The novelty of this study lies in its use of proteomics to explore factors related to FPIES pathogenesis up to symptom onset. FPIES is considered as triggered by specific antigens. After symptom onset, there is an increase in IL-17 levels7, indicating Th17 cell activation. However, the onset of FPIES symptoms within 1–4 h suggests that the initial response of FPIES may be due to an innate rather than a cellular immune response. Moreover, early ingestion of allergenic foods to prevent the development of IgE-mediated food allergies by inducing oral immune tolerance (induction of antigen-specific regulatory T cells) may conversely be related to a higher incidence of FPIES4,5. Further, the relatively early acquisition of tolerance18,19 compared with that in IgE-dependent allergy suggests that FPIES pathogenesis may be associated with abnormal innate immunity activation, while the T cell-mediated immune response may be a secondary event.
In our in-depth proteomic analysis, the levels of proteasome subunit- and neddylation-related proteins were significantly increased in the sera of the OFC-positive group before FPIES symptom onset, suggesting that pathways involving these proteins participate in symptom development. Both are involved in protein degradation and regulation of inflammation, suggesting that key pathway activation events occur early after exposure to the trigger food in FPIES patients.
The ubiquitin-proteasome system20, which labels unnecessary or defective proteins polyubiquitin for subsequent degradation to peptides via the proteasome, is responsible for selective non-lysosomal protein degradation. The biologically functional proteasome complex has been detected in normal human blood plasma/serum (known as the circulating proteasome) and is highly expressed in various diseases, including malignancies, autoimmune disorders, sepsis, and other conditions21. Notably, this is the first study to report the high expression of proteasomes in allergic disease.
Neddylation is a post-translational modification that occurs when NEDD8, a ubiquitin-like protein, is covalently bound to a target protein22. Neddylation is catalyzed by Cullin family and non-Cullin proteins. NEDD8 and neddylation-related proteins are often upregulated in various diseases, such as cardiac, metabolic, chronic liver, neurodegenerative, and immune-related diseases22. As with proteasomes, no reports exist on the high expression of neddylation-related proteins in allergic diseases; however, they play a regulatory role in inflammatory cytokine and interferon production during innate immune responses in various infectious diseases. Neddylation-related proteins may also be involved in the innate immune response to trigger food in FPIES.
Proteins related to neutrophil activation were upregulated in the serum and saliva after ingestion of the trigger food. In the OFC-positive group, we observed high expression of proteins related to neutrophil extracellular trap formation23 at symptom onset, which may trigger the previously reported increase in peripheral blood neutrophil count and activation after symptom onset. However, the high expression of proteins associated with neutrophil activation was also observed in blood and saliva samples of the OFC-negative group, suggesting that neutrophil activation may be inconsequential in the development of FPIES symptoms. Regardless, this is the first evidence in a proteomic study of neutrophil activation before the onset of symptoms.
The present study has some limitations. First, we compared positive and negative groups in the OFC using a specific quantity of trigger food (cooked egg yolk) rather than comparing healthy subjects and patients experiencing egg yolk-induced FPIES. This approach was ethically justified because it avoided oral challenge tests in healthy children and collecting blood and saliva samples. Although it was not feasible to examine protein variation in FPIES relative to healthy subjects, in-depth proteomic analysis allowed for the examination of a vast number of proteins, both in serum and saliva, revealing factors related to symptom induction. Second, MS-based proteomic analysis could not detect proteins of all molecular weights, such as cytokines and chemokines, owing to its limited measurement range, which precluded evaluation of the IL-17 inflammatory signature. Integrated analysis of the proteome using multiple advanced approaches for molecule and pathway analysis is needed to elucidate FPIES pathogenesis. Finally, this study was designed with a small sample size because only a few patients consented to collect both blood and saliva samples, as it is relatively invasive to take multiple blood samples from infants during OFC, when symptom induction can occur. However, as proteomic studies can be performed with high precision even with a small number of samples, we considered that our serum data do reveal the proteomic profiles of FPIES. Nevertheless, analysis of a larger sample is warranted to obtain detailed proteomic profiles and corroborate the results of the current study.
Taken together, our findings demonstrate that proteasome- and neddylation-related proteins were highly expressed before the onset of FPIES symptoms, which has not been previously reported. The presence or degree of this response to the trigger food may be related to FPIES onset, though further detailed investigation of their involvement in pathogenesis is warranted.