Microbiota and Cow’s Milk Tolerance

BACKGROUND The default immunological response to dietary antigen exposure via the gastrointestinal tract is oral tolerance, while failure to develop or breakdown of tolerance results in food allergy, but the mechanism leading to this outcome in some individuals is not fully understood. It has been suggested that failure to develop tolerance is linked to an imbalance in intestinal microbiota caused by environmental or lifestyle factors.

Cow’s milk allergy is the most common food allergy in infants and young children. The best way to prevent cow’s milk allergy in infants is by exclusive breastfeeding until introducing solid foods. However, in many cases breast milk must be supplemented with or replaced by infant formula, which is most often based on cow’s milk proteins.

The recommendation for allergy prevention in non-exclusively breastfed infants at high risk of developing allergy is highly debated. Infant formula based on hydrolysed cow’s milk have been suggested as a good option due to their reduced allergenicity, but concerns have been raised about the possible reduced preventive capacity of these products.

Alternative types of processing, such as heat-treatment, are being investigated with the aim of reducing allergenicity while maintain tolerogenicity, with the ultimate goal of producing safe and efficient products for prevention and treatment of cow’s milk allergy. To accomplish this, more knowledge on how tolerance development is influenced by both protein and host-related factors is essential.

AIM AND METHODS The aim of this project was to investigate whether and how (1) the physicochemical characteristics of whey products and (2) the gut microbiota composition of the host, independently and in combination, affect oral tolerance development, as well as the underlying mechanisms. This was investigated by inducing tolerance in Brown Norway rat models through oral administration of whey products with different physicochemical characteristics obtained by applying either heat-treatment (Manuscript I) or hydrolysis (Manuscript III).

The association between microbiota composition and acute immune regulation (Manuscript II) as well as tolerance development (Manuscript III) was assessed by manipulating the microbiota of rats by the antibiotic amoxicillin. The effect of amoxicillin on intestinal microbiota composition as well as on host intestinal permeability, morphology, humoral and cellular immune regulation was analysed.

Finally, the effect of the whey products on the growth of gut bacteria derived from healthy infant donors was evaluated in an in vitro incubation study (Manuscript III). RESULTS Results presented in Manuscript I, revealed that the capacity of whey to prevent sensitisation of naïve rats and to desensitise already sensitised rats was not reduced by mild heat-treatment.

Heat-treatment reduced the intraperitoneal sensitising capacity, but had no effect on oral sensitisation. However, oral provocation with heat-treated whey resulted in milder allergic symptoms compared to unmodified whey. Protein uptake studies showed that heat-treatment changed the uptake route of whey with less being absorbed through the epithelium but more into the Peyer’s patches.

Results presented in Manuscript III, revealed that moderate hydrolysis did not reduce the primary preventive capacity of whey. A very diverse response was observed in the group administered with intact whey, and in that group sensitisation was not significantly different from the control group, which did not receive any product for prevention.

To our knowledge, the present study is the first to show that moderately hydrolysed whey protein is superior to intact whey protein for preventing whey-specific IgE sensitisation. Results presented in Manuscript II, revealed that daily intra-gastric administration of amoxicillin resulted in immediate and dramatic shifts in microbiota composition, characterised by reduced within sample (α) diversity, reduced variation between animals (β diversity), increased relative abundance of Bacteroidetes and Gammaproteobacteria, with concurrent reduction of Firmicutes, compared to the control group.

After one week, the total fecal IgA level, relative abundance of small intestinal FoxP3+ regulatory T cells and goblet cell numbers were higher in the amoxicillin group compared to controls. Results presented in Manuscript III revealed that amoxicillin-induced perturbation of the gut microbiota one week prior to and during tolerance induction did not affect the development of tolerance.

In the group administered with the extensively hydrolysed whey (the product with the weakest tolerance inducing capacity) amoxicillin treated rats were actually better protected against allergic reactions than those with a conventional microbiota. In the light of epidemiological studies showing an association between early life antibiotic consumption and the development of food allergies, the observation that amoxicillin-induced perturbation of the gut microbiota promotes acute immune regulation (Manuscript II) and possibly tolerance development (Manuscript III) warrants further investigation.

Finally, In vitro incubation of infant faecal microbiota with whey products with different degree of hydrolysis included in Manuscript III indicated that moderately hydrolysed whey products promoted the expansion of the genus Enterococcus. CONCLUSION AND PERSPECTIVES Collectively, these results highlight both heat-treatment and moderate hydrolysis as potential methods for producing efficient and safe cow’s milk-based products intended to prevent cow’s milk allergy in infants at high risk of CMA, regardless of their gut microbiota composition.

However, possible effects of hydrolysed products on infant microbiota composition indicated by the in vitro incubation study warrants further investigation.