Human breast milk miRNA, maternal probiotic supplementation and atopic dermatitis (oral presentation)

Title: Human breast milk miRNA, maternal probiotic supplementation and atopic dermatitis in offspring. Presenting author: Melanie Rae Simpson Background: Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy. This has been observed in a number of randomised clinical trials using a variety probiotics strains and supplementation regimes, and the strongest evidence comes from studies which include both pre- and post-natal supplementation. Results from our own study, Probiotics in Prevention of Allergy among Children in Trondheim (ProPACT), suggest that maternal supplementation alone is sufficient to prevent AD in offspring. The mechanisms behind this preventative effect are incompletely understood and may be partially mediated by changes in breast milk composition. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. In this study, we investigated breast milk miRNAs as possible mediators of the observed preventative effect of probiotics using samples collected during the ProPACT trial. Objective: To describe the miRNA profile of human breast milk, and to investigate the association between miRNAs, maternal probiotic supplementation and the development of atopic dermatitis in offspring. Methods: Fifty-four breast milk samples were obtained 3 months postpartum from women participating in the ProPACT trial, a double blinded, placebo-controlled randomised trial of perinatal probiotic supplementation. The probiotic milk consumed by the women randomised to the probiotic arm of the trial contained 5 x 1010 CFU of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb-12 and 5 x 109 CFU of L. acidophilus La-5. Breast milk samples were enriched for extracellular vesicles using ExoQuick™, total RNA was isolated and small RNA sequencing was conducted on the Illumina HiSeq 2000 platform. Differential expression of miRNAs was assessed for the probiotic vs placebo and the AD vs non-AD groups. Target gene and functional predictions were conducted using TargetScan and DAVID bioinformatics resources, respectively. The ProPACT trial is registered at ClinicalTrials.gov (identifier NCT00159523). Results: The RNA isolates obtained from the human milk samples contained between 12.4 and 247.5ng/µL of RNA, and were found to contain RNA fragments up to 1000nt in length which aligned primarily to rRNA, tRNA and miRNA. The miRNA profile revealed a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of the target genes for the 20 most highly expressed miRNAs revealed enrichment of a broad range of biological processes and molecular functions and significant overlap with genes that are upregulated in brain and epithelial tissues. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences. Conclusion: Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period.