Background
Food Protein-Induced Enterocolitis Syndrome (FPIES) is a food allergy affecting predominantly infants, with cow’s milk (CM) being the most common causative food. Symptoms depend on frequency of food exposure [
1,
2]. Chronic exposure to CM results in chronic symptoms including diarrhoea and failure to thrive (FTT). Symptoms improve after CM exclusion and re-exposure leads to a typical acute digestive form including repetitive protracted vomiting starting 1–4 h after CM ingestion, often accompanied by pallor and lethargy [
3]. The acute form of FPIES differs from immediate IgE-dependent allergy, notably because there is no involvement of the skin or respiratory tract. In addition, skin prick testing and specific IgE levels are usually negative, although sensitization to the triggering food has been reported in some patients (atypical FPIES) and specific IgE to other foods may be present [
1,
3‐
5]. Resolution of FPIES occurs at different ages depending on the food and the geographical regions considered, with children presenting IgE sensitization at higher risk of a protracted course [
4‐
7].
The pathogenesis of FPIES remains poorly described and no biomarkers are available [
1]. Although a Th2-biased immunopathological response has been suggested, evidence of specific T cell activation is still lacking [
1,
2,
8]. Recent data suggest a role of systemic innate cells in mediating clinical reactivity [
9]. The paucity of IgG, IgG4 and IgA humoral response against casein was evidenced in patients with active FPIES vs resolved FPIES [
10]. However, these analyses were based on responses to CM and casein only, whereas other CM components were not studied, and other IgG subtypes or isotypes such as IgM or IgD were not analyzed. Moreover, the reactivity of neo-epitopes produced during gastroduodenal digestion was not assessed.
A further in-depth characterization of circulating cells and antibodies in patients with FPIES would improve our understanding of the pathogenesis of FPIES and could be useful for the development of new diagnostic tools. Moreover, processes such as glycolysis, fatty acid and mitochondrial metabolism are now recognized as crucial players in immune responses [
11,
12]. Pathophysiological changes associated with FPIES could then translate into a global modification of the plasma metabolome, providing a specific metabolomic signature that may help identify specific biomarkers. We investigated in depth the humoral and cellular immune response in FPIES and performed a non-targeted metabolomic analysis on plasma. We hypothesized that humoral and cellular responses are weak in FPIES and that FPIES is characterized by a specific metabolomic signature.
Methods
Subjects
In this descriptive study, children with a CM-FPIES were compared with age-matched control subjects. Children aged up to 10 years previously diagnosed with CM-FPIES [
1,
2] were recruited in our day care unit on the day of an open food challenge (OFC) with CM performed to evaluate natural resolution of CM-FPIES. All children with CM-FPIES had a history of chronic symptoms including diarrhoea, low weight gain and/or emesis that resolved within days on CM avoidance. All of them experienced at least one typical acute FPIES episode including recurrent emesis with pallor, lethargy and dehydration with or without hypotension, occurring 1–4 h after CM ingestion, and requiring intravenous fluid perfusion in an emergency department. Exclusion criteria were a history of immediate (< 2 h) reaction, reactions with skin or respiratory tract involvement, positive SPT to CM or CM specific IgE level > 0.1 KuA/L. Children with IgE-mediated CM allergy (IgE-CMA) were included as control subjects on the day of an OFC with baked milk, performed to evaluate baked milk tolerance. Children with IgE-CMA had a clear history of immediate reaction (< 1 h) occurring after CM ingestion, with CM specific IgE levels > 0.10 kuA/L and positive skin prick test to CM (wheal > 3 mm). In those patients, OFC was not mandatory to confirm CMA [
13]. All of them had recent immediate reaction following ingestion of raw CM, within the 3 months before the OFC to baked milk was performed.
Blood samples were taken before the OFC. Three other patients initially recruited as IgE-CMA children finally show no more clinical history of CM allergy, with no immediate reaction noticed after ingestion of CM, and where then considered as become tolerant (IgE-resolved). Additional plasma samples from children with an IgE-mediated peanut allergy were used as controls from allergic subjects tolerating CM.
