Diet, Microbiota and Epigenetics as target for innovative strategies against food allergy: deciphering the protective mechanism of butyrate as crucial human milk effector

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

Commentary: Raw Cow Milk Consumption and Atopic March

We have appreciated the interest of Dr Baars et al. in our paper describing dietary prevention of atopic march (AM) in children affected by cow milk allergy (CMA) (1). They claimed a lack of information on raw cow milk (unpasteurized cow milk) in our paper. In support of this point, they mentioned the result of a pilot study involving nine CMA children (2) that were able to tolerate up to 50 mL of raw milk (about 1,750 mg of cow milk proteins). This result was not confirmed by a similar study where five children with IgE-mediated CMA were orally challenged in a double-blind fashion with raw untreated cow milk, pasteurized cow milk, and homogenized/pasteurized cow milk. An extensively hydrolysed casein formula served as placebo. All patients presented significant allergic reactions from the consumption of the above three types of milk, whereas no adverse reactions to placebo were observed. The authors concluded that children with CMA cannot tolerate raw or pasteurized milk (3). Although, selected components of raw milk may potentially influence the immune system, proof based on controlled studies in children are still lacking (4). The authors of the PARSIFAL study concluded that raw cow milk may contain numerous disease-causing pathogens and that consumption of raw milk cannot be recommended as a preventive measure for allergy (5). Accordingly, none of the claims made by the raw milk advocates (including the postulated preventive effect against allergy) withstand the FDA scientific scrutiny (6)

Protective effects of the postbiotic deriving from cow's milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes

: Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow's milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age

Excretion of dietary cow’s milk derived peptides into breast milk

Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (ten samples) after receiving an oral load of cow’s milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at 5 time points after drinking cow’s milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow’s milk-derived β-Lg f(125-135) and β-casein f(81-92) in breast milk

Food allergies: Novel mechanisms and therapeutic perspectives

Childhood food allergy (FA) rates have rapidly increased with significant direct medical costs for the health care system and even larger costs for the families with a food-allergic child. The possible causes of food allergy become the target of intense scrutiny in recent years. Increasing evidence underline the importance in early life of gut microbiome in the development of allergic diseases. There are a range of factors in the modern environment that may be associated with changes to both the gut microbiome and risk of FA, such as mode of delivery, antibiotic exposure, infant feeding practices, farming environment, and country of origin. Knowledge of the relationship between early life gut microbiome and allergic diseases may facilitate development of novel preventive and treatment strategies. Based on our current knowledge, there are no currently available approved therapies for food allergy. More studies are needed to evaluate the safety and efficacy of allergen-specific and allergen-nonspecific approaches, as well as combination approaches

The Potential Immunonutritional Role of Parmigiano Reggiano Cheese in Children with Food Allergy

Parmigiano Reggiano (PR) is a ripened cheese with high nutritional value. Throughout ripening the bacteria contained in PR promote an extensive hydrolysis of cow’s milk proteins resulting in peptides that exhibit positive immunoregulatory activities. Additional modulatory activities on immune system are induced by butyrate, a short chain fatty acid widely expressed in PR. These findings suggest a possible immunonutritional role for PR able to stimulate oral tolerance in children with food allergy (FA)

New Insights and Perspectives in Congenital Diarrheal Disorders

Purpose of Review We highlight new entities of congenital diarrheal disorders (CDDs) and progresses in understanding of functionally related genes, opening new diagnostic and therapeutic perspectives. Recent Findings The more significant advances have been made in field of pathogenesis, encouraging a better under- standing not only of these rare diseases but also of more com- mon pathogenetic mechanisms. Summary CDDs represent an evolving group of rare chronic enteropathies with a typical onset early in the life. Usually, severe chronic diarrhea is the main clinical manifestation, but in other cases, diarrhea is only a component of a more complex systemic disease. The number of conditions has gradually increased, and many new genes have been indentified and functionally related to CDDs, opening new diagnostic and therapeutic perspectives. Advances in molecu- lar analysis procedures havemodified the diagnostic approach in CDDs, leading to a reduction in invasive and expensive procedures. Keywords Chronic diarrhe

Immunonutrition for Pediatric Patients With Cow's Milk Allergy: How Early Interventions Could Impact Long-Term Outcomes

Cow's milk allergy (CMA) is one of the most common food allergies and one of the main causes of food-induced anaphylaxis in the pediatric age. Moreover, up to 45% of CMA children develop other atopic manifestations later in life, a phenomenon commonly named atopic march. Thus, CMA imposes a significant cost to health care systems as well as to families, and has emerged as one of the most expensive allergic diseases. The immunonutrition strategy builds its foundation on the ability of selected dietary factors to modulate immune system development and function. Recent studies highlighted the potential of immunonutrition in the management of CMA. This review is focused on the mechanisms and long-term clinical outcomes of the immunonutrition approach in children with CMA. Introduction Much has changed during the recent decades regarding prevalence, persistence, and severity of clinical features and socio-economic burden of food allergy (FA) that currently affects up to 10% of children living in Western countries (1). Based on the immune mechanisms, FA may be classified as IgE-mediated, non-IgE-mediated, or a combination of both pathways (2). In addition, children presenting with FA in early life are at increased risk of developing other allergic manifestations later in life, such as allergic asthma and rhinitis, a phenomenon commonly named atopic march (AM) (3). With an estimated prevalence of up to 3%, cow's milk allergy (CMA) is one of the most common FA and one of the main causes of food-induced anaphylaxis in the pediatric age (4). This condition imposes a significant cost to health care systems as well as to families, and has emerged as one of the most expensive allergic diseases (4). Furthermore, early life CMA could be the first step of AM, which affects up to 45% of CMA children, also after the acquisition of immune tolerance to cow's milk proteins (CMP) (5–8). Both CMA and AM derive from a negative interaction between genetic and environmental factors (3) resulting in alteration in the gut microbiome (GM) and in immune system dysfunction. These modulatory effects are at least in part mediated by epigenetic mechanisms, and are now emerging as potential targets of intervention to facilitate the immune tolerance acquisition and to limit the occurrence of AM in CMA patients (5, 9–11). The traditional dietary management of CMA has greatly changed in the last few years, moving from a passive approach based on the strict elimination diet of CMP-containing foods, to a proactive one, able to change the CMA course (12). The discovery of the pivotal role of selected dietary factors in influencing immune system development and function has introduced the immunonutrition concept. The application of the immunonutrition approach in the management of CMA is paving the way to “active diet therapy,” an integrated dietary strategy able to facilitate the acquisition of immune tolerance and to prevent the occurrence of AM (5, 9, 13–15). The modern dietary management in CMA pediatric patients is focused on three targets: • Dietary education (allergen avoidance and healthy diet) • Ensure adequate intake of macro and micro-nutrients (stimulation of optimal body growth and development) • Active diet therapy (stimulation of immune tolerance and protection against AM occurrence). The immunonutrition approach could be promoted for all three of these targets. This review is focused on the objectives and long-term clinical outcomes of the immunonutrition approach in children affected by CM

Body growth assessment in children with IgE-mediated cow's milk protein allergy fed with a new amino acid-based formula

Amino acid-based formula (AAF) is a relevant dietary option for non-breastfed children. The present study was designed to evaluate the body growth pattern in cow's milk protein allergy (CMPA) children treated for 6 months with a new AAF