross-reactive shellfish allergen, but recent studies showed the presence of other clinically relevant allergens. This study aims at determining the allergic immune responses of mice sensitized with raw and boiled shrimp extracts in comparison to recombinant tropomyosin (rTM). Female Balb/c mice were intragastrically sensitized and challenged with raw, boiled shrimp or rTM. Systemic, cellular and humoral allergic responses were compared, while allergenicity of the extracts was also compared by skin prick test (SPT) and immunoblot on shrimp allergic subjects. We showed that rTM and shrimp extracts induced IgE- and Th2-mediated allergic responses in mice, distinguished by remarkable intestinal inflammation in small intestine across all regimens. Notably, boiled shrimp extract exhibited the highest sensitization rate (73.7% of mice developed positive TM-specific IgE response) when compared with raw extract (47.8%) and rTM (34.8%). Mice sensitized with boiled extract manifested the highest allergen-specific IgE and Th2 cytokine responses than the others. Immunoblot results indicated that tropomyosin remained the major allergen in extract-based sensitization and had stronger allergenicity in a heat-treated form comparing to untreated TM, which was in line with the SPT results that boiled extract induced larger wheal size in patients. Hemocyanin and glycogen phosphorylase were also identified as minor allergens associated with manifestation of shrimp allergy. This study shows that boiled extract enhanced sensitization and Th2 responses in agreement with the higher allergenicity of heat-treated TM. This study thus presents three shrimp allergy murine models suitable for mechanistic and intervention studies, and in vivo evidence implies higher effectiveness of boiled extract for the clinical diagnosis of shellfish allergy.

Keywords: Food allergy; Mouse model; Shrimp allergy; Skin prick test; Tropomyosin.

© 2024. The Author(s).

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1
Experimental design of shrimp allergy mouse model. 3–4 weeks old BALB/c mice (n = 16–23 in each group) were sensitized intragastrically with recombinant tropomyosin (rTM), raw shrimp extract and boiled shrimp extract respectively using cholera toxin as adjuvant on days 0, 12, 19 and 26, followed by a 5-fold challenge on day 33. Mice fed with PBS served as negative control. Mice were sacrificed on day 34 that blood sample, small intestine and spleen were harvested for analysis
Fig. 2
Fig. 2
Allergic responses after intragastric challenge. (A) The responses of allergic symptoms: mice were evaluated 30 to 40 min after challenge and scored. The number of animals per group is showed in parentheses. (B) Images of representative reaction of negative control (PBS) and allergen (boiled extract) sensitized mice after challenge. Note the swelling of snout and mouth puffiness in the sensitized mouse but not in the PBS control mouse. Specific levels of IgE against (C) rTM, (D) raw shrimp extract and (E) boiled shrimp extract measured by ELISA. Relative mRNA expression of IL-4 (F IL-5), (G), IL-13 (H) and GATA-3 (I) of control and experimental groups (n = 8 per group). Data were normalized to HPRT-1. Data are expressed as mean ± SD. Data is considered statically significant when p < .05 on one-way ANOVA followed by Kruskal–Wallis test. * p < .05; ** p < .01; *** p < .001 and **** p < .0001
Fig. 3
Fig. 3
Mast cell infiltration in small intestine of sensitized and challenged animals. Representative sections of duodenum, jejunum and ileum (arrows indicate mast cells stained with chloroacetate esterase) of PBS control, rTM, raw and boiled extract groups. Quantification of mast cells in the three intestinal sections in control and experimental groups (n = 8) is shown in the bottom. Data are expressed as mean ± SD. Different letters indicate statistically significant differences. Data is considered statically significant when p < .05
Fig. 4
Fig. 4
Goblet cell metaplasia in small intestine mucosa of sensitized and challenged animals. Representative sections of duodenum, jejunum and ileum (arrows indicate goblet cells stained in Periodic Acid-Schiff) of control and experimental groups. Percentage of goblet cells over epithelium cells in the three intestinal sections are also shown (n = 8). Data are expressed as mean ± SD. Data is considered statically significant when p < .05. Different alphabets indicate statistically significant differences
Fig. 5
Fig. 5
Protein and allergen profile. (A) 40 µg raw shrimp extract, 8 µg boiled shrimp extract, and 2 µg recombinant tropomyosin (rTM) were resolved on 10% SDS-PAGE. Note the significant loss of proteins in the boiled preparation. Immunoblot was performed with serum pool (n = 8) from PBS controls, rTM-, raw extract- and boiled-extract sensitized and challenge mice against (B) rTM (the 46 kDa protein band of his-tagged tropomyosin expressed in pET30a), (C) raw shrimp extract and (D) boiled shrimp extract. Note that IgE binding was only detected against tropomyosin in all experimental groups. (E) Pool of sera from raw extract-sensitized mice with higher IgE level to raw extract than to rTM (n = 5) were incubated against raw extract, and identified other shrimp allergens including hemocyanin (80 kDa) and glycogen phosphorylase (100 kDa). L, protein marker (New England Biolabs) with reference molecular weight (kDa) indicated
Fig. 6
Fig. 6
Comparison of tropomyosin allergenicity. (A) Immunoblot performed with sera from TM-sensitized mice against 40 µg raw shrimp extract, 8 µg boiled shrimp extract or 2 µg rTM. Immunoblot performed with sera from seven shellfish allergic subjects (1–7) against (B) raw shrimp extract and (C) boiled shrimp extract. Numbers in the lanes in (C) indicate the relative intensity of the IgE-binding bands in boiled extracted in comparison to the respective bands in raw extract (relative intensity = 1). Note the remarkably enhanced allergenicity of heat-treated TM.(D) SPT wheal diameter (mm) of ten DBPCFC-confirmed shrimp allergic patients. Statistical difference was determined by paired t-test (p = .044)
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