Reagents
\(\upbeta\)-Lactoglobulin (BLG), whole casein (cas) and its 4 constituents (
\(\upalpha\)s1-cas,
\(\upalpha\)s2-cas,
\(\upbeta\)-cas,
\(\upkappa\)-cas),
\(\upalpha\)-lactalbumin (
\(\upalpha\)-lact) and lactoferrin (LF) were purified from raw CM and characterized as previously described [
14‐
17]. Commercial infant formulas Pregestimil (extensive hydrolysate of caseins, Mead Johnson, Nijmegen, Netherlands), Pepti-junior (extensive hydrolysate of whey proteins, Laboratoires Picot, Laval, France) and Neocate (amino acid formula, SHS International, Liverpool, UK) were locally purchased.
Blood collection, PBMC and plasma separation
Blood samples were collected in BD Vacutainer® sodium heparin tubes (BD, Le Pont de Claix, France), kept at room temperature and processed within 3–4 h. Peripheral Blood Mononuclear Cells (PBMC) were obtained from peripheral blood diluted 1:2 in AIM V® Serum Free Medium (Thermo Fisher Scientific, Waltham, USA) by using Histopaque®-1077 (Sigma Aldrich, St Louis, USA) following the provider’s recommendations. After centrifugation (400×g, 30 min, + 20 °C), plasma was collected, aliquoted and kept at − 20 °C for antibody and metabolomics analysis. PBMC were collected, washed several times with PBS-EDTA 2 mM and finally suspended in RPMI-1640 medium supplemented with 5% autologous plasma, 2 mM l-glutamine, 100 U penicillin, 100 µg/mL streptomycin (All from GIBCO®, Thermo Fisher Scientific, Waltham, USA) for cellular analysis.
Analysis of humoral response
Total Ig
Concentrations of total IgE, IgG1, IgG2, IgG3, IgG4, IgM and IgA were determined using BioPlex Pro™ Human Isotyping Panel assays and BioPlex200 apparatus from Bio-Rad (Marnes-la-Coquette, France), following the provider’s recommendations.
Specific Ig
Specific antibodies against CM allergens were analyzed by using the direct Enzyme AllergoSorbant Test (EAST) as partially described in [
17,
18]. Assays were performed on allergen-coated 96-well microtitre plates (Immunoplate Maxisorb
®, Nunc, Roskilde, Denmark), using AutoPlate Washer and Microfill dispenser equipment from BioTek instruments (Avantec, Rungis, France). Pre-selected and labelled (biotinylated or acetylcholinesterase-labelled) anti-human IgE (clone BS17, [
19]), or anti-human IgG1 (clone JDC1), IgG2 (clone HP6002), IgG3 (clone HP6050), IgG4 (clone HP6023), IgD (clone IADB6) and IgM (clone UHB) (all from Southern Biotech, Birmingham, AL, USA), or anti-human IgA (AffiniPure F(ab’)
2 Fragment, Jackson ImmunoResearch Laboratories, Wets Grove, USA) were used for staining. Preliminary experiments allowed selection of these antibodies, based on their specificity and sensitivity using purified standard isotypes. Biotinylation was performed in 20 mM borate buffer using an antibody:biotin molar ratio of 40 (EZ-link
® Sulfo-NHS-LC-LC-biotin, Thermo Scientific). Labelling of streptavidin or direct labelling of antibodies with acetylcholinesterase (AChE) was performed as previously described [
19,
20]. Solid phase-bound AChE activity was determined by addition of 200 µL/well of Ellman’s reagent as an enzyme–substrate and the absorbance was measured at 414 nm using automatic reader plates (MultiskanEx, Thermo Electron Corporation, Vantaa, Finland). Plasma was tested at 3 dilutions, from 1/2 to 1/200 depending on the isotype. A signal greater than the mean of nonspecific binding + 3σ (obtained with buffer instead of plasma) was considered positive. Standard curves obtained with anti-human IgE [
19] or anti-human IgG (F(ab′)2 Fragment specific, Pierce
®, Thermo Scientific, Rockford, USA) coated plates and standard human IgE (World Health Organization; concentrations ranging from 10 to 0.08 IU/mL) or commercial isotype standard IgGs (all from AbD-Serotec, Bio-Rad, concentrations ranging from 1 µg/mL to 50 ng/mL) were used as reference to quantify antibody concentrations. Specific IgE against CM purified proteins were also assayed using reverse EAST, a more sensitive method where IgE were first captured on anti-human IgE coated plates and labelled CM-allergens were used for staining [
21].
