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.