Food-allergic patients: from phenotype to endotype for biomarker discovery. (Doctoral thesis)
- Molecular and Translational Allergology
Food allergy (FA), a pathology summarizing immune-mediated responses directed against specific foods, affects more than 250 million people around the world. Rising case figures, exacerbated disease severity and escalating socioeconomic burdens are calling for effective countermeasures to break this serious progression. The present PhD thesis focused on IgEmediated FA, commonly characterized by Th2-skewed and inflammatory responses. Patients differ markedly based on their clinical phenotypes, such as levels of clinical signs, threshold doses, reaction delay and cross-reactivity, pointing to entities of disease endotypes. The aim of this translational project was to improve existing and identify novel ex-vivo FA biomarkers. In study 1, a new method to calibrate functional biological assays resulted in predictive tool for clinical reactivity in FA patients. The clinical model was on shrimp allergy (n=20 patients) because the main allergen, tropomyosin, has striking similarity to other non-allergenic homologs. Allergenic shrimp and non-allergenic chicken tropomyosin qualified as clinically validated opponents. The activation of both human basophils (BAT, flow cytometry) and humanized rat basophil leukemia (RBL) cells, titrated on the pair of tropomyosins, allowed scaling of the effector cell reactivity in a semi-quantitative way. This calibration enabled to predict reliably the allergenicity of mealworm, a new food allergen. Molecule-calibrated, basophil assays represent an advanced biomarker approach towards predicting clinical reactivity and cross-reactivity, while outperforming established IgE-binding assays. In study 2, scaling basophil responses towards food allergen metabolites yielded a new biomarker approach to predict clinical outcome of FA events. Peanut allergy (PA) served as suitable model (n=40 patients), as it is one of the most prevalent and severe FA but also, as it involves important allergens of pronounced stability. A healthy discovery cohort represented the basis for this innovative study. For the first time, peanut peptides appeared in human blood upon ingestion, identified after co-immunoprecipitation and mass spectrometric (MS) analysis. Simulated digests validated the peptides identified in-vivo, by further MS-analyses. BAT, calibrated with ex-vivo digest peptides, surpassed IgE-assays in stratifying patients clustered by threshold dose reactivity as well as reaction delay. Thus, food allergen digestion and bioavailability contributes to clinical outcome. Calibrating BAT using digest peptides provides a novel way to endotype FA patients, based on metabolite-induced effector cell reactivity. In study 3, deep blood immune signatures in peripheral samples during diagnostic food challenges (OFC) emerged as a novel biomarker area to predict clinical symptoms and threshold levels. The used FA model was on PA (n=26 patients) because there, OFC are often necessary to diagnose the disease/progression and since recently, to monitor immunotherapy treatment.
During peanut-OFC, immune alterations along with symptom development appeared from single-cell mass cytometry analyses, based on a comprehensive panel of 35 surface markers. No immune signature changes occurred in healthy controls (n=7). Immunophenotypes developing during OFC discriminated patients with a positive outcome from those who did not reach threshold reactivity. A positive OFC correlated to a great homing marker expression, pointing to immune cell migration along with symptom onset. Immune signatures also identified subgroups of clinical reactivity, thresholds and organ involvement. Circulating signatures provide a basis for understanding immune markers of acute FA reactions, with an important outlook on novel predictive endotype marker to reduce OFC.
The present thesis provides novelty in FA biomarker research. Findings contribute to the movement “from phenotype to endotype”, with key perspectives on translational solutions regarding patient management, treatment and prevention.