Additionally, specific IgE and IgG4 were analyzed against hydrolyzed proteins. Raw milk was digested under “physiological conditions” using pepsin and then trypsin/chymotrypsin, as previously described [
22,
23]. Pepsin (from porcine gastric mucosa, 3440 U/mg of protein calculated using haemoglobin as substrate; Sigma-Aldrich) was used at an enzyme-to-substrate ratio of 172 U/mg, and trypsin (from bovine pancreas, 11,886 U/mg of protein calculated using BAEE as substrate, Sigma-Aldrich) and chymotrypsin (
\(\upalpha\)-type VII from bovine pancreas, 52 U/mg of protein calculated using BTEE as substrate, Sigma Aldrich) were added at an enzyme-to-substrate ratio of 34.5 U/mg and 0.4 U/mg, respectively. Hydrolysates were passively immobilized on microtitre plates and specific IgE and IgG4 stained as for specific Ig.
IgE immunoblot
SDS-PAGE and IgE immunoblot analyses of defatted CM were performed under reducing conditions using reagents and recommendations from the provider (Invitrogen, Life Technologies, Carlsbad, USA). Defatted CM and molecular weight markers (Novex® Sharp prestained protein standard) were loaded on NuPage Novex Bis–Tris Gels. Electrophoresis was performed using XCell SureLock Mini-Cell with a constant voltage of 200 V for 40 min. After electrophoresis, gels were stained with GelCode Blue Stain Reagent (Pierce, Thermo Scientific) or proteins were transferred to PVDF membranes (Hybond-P, GE-Healthcare Life Sciences) for 90 min at 25 V using a XCell II blot module. Membranes were saturated for 1 h at 20 °C with TBST (20 mM Tris, pH 7.6, 0.25 M NaCl, 0.5% Tween) supplemented with 5% BSA (Sigma-Aldrich). Plasma diluted 1:4 were incubated with slight shaking for 18 h at 4 °C. After several washings with TBST, secondary antibody (goat anti-human IgE peroxidase conjugated STAR147P, AbDSerotec-Bio-Rad) was incubated with slight shaking for 2 h at 20 °C. Membranes were then revealed with ECL Prime Western blotting detection reagent (GE-Healthcare Life Sciences) for 5 min and then analyzed using ChemiDoc™Touch Imaging System from Bio-Rad.
Analysis of cellular immune response
In vitro reactivation of PBMC
After isolation of PBMC, cell count was performed using TC-10 apparatus (Bio-Rad, Marnes-la-Coquette, France). Cellular concentration was adjusted to 1 × 10
6 cells/mL and 225 µL/well was added to 96-well culture plates. Purified proteins (final concentrations 10 and 50 µg/mL) or infant formula (final protein concentrations 500 and 100 µg/mL) was then added, and cell cultures were incubated for 6 days at 37 °C in a humidified 5% CO
2 atmosphere. LPS content in purified proteins and formulas was checked using the Pierce™ LAL chromogenic endotoxin quantification kit (Thermo Fisher Scientific), following the provider’s recommendations. LPS contents in BLG,
\(\upalpha\)s1-cas,
\(\upbeta\)-cas,
\(\upkappa\)-cas and LF were below 30 pg/mg of protein. LPS contents in whole caseins and
\(\upalpha\)-lact was 70 pg/mg of protein, and 125 pg/mg for
\(\upalpha\)s2-cas. Endotoxin levels were then considered as acceptable taking into account the amount of protein added during in vitro reactivation and the amount of endotoxin that will not induce non-specific activation of PBMC [
24]. Pregestimil, Pepti-junior and Neocate solutions contained respectively 135, 215 and 205 pg of endotoxin/mg of protein. Medium alone (PBS) was used as a negative control, whereas phytohaemagglutinin (PHA-L, lectin from
Phaseolus vulgaris, Sigma Aldrich, St Louis, USA) or bacterial lipopolysaccharide (LPS from
E. coli serotype 0127:B3, Sigma Aldrich) was used as positive control (1–10 µg/mL). In some experiments, cells were stained with 1 µM CFSE (CFSE Cell Division Tracker kit, Biolegend, San Diego, USA) following the provider’s recommendation before reactivation.
Cytokine production analysis
After in vitro reactivation, plates were centrifuged and supernatants collected, aliquoted and kept at − 80 °C until further assay. Cytokines (IL-1\(\upbeta\), IL-2, IL-3, IL-5, IL-6, IL-10, IL-13, IL-17A, IFN\(\upgamma\), TNF-\(\upalpha\)) were assayed using BioPlex Pro™ Human cytokine kits and BioPlex200® apparatus, following the provider’s recommendations (Bio-Rad, Marnes-la-Coquette, France).
