Gut microbiota from infant with cow’s milk allergy promotes clinical and immune features of atopy in a murine model

Numerous clinical and epidemiological studies suggest an associ‐ ation between abnormal development of the gut microbiota in early life and development of clinical manifestations related to allergy including cow's milk allergy (CMA). Despite advances, at present there is no consensus on a clear signature of a CMA microbiota due to variations in allergic phenotypes and methods used. Moreover, it remains unclear whether the observed microbial alterations are a cause or a consequence of allergy. Here, we report the effects of fecal microbiota transfer (FMT) of a healthy control (HC) and CMA infant in a gnotobiotic murine model of CMA. Detailed methods are provided in Appendix S1.


Gut microbiota from infant with cow's milk allergy promotes clinical and immune features of atopy in a murine model
Infants with CMA (n = 5), under the care of Great Ormond Street Hospital, London, UK, were recruited alongside healthy controls (n = 6) from the community (Tables S1 and S2) (REC 14/LO/0364).
Stool samples from the infants with CMA analyzed by 16S rRNAgene sequencing showed significant increased bacterial diversity, decreased abundances of Bifidobacterium spp., and increased abundances of Lachnospiraceae spp., and one of its genera, that is, Eisenbergiella (FDR < 0.05), was compared with HC ( Figure 1A-C).
These specific gut microbiota signatures corroborate previous findings comparing CMA infants with breastfed HC. 1,2 Significant increased levels of bacterial-derived short chain fatty acids (butyrate, iso-valerate, and iso-butyrate) were observed in infants with CMA compared with HC ( Figure S1). No significant differences in stool pH, acetate, D-lactate, L-lactate, sIgA, calprotectin, and eosinophil-derived neurotoxin levels were recorded ( Figure S1). All these parameters were supplemented into a principal component analysis (PCA) of microbial compositions that revealed distinct patterns between CMA and HC ( Figure 1D).
We selected 2 infants, 1 with CMA and the other with HC (infants 3 and 4), matched for age (9 and 10 months old), gender (female), and delivery mode (cesarean section) for FMT into threeweek-old germ-free mice (C3H/HeN). Following 12 days of microbiota establishment, mice received, once a week for 5 weeks, Following oral allergen sensitization, CMA microbiota was associated with diarrhea-related symptoms (Figure 2A), with higher fecal scores (reflecting softer to diarrheic stools/ anal inflammation) that persisted at least 24 hours for the CMA-S group compared with HC-S group (P < 0.001). No significant differences in fecal scores were recorded between HC-S and HC-NS, which may indicate a protective effect of healthy microbiota upon allergen exposure as we and others observed previously. 3,4 In addition, clinical scores (scratching/ puffiness/ loss of mobility) after oral challenge with β-lactoglobulin (BLG) were significantly higher in CMA-S group versus CMA-NS and HC-S groups ( Figure 2B). Minimal differences in mouse mast cell protease 1 (mMCP-1) and allergen-specific sensitization markers were observed between the two fecal transfers ( Figure 2C-E), which contrasted with the increases observed in total IgE levels and total IgG1/IgG2a ratio in CMA-S and CMA-NS groups compared with HC-S and HC-NS groups, respectively ( Figure 2F,G). The latter observations were consistent with increased gata3 mRNA expression in the colon ( Figure 2H), which is a marker of Th2 lymphocytes. Interestingly, despite nonsignificant, colonic fcγRIII mRNA expression was increased in CMA-S mice ( Figure 2H, P = 0.07). FCγRIII is an IgG1 binding/activating receptor, and its increased expression may imply a pathway linked to IgG1 and basophils and the potential development of anaphylaxis. 5 Literature indicates that germ-free mice have increased total IgE, which can be normalized upon colonization with commensal microbiota until 8 weeks of age. 6 We found that only colonization with HC-associated microbiota led to low total IgE levels similar to that of nonsensitized control. This may be due to the observed enrichment of protective bacteria including bifidobacteria and Anaerostipes spp. Our findings confirm a Th2-type immune orientation following FMT with CMA microbiota. This Th2 profile was associated with minimal differences in mMCP-1 levels or allergen-specific immunoglobulin levels between the CMA-S and HC-S groups, raising the hypothesis that non-IgE mediated immunity might be at play. Indeed, patients with non-IgE-dependent food-allergy predominantly  We noted an increase in colonic foxp3 mRNA gene expression in CMA-S group compared with HC-S group. Foxp3 has been associated with the production of Th2 cytokines in several cell lines, including Foxp3 + Gata3 + cells, as well as with regulatory T-cells (Treg).
If it is associated with Treg cells, the increased foxp3 expression may reflect a regulatory mechanism in response to the enhanced Th2 profile of the CMA-S group. Another explanation of the increased foxp3 expression could be linked to the increase in Lachnospiraceae spp. and the associated increase in butyrate ( Figure S3), which has been implicated in the induction of Foxp3 Treg cells. 10 However, in our study, this was not associated with protection against allergic sensitization.
In summary, we demonstrate for the first time that infant microbiota with a low bifidobacteria/Lachnospiraceae ratio orients the