Paparo, Lorella (2018) Diet, Microbiota and Epigenetics as target for innovative strategies against food allergy: deciphering the protective mechanism of butyrate as crucial human milk effector. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: Diet, Microbiota and Epigenetics as target for innovative strategies against food allergy: deciphering the protective mechanism of butyrate as crucial human milk effector
Date: 2 December 2018
Number of Pages: 98
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Mediche Traslazionali
Dottorato: Medicina clinica e sperimentale
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
Berni Canani, RobertoUNSPECIFIED
Date: 2 December 2018
Number of Pages: 98
Keywords: : immune tolerance, gut microbiota, breast milk, dysbiosis, gut microbiota metabolites, short chain fatty acids, butyrate
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 05 - Scienze biologiche > BIO/12 - Biochimica clinica e biologia molecolare clinica
Area 06 - Scienze mediche > MED/38 - Pediatria generale e specialistica
Date Deposited: 19 Dec 2018 12:44
Last Modified: 30 Jun 2020 08:50

Collection description

Objectives and study: The dramatic increase in food allergy prevalence and severity globally requires effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota function and structure , and alterations in the gut microbiome composition (dysbiosis) early in the life have a pivotal role in the development of food allergy. Many environmental factors, including a low-fibre/high-fat diet, caesarean delivery, antiseptic agents, lack of breastfeeding, and drugs can induce gut microbiome dysbiosis, and have been associated with food allergy. New technologies and experimental tools have provided information regarding the importance of metabolites generated from dietary nutrients and selected probiotic strains that could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence has provided useful information on optimal bacterial species/strains, dosage and timing for intervention. The increased knowledge of the crucial role played by nutrients and gut microbiota-derived metabolites is opening the way to a post-biotic approach in the stimulation of immune tolerance through epigenetic regulation. The mechanisms of the preventive effect of breast milk (BM) against food allergy (FA) are still largely undefined. The short chain fatty acid butyrate has a pivotal role in immune tolerance. We aimed to see whether BM butyrate concentrations are able to exert immune and non-immune tolerogenic effects in human enterocytes, peripheral blood mononuclear cells (PBMCs) from children affected by FA, and in FA animal model. Methods: Mature BM butyrate concentrations from 98 healthy women (aged 21-42 yrs) were assessed by gas-chromatography. Dose-dependent effects of butyrate in human enterocytes (Caco-2 cells) on immune (beta-defensin-3, HBD-3) and non-immune (mucus production; mucin 2, MUC2; tight-junction proteins, zonulin and occludin) were analyzed. PBMCs from 6 children with challenge-proven FA (2 cow milk allergy, 2 peanut allergy, 2 egg allergy; age range 1-5 yrs) were stimulated with b-lactoglobulin (BLG;100μg/ml), peanut extract (PE;200μg/ml) or ovalbumin (OVA;200μg/ml) in the presence or absence of butyrate. Expression and DNA methylation rate of IL4, IL5, IL-10, IFN-γ and Treg-specific-demethylated region (TSDR) Forkhead box Protein 3 (FoxP3) were assessed. Four-weeks-old female C3H/HeJ mice were used in FA animal model. Two weeks before first sensitization, oral gavage with 30 mg/kg/d of butyrate was started and continued during the whole study. Mice were sensitized orally on day 0, 7, 14, 21, 28 with 20 mg of BLG or 1 mg of OVA or 12 mg of PE mixed with 10 µg cholera toxin (CT) as adjuvant. Control mice receive CT only. On day 35 mice were challenged by gavage with BLG (50mg) or OVA (5mg) or PE (36mg). Anaphylaxis score and rectal temperature were assessed for 1 h after challenge and blood samples were collected to measure MCPT-1 and sIgE. After 24h, mice were sacrificed, colon, ileum and spleen were collected. Results: Mean butyrate concentration in BM was 0.75 mM (SD±0.15). Butyrate stimulates HBD-3, mucus production and MUC2, zonulin and occludin expression with maximal effective doses between 0.75 and 1 mM in human enterocytes. PBMCs stimulation with BLG, PE, OVA resulted in a significant increase in IL-4 and IL-5 production. A significant inhibition of IL-4 and IL-5 production was observed with 0.75 mM butyrate. Butyrate stimulated, in a dose-dependent manner (maximal effective dose 0.75 mM), IL-10 and IFN-γ production through a demethylation of respective genes and TSDR FoxP3 demethylation. Pre-treatment with butyrate significantly reduced anaphylactic score, body temperature decrease, serum MCPT-1 and sIgE levels. Butyrate stimulated mucus and IL-10 and IFN-γ production and inhibited IL-4, IL-5 and IL-13 production. Conclusion: Our data support the role of butyrate as effective human milk component able to prevent food allergy through a wide range of immune and non-immune tolerogenic mechanisms. Understanding how nutrients and metabolites, or probiotics could influence gut bacteria communities and the immune system will contribute to building up a precision medicine approach for food allergy care.


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