Flow cytometry analysis
In some patients, cultured cells obtained after in vitro reactivation were suspended in PBS Ca
−/Mg
−, 2 mM EDTA, 5% heat-inactivated foetal calf serum (FCS) and extracellular and intracellular labelling of Th and Treg cells was performed using optimized antibody panels. For Th cells, analysis was performed as described in [
25], using anti-human CD4 (clone OKT4, Brilliant Violet 785™), CD25 (clone M-A053, PE/Dazzle™ 594), CD45RA (Clone HI100, Brilliant Violet 510™), CCR6 (CD196, clone G034E3, PE-Cy7), CXCR3 (CD183, clone G025H7, Brilliant Violet 421™) and CCR4 (CD194, clone L291H4, Brilliant Violet 605™); for Treg analysis, we used anti-human CD3 (clone UCHT1, Brilliant Violet 605™), CD25 (clone M-A251, Brilliant Violet 421™), CD4 (clone RPA-T4, Brilliant Violet 785™), Helios (clone 22F6, PE) and Foxp3 (clone 206D, Alexa Fluor
® 647). All antibodies were from Biolegend (San Diego, USA). Intracellular labelling of Foxp3 and Helios was performed after fixation/permeabilization using the Foxp3 staining kit from Miltenyi Biotec (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the provider’s recommendations.
Blood and biopsies of the jejunum, sigmoid and rectum were obtained from two brothers suffering from FPIES, and not initially included in our cohort: one resolved under a strict elimination diet (Neocate; age 38 months), the other one had an active FPIES with typical acute symptoms few days before the endoscopy, after ingestion of wheat and corn. Plasma and PBMC were isolated as above. Both demonstrated plasma humoral responses in line with that observed in our CM-FPIES population (not shown). Biopsy material was immediately placed in tissue storage solution (Miltenyi Biotec GmbH) and processed within 24 h. Biopsy material was washed in RPMI-1640 medium and tissue was digested using Liberase™ (Research Grade, 1 mg/mL, Roche Diagnostics GmbH, Sigma-Aldrich) and DNase I (0.02 mg/mL, Invitrogen, Life Technologies) for 45 min at 37 °C followed by mechanical dissociation using gentleMACS® C tubes and the gentleMACS™ Dissociator (Miltenyi Biotec GmbH). After washing, cells were suspended in PBS, 5% FCS, 2 mM EDTA and staining for flow cytometry was performed as above and using the following reagents and antibodies for viability assessment and extracellular or intracellular labelling: Fixable Yellow Dead Cell Stain Kit (Thermo Fisher Scientific), lineage (lin; anti-human CD3, CD11c, CD14, CD16, CD19, CD56 (NCAM), Fc\(\upvarepsilon\)RI\(\upalpha\), CD1a, CD123; APC-Vio770™), and anti-human CD127 (IL-7R\(\upalpha\), PE-Vio615™), CD4 (VioGreen®), CD45 (PerCP-Vio700™), CD294 (CRTH2; PE-Vio770™), T-bet (PE), ROR\(\upgamma\)t (APC), GATA3 (FITC) all from Miltenyi Biotec and anti-human-IL-13 (BV711, BD Biosciences, Le Pont de Claix, France), anti-human IL-22 (eFluor 450, Affymetrix eBiosciences, Thermo Fisher Scientific), anti-human-IFN\(\upgamma\) (BD Bioscience).
Approximately 70,000 cells were collected using a NovoCyte flow cytometer (ACEA Bioscience, Inc.) and analysis was performed using NovoExpress™ Software (Version 1.2.1, ACEA Biosciences, Inc.). Samples were first inspected in all light scatter patterns and fluorescence channels to confirm quality and abnormal cells (dead cells, aggregates…) were excluded. Each acquisition contained unlabelled samples, single-stained cells and/or an FMO strategy for reporting percentage of positive cells and compensation.
Statistical analysis for humoral and cellular analysis
Non-parametric tests were performed using the Mann–Whitney t test (comparison between specified groups), the Kruskal–Wallis test and Dunn’s multiple comparison post-test to compare all the groups together, or the Wilcoxon sign rank test (comparison of different treatments/Assays for patients in the same group). A p value < 0.05 was considered to be significant. All statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA).
