Deep analysis of immune response and metabolic signature in children with food protein induced enterocolitis to cow's milk

Karine Adel-Patient, Guillaume Lezmi, Florence Anne Castelli, Sibylle Blanc, Hervé Bernard, Pascale Soulaines, Pascale Dumond, Sandrine Ah-Leung, Florence Lageix, Delphine de Boissieu, Naima Cortes-Perez, Stéphane Hazebrouck, François Fenaille, Christophe Junot, Christophe Dupont, Karine Adel-Patient, Guillaume Lezmi, Florence Anne Castelli, Sibylle Blanc, Hervé Bernard, Pascale Soulaines, Pascale Dumond, Sandrine Ah-Leung, Florence Lageix, Delphine de Boissieu, Naima Cortes-Perez, Stéphane Hazebrouck, François Fenaille, Christophe Junot, Christophe Dupont

Abstract

Background: Food Protein-Induced Enterocolitis Syndrome (FPIES) is considered to be a non-IgE mediated food allergy. However, its pathogenesis remains poorly understood and biomarkers are lacking. We aimed to perform in-depth characterization of humoral and cellular immune responses in children with cow's milk (CM)-FPIES and investigated whether there is a FPIES metabolomic signature.

Methods: Children with CM-FPIES and control subjects with an IgE-mediated CM allergy (IgE-CMA), both avoiding CM, were recruited on the day of an oral food challenge. Blood samples were collected before the challenge. Total and specific levels of IgE, IgG1-4, IgA, IgM and IgD to various whey and casein allergens and to their gastroduodenal digestion products were measured in plasma, using plasma from CM-tolerant peanut allergic patients (IgE-PA, not avoiding CM) as additional controls. Cytokine secretion and cellular proliferation were analyzed after stimulation of PBMC with different CM allergens. Metabolomic profiles were obtained for plasma samples using liquid chromatography coupled to high-resolution mass spectrometry.

Results: Nine children with CM-FPIES and 12 control subjects (6 IgE-CMA and 6 IgE-PA) were included. In children with CM-FPIES, total Ig concentrations were lower than in control subjects, specific Ig against CM components were weak to undetectable, and no specific IgE against CM digestion products were detected. Moreover, in CM-FPIES patients, we did not find any Th cell proliferation or associated cytokine secretion after allergen reactivation, whereas such responses were clearly found in children with IgE-CMA. Plasma metabolic profiles were different between CM allergic patients, with significantly lower concentrations of various fatty acids and higher concentrations of primary metabolites such as amino acids in CM-FPIES compared to IgE-CMA patients.

Conclusions: In CM-FPIES, both humoral and cellular specific immune responses are weak or absent, and this is not related to CM avoidance. A metabolomic signature was identified in patients with CM-FPIES that may be useful for the diagnosis and management of this disease.

Keywords: Cellular immunity; Cow’s milk; FPIES; Food allergy; Humoral immunity; Mechanisms; Metabolomics; Non-IgE.

