Project description:<h4><strong>BACKGROUND: </strong>IgE-mediated cow's milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which β-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies.</h4><h4><strong>METHODS: </strong>We report a longitudinal <em>in vivo</em> murine model, in which two mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n = 6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further extrapolated to fecal microbiome-metabolome data from our population of IgE-CMA (n = 22) and healthy (n = 23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes.</h4><h4><strong>RESULTS: </strong>During mice sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. We confirmed microbial dysbiosis, and its associated effect on metabolic alterations in our patient cohort, through <em>in vitro</em> digestions and multi-omics, which was accompanied by metabolic signatures of low-grade inflammation.</h4><h4><strong>CONCLUSION: </strong>Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.</h4>

Project description:Cow milk (CM) allergy is the most prevalent food allergy in young children in the US and Great Britain. Current diagnostic tests are either unreliable (IgE, skin prick test), or resource-intensive with risks (food challenges). Here we set out to determine if allergen-specific T cells in cow milk allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. Starting from cow milk extract, we mapped T cell responses to a set of reactive epitopes that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing allergic from non-allergic subjects. Using single-cell RNA and paired TCR sequencing we detected significant changes in the transcriptional program and clonality of cow milk antigen-specific (CM+) T cells elicited by the pool in allergic vs. non-allergic subjects ex vivo. CM+ T cells from allergic subjects had increased percentages of FOXP3+ over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells (Tregs) that have suppressive activity, but CM+ FOXP3+ cells from allergic subjects showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared to non-allergic subjects, suggesting that these are not conventional Tregs. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to utilize surface markers (CD25, CD127, CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from CMA subjects in an independent cohort, which could be used for diagnostic purposes. Looking specifically for Th2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in CMA subjects and that had high expression of Th2 cytokines and pathogenic Th2/Tfh markers. Overall, these findings suggest that there are several differences in the phenotype of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.

Dataset Information

Specific Gut Microbiome Signatures in Children with Cow’s Milk Allergy

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