Metabolites were extracted from 50 µL of plasma as previously described [
26]. Briefly, for each sample, 2 aliquots of 50 μL of plasma were treated with 200 μL of methanol, vortexed for 20 s and left on ice for 30 min to allow protein precipitation. Samples were then centrifuged for 20 min at 15,000×
g. Supernatants were collected and dried under nitrogen. Dried extracts were dissolved in 150 µL of H
2O/ACN (95/5%) for C18 analysis or ammonium carbonate 10 mM pH 10.5/ACN (40/60%) for HILIC analysis.
Extracts were analyzed by liquid chromatography (LC) coupled to mass spectrometry, as previously described [
26] using a Dionex Ultimate chromatographic system coupled to an Exactive (Orbitrap) mass spectrometer from Thermo Fisher Scientific (Courtaboeuf, France) fitted with an electrospray source operated in the positive and negative ion modes. The software interface was Xcalibur (version 2.1) (Thermo Fisher Scientific, Courtaboeuf, France).
Ultra high-performance LC (UHPLC) separation was performed on a Hypersil GOLD C18 (1.9 μm, 2.1 mm × 150 mm) column at 30 °C (Thermo Fisher Scientific, les Ulis, France). Mobile phases for reverse phase columns were 100% water in A and 100% ACN in B, both containing 0.1% formic acid. Chromatographic elution was achieved with a flow rate of 500 μL/min. After injection of 10 μL of sample, elution consisted of an isocratic step of 2 min at 5% phase B, followed by a linear gradient from 5 to 100% of phase B for the next 11 min. These proportions were kept constant for 12.5 min before returning to 5% B for 4.5 min. The column effluent was directly introduced into the electrospray source of the mass spectrometer and analyses were performed in the positive ion mode. Source parameters were as follows: droplet evaporation temperature 280 °C; capillary voltage, 5 kV; sheath gas pressure and the auxiliary gas pressure, respectively at 60 and 10 arbitrary units with nitrogen gas; mass resolution power, 50,000 m/Δm; full width at half maximum (FWHM) at m/z 200, for singly charged ions; detection from m/z 85 to 1000.
The high-performance LC (HPLC) separation was performed on a Sequant ZICpHILIC column (5 μm, 2.1 × 150 mm) at 15 °C (Merck, Darmstadt, Germany). Mobile phase A consisted of an aqueous buffer of 10 mM ammonium carbonate pH 10.5, and mobile phase B of 100% ACN. Chromatographic elution was achieved with a flow rate of 200 μL/min. After injection of 10 μL of sample, elution started with an isocratic step of 2 min at 80% B, followed by a linear gradient from 80 to 40% of phase B from 2 to 12 min. The column effluent was directly introduced into the electrospray source of the mass spectrometer, and analyses were performed in the negative ion mode. Source parameters were as follows: droplet evaporation temperature, 280 °C; capillary voltage, − 3 kV; sheath gas pressure and the auxiliary gas pressure, respectively at 60 and 10 arbitrary units with nitrogen gas; mass resolution power, 50,000 m/Δm; full width at half maximum (FWHM) at m/z 200, for singly charged ions; detection from m/z 85 to 1000.
Data processing and statistical analysis
Data processing workflow and statistical analyses were performed on the open web-based platform workflow4metabolomics (W4M:
http://workflow4metabolomics.org), a collaborative research infrastructure for computational metabolomics [
27]. Automatic peak detection and integration were performed using the matched filter algorithm in the W4M pre-processing package (including XCMS software). All raw data were manually inspected using the Qualbrowser module of Xcalibur, while the Quanbrowser module was used for peak detection and integration of internal standards. To remove analytical drift induced by clogging of the ESI source observed in the course of analytical runs, chromatographic peak areas of each variable present in the XCMS peak lists were normalized using the LOESS algorithm (W4M package). Features generated from XCMS were filtered according to the following criteria: (i) the correlation between QC dilution factors and areas of chromatographic peaks (filtered variables should have coefficients of correlation above 0.7 to account for metabolites occurring at low concentrations and which are no longer detected in the most diluted samples) (ii) repeatability (the coefficient of variation obtained on chromatographic peak areas of QC samples should be below 30%), and (iii) ratio of chromatographic peak areas of biological to blank samples above a value of 3.
