In‐Depth Proteomic Profiles Prior to Symptom Development in Food Protein‐Induced Enterocolitis

Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-dependent food allergy that causes vomiting, extreme lethargy, marked pallor, diarrhoea and hypotension following ingestion of food allergens [1, 2]. Although cytometric and transcriptome analysis of peripheral blood after the onset of symptoms in patients with FPIES has revealed activation of systemic innate immune responses [3, 4], few studies have explored these immune responses at earlier time points. Therefore, in this study, we aimed to elucidate the pathogenic mechanisms underlying FPIES symptom development by examining serum and salivary proteomic profiles of individuals with FPIES before symptom onset. This article's Online Repository at Zenodo (https://doi.org/10.5281/zenodo.14736098) contains a full description of methods, results, including saliva analysis data, and discussion. We enrolled 17 patients previously diagnosed with egg yolk FPIES. The oral food challenge (OFC) test was performed by administering a single (5 g) dose of heated egg yolk. Whole blood was collected before OFC, at 1 and 2 h after egg yolk ingestion, and, when possible, at the onset of acute FPIES symptoms from nine participants who consented to both serum and saliva collection; eight participants consented only to saliva collection. We did not find any difference in the characteristics of OFC-positive and OFC-negative patients (Tables S1 and S2). We identified 4883 proteins via serum proteomic analysis and analysed 4138 after selecting those with at least two detected peptide counts. In subsequent group comparisons, the number of proteins for analysis was filtered to ~2000 to ensure that at least one group contained a minimum of 70% valid values for each protein. First, we assessed differences in protein levels between the OFC-positive and OFC-negative groups before the OFC (Figure S1). We found significant differences in only 1.3% (23 proteins) of the 1723 comparable proteins, suggesting that pre-OFC serum proteome profiles may not differ between OFC-positive and OFC-negative groups. Proteins with significantly altered expression 1 and 2 h after OFC and symptom onset compared with that before OFC in each group are shown in Figure S2. At 1 h after OFC, 25 proteins (15 upregulated and 10 downregulated) in the OFC-positive group (Figure S2A) and 34 proteins (29 upregulated and five downregulated) in the OFC-negative group (Figure S2D) were detected. Contrastingly, 2 h after OFC, 614 proteins (609 upregulated and five downregulated) were observed in the OFC-positive group (Figure S2B), and 559 proteins (549 upregulated and 10 downregulated) were observed in the OFC-negative group (Figure S2E). The number of proteins with altered expression decreased to 309 (304 upregulated and 5 downregulated) at symptom onset in the OFC-positive group (Figure S2C). The upregulated proteins overlapped 2 h after OFC in the OFC-positive group (Serum-UpPOS2), 2 h after OFC in the OFC-negative group (Serum-UpNEG2) and at symptom onset in the OFC-positive group (Serum-UpS) (Figure S2F). To further investigate the differences between Serum-UpPOS2 and Serum-UpNEG2, we performed a cluster analysis of enriched ontologies (Figure 1). Several terms and pathways, including neutrophil degranulation (R-HAS-6798695), were shared by the enriched ontologies in Serum-UpPOS2 and Serum-UpNEG2, suggesting that neutrophil degranulation may occur with or without symptom onset. The enriched ontologies in Serum-UpPOS2, but not in Serum-UpNEG2, included neddylation (R-HSA-8951664) and protein modification by small protein conjugation (GO: 0032446). In contrast, the enriched ontologies in Serum-UpNEG2, but not in Serum-UpPOS2, included focal adhesion (WP306) and platelet activation (hsa04611). The protein–protein interaction network and MCODEs of merged proteins of Serum-UpPOS2 and Serum-UpNEG2 (Figure S3) suggest that proteins contained in MCODE1 (PA700 [proteasome] complex, Table S3) and MCODE4 (neddylation, Table S3) were predominantly upregulated in Serum-UpPOS2 but not in Serum-UpNEG2. Similarly, volcano plots of differentially expressed proteins relative to the time before OFC indicated transient upregulation of proteasome subunits and neddylation-related proteins 2 h after OFC in the OFC-positive group (Figures S4, S5). To verify these results, we measured the levels of serum proteasome 20S and NEDD8—protein modification molecules involved in neddylation—using ELISA and compared their levels before and 2 h after OFC (Figure S6). Changes in both serum proteasome 20S and NEDD8 levels were greater in the OFC-positive than in the OFC-negative group (p < 0.05). To focus on disease pathogenesis at symptom onset, we performed enrichment analysis (GO terms and KEGG pathways) using Enricher-KG on 73 proteins in Serum-UpS but not in Serum-UpPOS2 and Serum-UpNEG2 (indicated by a white star in Figure S2F). We identified genes related to neutrophil extracellular trap formation (KEGG: hsa04613), neutrophil activation pathways such as neutrophil degranulation (GO: 0043312), neutrophil activation involved in the immune response (GO: 0002283) and neutrophil-mediated immunity (GO: 0002446) (Figure S7). We aimed to clarify the pathogenesis of FPIES symptoms based on the proteomic profiles of serum and saliva before symptom onset. We observed a transient increase in serum proteasome- and neddylation-related protein levels before the onset of FPIES symptoms, possibly triggering the activation of various innate immune cell types at symptom onset. Regardless of symptom onset, expression of proteins related to neutrophil activation was high in the serum and saliva prior to symptom onset, suggesting that initial neutrophil activation might only be associated with FPIES pathogenesis and not with symptom development. However, this activation may be prolonged and might exacerbate symptoms in patients who develop FPIES. Taken together, our findings demonstrate that proteasome- and neddylation-related proteins were highly expressed before the onset of FPIES symptoms, which have not been previously reported. The presence or degree of this response to the trigger food may be related to FPIES onset, although detailed investigation of their involvement in pathogenesis is warranted. Y.I. designed the study. H.S. and Y.Y. supervised the study. M.I. and Y.K. performed experiments. Y.I. and H.S. analysed the data. H.S., H.K., M.E., Y. Osaki, S.K., M.A., A.I., E.H., T.Y., T.N., S.T., Y. Okada, H.I., D.S., Y.T., M.S., M.T., and Y.Y. provided the patient samples and clinical information. Y.I. wrote the manuscript. All authors read and approved the final manuscript. We would like to thank Editage [http://www.editage.com] for editing and reviewing the language of this manuscript. This work was supported by a Grant from the Nipponham Foundation for the Future of Food. The authors declare no conflicts of interest. The data that support the findings of this study are openly available in Zenodo at https://doi.org/10.5281/zenodo.14736098. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Table S1. Table S2. Table S3. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.