Figures

Fig. 1
Fig. 1
Total IgE, IgGs, IgM and IgA (ng/mL) in children with CM-FPIES (red), IgE-CMA (blue) or IgE-PNA (green). *p < 0.05, **p < 0.01, ***p < 0.001 using Kruskal–Wallis and Dunn’s multiple comparison test
Fig. 2
Fig. 2
a Specific IgE (IU/mL) in patients with CM-FPIES (red), IgE-CMA (blue) or IgE-PA (green) assayed using immunoenzymatic assays. *p < 0.05, **p < 0.01, ***p < 0.001 using the Kruskal–Wallis test and Dunn’s multiple comparison test. b Representative IgE immunoblot of CM proteins using plasma from children with IgE-CMA (left) or FPIES-CMA (right). Lane 1: MW markers, Lane 2: CM proteins
Fig. 3
Fig. 3
Specific IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D) and IgA (E) in patients suffering from CM-FPIES (red), IgE-CMA (blue) or IgE-PNA (green). Values are given as µg/mL except for IgA where values are given as milli-Absorbance Unit at 414 nm. Bars indicate medians. Statistics *p < 0.05, **p < 0.01, ***p < 0.001 using Mann–Whitney test. “a” indicates a statistical difference using the Kruskal–Wallis test and Dunn’s multiple comparison test
Fig. 4
Fig. 4
Specific IgG4 against gastroduodenal digestion products. a Electrophoresis of gastroduodenal CM protein digestion products: CM was digested for 0 to 120 min under physiological conditions using pepsin (T0 to T120, gastric digestion). Digestion products obtained after 60 min of pepsin digestion (T60 gastric, TO’) were then submitted to trypsin/chymotrypsin physiological digestion for 1 to 30 min (T1′ to T30′, gastroduodenal digestion). b Undigested CM proteins (T0), gastric digestion products obtained at 5 (T5) and 60 min (T60) and gastroduodenal digestion products obtained after 60 min of gastric digestion and 30 min of duodenal digestion (T30′) were immobilized on plates, and specific IgG4 were assayed using individual plasma (FPIES-CMA: red, IgE-CMA: blue, IgE-PNA: green). Absorbance obtained at T0 was used as an internal reference for each patient (100%). Statistics: bars and asterisks indicate statistical differences between specified time points and T0 in one group of patients (*p < 0.05, **p < 0.01; Wilcoxon sign rank test) or significant differences between groups at a given time of digestion (*p < 0.05, **p < 0.01; Mann–Whitney t test)
Fig. 5
Fig. 5
IL-13 (a), IFNγ (b) and IL-6 (c) secreted after specific reactivation of PBMC from IgE-CMA (blue) or FPIES-CMA (red) patients. PBMC were stimulated for 6 days with 50 µg/mL of purified allergens and cytokines were assayed in supernatants. Results are expressed as individual values and medians (bar) obtained for the PBMC from IgE-CMA (n = 6) or FPIES-CMA (n = 9) patients. * indicates a difference between control (PBS and/or Ara h 2) and stimulated PBMC in the same group of patients (p < 0.05 using Wilcoxon sign rank test); Significant difference or trends between IgE-CMA and FPIES-CMA patients are also mentioned with the associated p value (Mann–Whitney t test)
Fig. 6
Fig. 6
Analysis of proliferative T cells in CMA patients after allergen reactivation. PBMC from IgE-CMA (a) or FPIES-CMA (b) patients were labelled with CFSE and then cultured for 6 days with PBS or allergens purified from cow’s milk. Cells were then recovered and labelled with anti-human CD4. Among SSC-FSC gated cells, single cells were selected and analyzed for CD4 expression and CFSE signal. Proliferative T cells are defined as CD4+CFSElow cells within a selected population (red square). Proliferative cells after reactivation with PBS, BLG, caseins or α-lact are shown
Fig. 7
Fig. 7
Cellular analysis in intestinal biopsies and PBMC from active versus controlled FPIES. Cellular analysis was performed on cells extracted from sigmoid biopsies obtained from children presenting controlled FPIES (non-active) or active FPIES. Cells were labelled and analyzed by flow cytometry using a NovoCyte flow cytometer and analysis was performed using NovoExpress™ software (Version 1.2.1; ACEA Biosciences, Inc.). Percentages of ILC1; ILC2, or ILC3 cells among parent cells (CD45+CD127+) and grandparent cells (Lin−SSClow) are indicated in colour and in brackets, respectively. Lin: mix of labelled anti-human CD3, CD11c, CD14, CD16, CD19, CD56, FcεRIα, CD1a, CD123
Fig. 8
Fig. 8
Plasma metabolites in samples from CM-FPIES, IgE-CMA or resolved IgE-CMA patients. a Discriminant metabolites between CM-FPIES (red symbols), IgE-CMA (dark blue symbols) and IgE-resolved (light blue symbols). a Fatty acids, b amino acids and their derivatives, c purine metabolites or vitamins. The p values of nonparametric Mann–Whitney statistical test are indicated

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