Statistical analyses were performed with W4M (multivariate and univariate statistical tests), Simca P (multivariate PLS-DA models) or Prism (univariate tests) software tools. The discriminant metabolites were selected by combining multivariate variable importance in the projection (VIP) obtained from the PLS-DA model and univariate p values (nonparametric Mann–Whitney statistical test). The metabolites were considered as discriminant when VIP > 1.5 and p value < 0.1.
Feature annotation was performed considering a ± 10 ppm mass tolerance and using our in-house spectral database [
26,
28], as well as the publicly available databases KEGG [
29], HMDB [
30] and METLIN [
31]. To be identified, ions had to match at least two orthogonal criteria among accurate measured mass, isotopic pattern, MS/MS spectrum, and retention time; and to those of an authentic chemical standard analyzed under the same analytical conditions, as proposed by the Metabolomics Standards Initiative [
32]: level 1 (identified): based on accurate mass, column retention time similarity with a standard and MS/MS spectrum. Level 3 (putatively characterized): based on accurate mass, and interpretation of MS/MS spectra. Metabolite identification was further confirmed by additional LC/ESI–MS–MS experiments, performed using a Dionex Ultimate chromatographic system combined with a Q-Exactive Plus mass spectrometer (Hilic) or a Fusion mass spectrometer (C18), under nonresonant collision-induced dissociation conditions using higher-energy C-trap dissociation (HCD), at normalized collision energies (NCEs) 10, 20, 40 and 80%.
Discussion
In this study, we show that both humoral and cellular responses to relevant CM components are poor in children with CM-FPIES. In these patients, the levels of total and specific Ig differed from those of both milk-tolerant peanut allergic children and children with IgE-CMA who were avoiding milk. Thus, these differences cannot be related to CM consumption or avoidance. Moreover, we found no specific IgE against polypeptides derived from gastric and gastroduodenal digestion of CM proteins. This finding does not support the hypothesis that patients with FPIES have specific IgE against neo-epitopes generated during gastroduodenal digestion. Our findings confirm previous data showing a poor humoral response in FPIES, and extend these results to other CM components and their digestion products, and to all Ig types [
10,
33,
34]. Our findings also further support that this low level of humoral response is a feature of the disease itself, and is not dependant on milk avoidance.
Interestingly, IgG4 binding to gastroduodenal digestion products of CM proteins differed between children with CM-FPIES and IgE-CMA. We can therefore speculate that IgG4 epitopes might differ between CM-FPIES and IgE-CMA, being more resistant to digestion in IgE-CMA.
Our in-depth cellular analyses showed that in children with CM-FPIES, isolated PBMC were unable to secrete significant amounts of Th cytokines upon CM stimulation, and that Th memory cell proliferation was not detectable after stimulation with CM. Conversely, in children with IgE-CMA, significant secretions of IL-5, IL-13, IFN
\(\upgamma\) and to a lesser extent of IL-17 were found, and proliferation of Th cells was observed. Our results are not in line with those of Morita et al. showing that reactivated PBMC from patients with non-IgE CMA produce high levels of Th2 and Th1 cytokines [
24]. However, their patients were younger and had heterogeneous phenotypes, with 52% having FPIES, and 41.5% and 6.5% presenting food protein-induced proctocolitis or enteropathy syndrome, respectively. In addition, the authors did not report any CM-induced cytokine secretion in their IgE-CMA patients. Caubet et al. found significant secretion of IL-5, IL-13 and IFN
\(\upgamma\) after casein stimulation in children with both CM-FPIES and IgE-CMA, and low secretion of IL-10. However, Th2 cytokine secretion was comparable between children with CM-FPIES and control subjects tolerating CM [
10]. Differences in the age or phenotype of the patients, or the fact that PBMC were obtained after the OFC in the study by Caubet et al., may explain the differences with our results. In addition, our patients with CM-FPIES reacted during the OFC without having any detectable specific memory Th cells before the OFC. This suggests that these cells may not be involved in clinical reactivity, supporting that subjects with active FPIES do not have elevated numbers of food responsive T-cells compared to healthy control subjects before or after the OFC [
9]. In line with previous studies, we found significant allergen-specific secretion of the pro-inflammatory cytokines IL-6 and TNF-
\(\upalpha\) in children with CM-FPIES, albeit lower than that observed in IgE-CMA patients [
10].
