Fig 1.
Allergen identification in shrimp allergen extract (SAE).
A total of ten known allergens were identified in SAE. TPM, the primary shrimp allergen, is recognized in its isoforms.
Table 1.
Sites and types of TPM modifications in SAE identified using proteomic approach.
Fig 2.
Shrimp allergen extract (SAE) immunotherapy affects systemic allergy reactions in allergic mice.
(A) Experimental protocol. (B) The desensitized mice after SAE immunotherapy on days 53 and 58. Systemic allergy symptoms were observed for 30 minutes following challenge with 400 μg SAE (i.g.). SAE immunotherapy reduced systemic allergy symptoms in allergic mice on day 53 and showed a sustained effect until day 58. Each bar represents the mean ± SEM (n = 6 mice per group). p-values were derived from Kruskal-Wallis test (####, p≤0.0001; ###, p≤0.001; ##, p≤0.01 were significant against Untreated and ***, p≤0.001; **, p≤0.01; *, p≤0.05 were significant against NC).
Fig 3.
Shrimp allergen extract (SAE) immunotherapy preventing the development of allergies.
Sensitized mice, but not the NC group, were treated (i.p.) with high- to low-dose immunotherapy (10 μg SAE for LI, 50 μg SAE for MI, and 100 μg SAE for HI). All mice were euthanized on day 59; blood serum and ileum tissues were collected and stored at −80°C for further analysis. SAE desensitization successfully decreased transcription of (A) FcεR1α mRNA (n = 6 mice per group) and (B) IL-4 mRNA—relative to NC—in the ileal tissue (n = 3 mice per group), and suppressed the production of the pro-inflammatory cytokine (C) IL-4 in serum (n = 6 mice per group). Each bar represents the mean ± SEM. p-values of FcεR1α and IL-4 mRNA expression were derived from one-way ANOVA test (###, p≤0.001; ##, p≤0.01; were significant against Untreated and ***, p≤0.001; **, p≤0.01; *, p≤0.05 were significant against NC) while p-values of IL-4 serum levels were derived from Kruskal-Wallis test (#, p≤0.05 were significant against Untreated and ****, p≤0.0001; *, p≤0.05 were significant against NC).
Fig 4.
Shrimp allergen extract (SAE) immunotherapy affects mast cell (MC) degranulation in allergic mice.
Sensitized mice, but not the NC group, were treated (i.p.) with high- to low-dose immunotherapy (10 μg SAE for LI, 50 μg SAE for MI, and 100 μg SAE for HI). All mice were euthanized on day 59; ileum tissues were collected and preserved in Carnoy’s solution. Degranulated MCs were stained using 0.5% toluidine blue (TB), and (A) the percentage of degranulated MCs in the intestinal tissues were determined. Each bar represents the mean ± SEM (n = 6 mice per group). p-values were derived from one-way ANOVA test (####, p≤0.0001 were significant against Untreated and ****, p≤0.0001; ***, p≤0.001; **, p≤0.01; were significant against NC). (B) Representative MCs in ileum specimens (1000× magnification). Degranulated MCs have blurred cell membrane boundaries and increased cell membrane shrinkage and granules scattered around the cells (red arrow). Intact MCs have several viscous intracellular granules that stain intensely with TB and appear violet in the cytoplasm (black arrow).
Fig 5.
Shrimp allergen extract (SAE) immunotherapy inducing allergy tolerance.
Sensitized mice, but not the NC group, were treated (i.p.) with high- to low-dose immunotherapy (10 μg SAE for LI, 50 μg SAE for MI, and 100 μg SAE for HI). All mice were euthanized on day 59; ileum tissues were collected and stored at −80°C for further analysis. SAE desensitization successfully increased transcription of (A) Foxp3 mRNA (n = 6 mice per group) and (B) IL-10 mRNA (n = 3 mice per group)—relative to NC—in the ileal tissue. An increased (C) IL-10/ IL-4 mRNA ratio indicates a shift towards a stable tolerogenic phenotype following SAE desensitization. Each bar represents the mean ± SEM. p-values were derived from one-way ANOVA (##, p≤0.01; were significant against Untreated and ****, p≤0.0001; ***, p≤0.001; *, p≤0.05 were significant against NC).
Fig 6.
Schematic presentation of SAE immunotherapy-induced allergy tolerance in shrimp allergy.
During the desensitization phase, SAE administration increases systemic allergen concentration, shifting the Treg/Th2 immune response. Subsequently, tolerogenic DC activates Treg cells through the transcription factor Foxp3. Activation of Treg cells induces IL-10 production, which inhibits IgE production from B cells, suppresses ILC2 activation via alarmins, and prevents cross-linking formation. This ultimately reduces MC degranulation and the release of pro-inflammatory cytokines by MCs.