The presence of increased numbers of eosinophils and other plasma cells in the lamina propria of patients with FPIES supports the presence of neutrophils, eosinophils and other mononuclear cells found in faecal mucus of positive challenge cases [
35,
36]. Faecal extracts obtained after milk challenge have shown a high concentration of eosinophil-derived neurotoxin that might be due to a reduction in epithelial barrier function and increased eosinophil degranulation [
37]. All these observations may question the role of the adaptive immune response in FPIES, in line with a recent hypothesis suggesting that FPIES resembles the innate response to bacterial infection and may result from abnormalities in the innate immune system, which misrecognizes specific foods [
38] and/or which may over-respond to a dysbiotic microbiota, a question that has not been assessed to date. However, our preliminary cytometry analysis of biopsy samples found activated Th1, Th2 and Th17 cells in the mucosa of active FPIES patient, suggesting involvement of the local adaptive immune system in the pathophysiology of FPIES. This may largely explain the specificity of the clinical response, which is triggered only by specific foods, but also the occurrence of symptoms restricted to the gastrointestinal tract and their delayed onset. Cellular analysis of biopsy samples also revealed the presence of other cell types in the mucosa of patients with active FPIES, including ILC. These mucosal ILC mainly have a mixed ILC2/ILC1 phenotype, probably induced by the local inflammatory microenvironment, reflecting ILC plasticity in tissue [
39]. These results are in line with the recently reported systemic antigen-specific activation of innate cells associated with positive food challenge [
9], but ILC were not assessed. In this latter study, a systemic antigen-specific activation of innate cells, involving monocytes, neutrophils, eosinophils, and NK cells, was associated with a positive food challenge. In this study and ours, the small sample sizes preclude definitive conclusions, and the results need to be confirmed.
No predictive biomarkers are available to date to improve the diagnosis of active FPIES, and to avoid a stressful OFC. A trend to increased serum glutamic oxaloacetic transaminase and lactate dehydrogenase levels in resting conditions was noted, compared with normal ranges in most of the patients who further experienced a positive OFC or acute accidental episodes, which may suggest some intestinal cellular damage [
38]. However, the predictive value of this increase has to be validated. Our non-targeted metabolomics approaches performed using plasma collected before the OFC show that CM-FPIES patients are characterized by a specific metabolic profile, for example with lower concentrations of some fatty acids in plasma. Interestingly, besides the role of fatty acids in membrane biosynthesis and energy supply, interference with their endogenous synthesis has profound effects on the metabolic programming of T cells and finally on the development of Th, notably Th17, and Treg cells. In fact, the glycolytic-lipogenic axis is crucial for Th17 development, but not for that of Treg cells, which require exogenous fatty acids [
40‐
42]. Moreover, protein acetylation, N-myristoylation and palmitoylation, which depends on the corresponding availability of fatty acids, are crucial for many T cell functions, as for example palmitoylation of Ras [
43] or N-myristoylation LcK [
44] that were shown to be necessary for T-cell activation after TCR engagement. This, like altered metabolism of amino acids, purine compounds or vitamins, clearly warrants further investigation with a larger well-characterized cohort.
Our study has several limitations. First, the sample size is small and we couldn’t include healthy non-atopic age-matched control children due to ethical reasons. However, our analyses were robust and included all major CM constituents in a well-phenotyped population. In addition, most of our children with CM-FPIES tolerated small amounts of CM, and thus may be outgrowing the disease. Their immunologic response may therefore differ from that of children with active disease. It is not recommended to perform an OFC for the diagnosis of FPIES, so further analysis should be performed shortly after an acute episode in patients with active CM-FPIES. Finally, children with IgE-CMA tolerated baked milk on the day of their OFC, thus their allergy was probably less severe than those who cannot tolerate either raw or baked milk. These children may have higher levels of Ig, particularly IgG4 subtypes, than those who are allergic to both forms of CM.
Authors’ contributions
KAP, GL, CD designed the study, analyzed the data, interpreted the results, drafted the manuscript. SB, DdB, FL, PS, PD recruited and diagnosed the patients, performed OFC and collected blood samples. KAP, HB, SAL, NC, SH produced the reagents and analyzed the cellular and humoral responses. FC, CJ and FF performed and interpreted the metabolomics analysis. All authors read and approved the final manuscript.